BMP - a key signaling molecule in specification and morphogenesis of sensory structures

Vijay Kumar Jidigam

Umeå center for molecular medicine

Umeå 2016

Responsible publisher under swedish law: the Dean of the Medical Faculty

This work is protected by the Swedish Copyright Legislation (Act 1960:729)

ISBN: 978-91-7601-468-4

ISSN: 0346-6612

Cover page: Confocal image of elecrtroprated olfactory invaginated placode

showing GFP+ cells in green and nuclei stained with DAPI in grey.

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Umeå, Sweden, 2016

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i

Table of Contents

Table of Contents i Abstract iii Papers included in this thesis iv Abbreviations v Introduction 1

Cranial sensory placodes and their derivatives 1 Placode precursors in the neural plate border 2 Placode precursors are derived from a common pre-placodal ectoderm 4 Individual placode identity 7 Lens placode 7

Specification of lens placodal cells 8 Olfactory placode 10

Neurogenesis in the olfactory epithelium 11 Otic placode 12 Placode Invagination 14 Ectodermal thickening (placode) 14 Retina 16

Aims 19 Materials and methods 20

Functional experiments: 20 Electroporation 20 Chick explant assay 20 Whole embryo culture 21

Results 23 Paper I: BMP-Induced L-Maf Regulates Subsequent BMP-Independent

Differentiation of Primary Lens Fibre Cells 23 Paper II: Neural retina identity is specified by lens-derived BMP signals 27 Paper III: Apical constriction and epithelial invagination are regulated by BMP

activity 33 Discussion 39

Paper I: Lens 39 Paper II: Retina 42 Paper III: Placode invagination 46

Conclusions 54 Acknowledgements 55 References 58

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iii

Abstract

Cranial placodes are transient thickenings of the vertebrate embryonic head

ectoderm that will give rise to sensory (olfactory, lens, and otic) and non-sensory

(hypophyseal) components of the peripheral nervous system (PNS). In most

vertebrate embryos, these four sensory placodes undergo invagination. Epithelial

invagination is a morphological process in which flat cell sheets transform into

three-dimensional structures, like an epithelial pit/cup. The process of invagination

is crucial during development as it plays an important role for the formation of the

lens, inner ear, nasal cavity, and adenohypophysis. Using the chick as a model

system the following questions were addressed. What signals are involved in

placode invagination? Is there any common regulatory molecular mechanism for all

sensory placode invagination, or is it controlled by unique molecular codes for each

individual placode? Are placode invagination and acquisition of placode-specific

identities two independent developmental processes or coupled together? To address

this we used in vivo assays like electroporation and whole embryo culture. Our in

vivo results provide evidence that RhoA and F-actin rearrangements, apical

constriction, cell elongation and epithelial invagination are regulated by a common

BMP (Bone morphogenetic protein) dependent molecular mechanism. In addition,

our results show that epithelial invagination and acquisition of placode-specific

identities are two independent developmental processes.

BMP signals have been shown to be essential for lens development and patterning of

the retina. However, the spatial and temporal requirement of BMP activity during

early events of lens development has remained elusive. Moreover, when and how

retinal cells are specified, and whether the lens plays any role for the early

development of the retina is not completely known. To address these questions, we

have used gain- and loss-of-function analyses in chick explant and intact embryo

assays. Here, we show that during lens development BMP activity is both required

and sufficient to induce the lens specific marker, L-Maf. After L-Maf upregulation

the cells are no longer dependent on BMP signaling for the next step of fiber cell

differentiation, which is characterized by up-regulation of δ-crystallin expression.

Regarding the specification of retinal cells our results provide evidence that at

blastula stages, BMP signals inhibit the acquisition of eye-field character.

Furthermore, from optic vesicle stages, BMP signals emanating from the lens are

essential for maintaining eye-field identity, inhibiting telencephalic character and

inducing neural retina cells.

iv

Papers included in this thesis

This thesis consists of following papers

I. Pandit, T., V. K. Jidigam, Gunhaga, L. (2011). "BMP-induced L-Maf

regulates subsequent BMP-independent differentiation of primary lens

fibre cells." Developmental dynamics. 240(8):1917-28.

II. Pandit, T., Jidigam, V. K., Patthey, C., Gunhaga, L. (2015). "Neural

retina identity is specified by lens-derived BMP signals." Development

(Cambridge, England) 142(10): 1850-1859.

III. Jidigam, V.K., Srinivasan, R. C., Patthey C., Gunhaga L. (2015).

"Apical constriction and epithelial invagination are regulated by BMP

activity." Biology Open: bio. 015263.

v

Abbreviations CNS-Central nervous system

PNS-Peripheral nervous system

BMP-Bone morphogenetic protein

FGF-Fibroblast growth factor

PPE-pre-placodal ectoderm

EMT-Epithelial mesenchymal transition

Six- sine oculis homeobox

Eya-eye absent

Dach-Dachshund family transcription factor

St-Stage

Otx2-orthodenticle homeobox2

PMLC-phosphorylated myosin light chain

ROCK-Rho associated coiled-coil containing protein kinases

RhoA-Ras homolog gene family A

Maf-musculoaponeurotic fibro sarcoma oncogene homolog

Irx3- Iroquois homeobox gene

Pax- Paired box

Rx- Retinal anterior neural fold

Sox-SRY related HMG box

Dlx- Distal-less homeobox

Gbx2-Gastrulation brain homeobox 2

OV-optic vesicle

OVL-optic vesicle lens

LR-lens retina

RPE-retinal pigmented epithelium

GFP-green fluorescent protein

Ngn-neurogenin

Hes5-Hairy and enhancer of split5

MSX- muscle segment homeobox gene

Id3-inhibitor of DNA binding 3

SoHO1-sensory organ homeobox protein

IKNM-interkinetic nuclear migration

N-CAM-Neural cell adhesion molecule

Mitf- micropthalmia associated transcription factor

FoxG1-Forkhead boxG1

Smad-Sma-small body size, Mad-mother against decapentaplegic

Vsx2-visual system homeobox 2

Emx2-Empty spiracles homeobox 2

Gastrula stage= stage 4

Blastula stage=stage 2

Neural fold stage= stage 8

Neural tube stage=around stage 11

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1

Introduction

During early development, the vertebrate ectoderm becomes regionalized into

neural, neural plate border and epidermal domains. The neural plate border region is

located at the junction between the neural and epidermal ectoderm, and the

derivatives of this intermediate domain include cranial sensory placodes and neural

crest cells (Patthey, Edlund et al. 2009). The cranial placodes will give rise to

several structures with various cell types of functional diversity, including our

sensory organs. Although placode-derived structures are different in their functional

properties, they do share certain morphological similarities during early

development like columnar epithelia, invagination and delamination of neuroblasts

(Begbie and Graham 2001). The focus of my thesis has been i) to understand at what

stage and how the lens becomes independent of its initial inductive signals, ii) to

understand when and how the retinal cells are specified, and iii) to identify

mechanisms of placode invagination, i.e. whether it is regulated by common

molecular machinery or is controlled by unique molecular codes for each individual

placode.

Cranial sensory placodes and their derivatives

The cranial sensory placodes give rise to several important sensory structures in the

vertebrate head (Streit 2004; Jidigam and Gunhaga 2013; Saint-Jeannet and Moody

2014). The most anterior placodes consist of the single midline adenohypophyseal

and the two-sided olfactory and lens placodes. The hypophyseal placode gives rise

to Rathke’s pouch, a dorsal out-pocketing in the roof of the oral ectoderm, which is

located ventral to the hypothalamus. Within the adenohypophysis several hormone

secreting cell types are generated that regulate gonadal, thyroid and adrenal

functions, growth and metabolism (Baker and Bronner-Fraser 2001; Jidigam and

Gunhaga 2013; Saint-Jeannet and Moody 2014). Lateral to the adenohypophyseal

placode and close to the ventral telencephalon, the paired olfactory placodes develop

and invaginate as pits. The olfactory placode will give rise to sensory epithelium and

respiratory epithelium, and the latter eventually lines part of the nasal cavity.

Respiratory epithelial cells are non-neural and mucus-generating cells that eliminate

2

dust particles from inhaled air. The sensory epithelium gives rise to several cell

types including glial cells, horizontal cells, sustentacular cells, and globose cells as

well as primary sensory neurons that transduce odour signals via axons of the first

cranial nerve to the olfactory bulb in the forebrain. In some animals a separate

domain of the olfactory placode contributes to the development of the vomeronasal

organ, which is specific for pheromone detection (Akutsu, Takada et al. 1992;

Norgren and Gao 1994). The lens placode is the only sensory placode that will not

give rise to any neuronal cell types. Like other anterior sensory placodes, lens

placodes also invaginate and later form a vesicle in response to signals within the

placodal ectoderm and the adjacent growing optic cup. The anterior layer of the lens

vesicle comprises lens epithelial cells that provide homeostatic support for the lens

and act as stem cells to produce new fiber cells. The posterior layer of the lens

vesicle differentiates into highly specialized lens fibers filled with crystallin

proteins, which makes the organ transparent with the proper curvature. The lens is

responsible for projecting the visual image on the central retina, and it is therefore

important for vision (Simpson, Moss et al. 1995).

The more posterior located otic placode is formed at the level of rhombomere 5/6 of

the hindbrain (Layer and Alber 1990). As the tissue develops, the otic placode

invaginates and forms into a cup like structure, and the gradually invaginated cup

separates from the overlying surface ectoderm and develops into an otic vesicle. The

otic tissue undergoes complex morphogenetic movements to form the entire inner

ear including supporting cells like mechanosensory hair cells that transmit balance

and auditory information. The otic placode also gives rise to the neurons of the

vestibuloacoustic ganglion of cranial nerve VIII (Fekete 1999; Brigande, Iten et al.

2000; Baker and Bronner-Fraser 2001).

Placode precursors in the neural plate border

Several signaling molecules have been shown to be vital for placode induction

including members of the fibroblast growth factor (FGF), bone morphogenetic

protein (BMP) and Wnt families, as well as sonic hedgehog (Shh), and Retinoic

acid. Of these, two stand out (BMP and FGF) for having roles in the progression of

3

diverse placodes (Faber, Dimanlig et al. 2001; Sjodal, Edlund et al. 2007; Pandit,

Jidigam et al. 2011). BMP signals are involved in the specification of olfactory and

lens placodal cells at the gastrula stage in chick embryos. In vitro experiments from

rostral neural plate border explants from the late gastrula stage (stage 4) show that

short time exposure to BMP promotes olfactory identity and prolonged exposure to

BMP signaling promotes lens character (Sjodal, Edlund et al. 2007; Saint-Jeannet

and Moody 2014). Olfactory/lens placodal explants isolated from gastrula stage

embryo confirm that the balance of both FGF and BMP signals is important for the

generation of placodal cells, while FGF prevents from acquiring epidermal cell fate

and BMP from acquiring neural fate (Sjodal, Edlund et al. 2007).

An interesting part of the caudal placode field is that at stage 6 otic and epibranchial

placodal cells are intermingled in a single domain (Fig. 1), and both placodal cells

require FGF signaling for their induction. Thus, although initially both require FGF

signaling, other factors combine and give unique fate (identity) to intermingled

placode precursors. One such factor is Wnt, which is inductive for otic placode and

repressive for epibranchial placode (Ohyama, Mohamed et al. 2006; Freter, Muta et

al. 2008).

Figure 1: Schematic picture of a St 7 chicken embryo

showing rostral placode territory in purple color and OEPD

domain in the caudal region, indicated by red. OEPD, otic

epibranchial placode domain.

4

Placode precursors are derived from a common pre-placodal ectoderm

Patterning events during gastrula and early neurula stages lead to the subdivision of

the ectoderm into at least four distinct domains – neural plate, neural crest, pre-

placodal ectoderm (PPE), and future epidermis. During the gastrula stage, a specific

gene expression profile indicative of the PPE has not been observed (Bailey and

Streit 2005). But by the early neurula stage, the PPE domain for the first time is

detected by gene expression patterns (Streit 2002; Bhattacharyya, Bailey et al.

2004). The PPE is the precursor region of the placodes, which is identified as a U-

shaped domain restricted to the rostral border of the neural plate (Fig. 2) (Schlosser

2010). Within this domain, precursors for different placodes are intermingled,

although there is a rough subdivision along the rostral caudal axis into precursors

that contribute to more rostral and caudal placodes, respectively (Streit 2002;

Bhattacharyya, Bailey et al. 2004). The rostral part of the domain is divided into

adenohypophyseal, olfactory and lens placodal precursors. The caudal part of the

domain is further divided into trigeminal, otic and epibranchial placodes. At this

stage, the rostral and caudal domains are made evident by differential expression of

distinct transcription factor encoding genes like Pax6, Six3 and Otx2 in the rostral

domain, and Pax2, Irx3 and Gbx2 in the caudal domain (Schlosser 2006; Patthey

and Gunhaga 2011). Otx2 and Gbx2 mutually repress each other (Katahira, Sato et

al. 2000). Otx2 target activation is required for olfactory and lens placodal

precursors, whereas Gbx2 is required for otic placode specification (Steventon,

Mayor et al. 2012). In the later stages of the embryonic development, the PPE

successively separates into discrete patches of thickened ectoderm called placodes.

5

Numerous scientific studies have shown that the induction of sensory placodes at

appropriate positions around the developing neural tube is a multi-step process that

involves inductive signals from more than one tissue and the sequential or parallel

action of several signaling pathways (Baker and Bronner-Fraser 2001; Streit 2004;

Litsiou, Hanson et al. 2005). Attenuation of BMP and Wnt, together with activation

of FGF signals are required for induction and positioning of the PPE (Litsiou,

Hanson et al. 2005; Bailey, Bhattacharyya et al. 2006; McCabe and Bronner-Fraser

2009). Alterations in these signaling molecules will change the location of the neural

plate border and further affect the development of the pre-placodal region.

On the other hand, Six, Eya and Dach families are generalized preplacodal markers

commonly expressed both in the rostral and caudal placodal domain (Bailey and

Streit 2005). These genes are known to be involved in preplacodal formation and in

the control of several aspects of sensory organ development. In chick, the head

mesoderm below the preplacodal region expresses FGF together with inhibitors of

BMP and Wnt signals, which is sufficient and required for inducing preplacodal

characteristic markers (Litsiou, Hanson et al. 2005). These factors act to guard

preplacodal cells from antagonistic effects emanating from surrounding tissues.

Mesoderm is not the only source of signals to induce an ectopic PPE (Seifert, Jacob

et al. 1993). Experiments in Xenopus and chick support the idea that neural plate

grafts induce Six1 and Xiro1 expression in the ventral epidermis (Glavic, Honoré et

al. 2004; Bailey and Streit 2005; Litsiou, Hanson et al. 2005). It is also shown that

the rostral region of the PPE is in close contact with underlying endoderm and the

Figure 2: At 0-1 somite stage precursor region for all the placodes

(pre-placodal ectoderm) is identified as a U-shaped domain

detected by expression of Six1 and Eya genes, shown as blue

region.

6

prechordal plate (Seifert, Jacob et al. 1993), which might also play a role in the

formation of the olfactory placode and to lesser extent for the induction of the lens

(reviewed in Bailey and Streit 2005). With this it is clear that the induction of PPE is

a multi-step process which is tightly involved with regulation of other derivatives

from mesoderm, ectoderm and endoderm.

Even though the pre-placodal region displays rostral-caudal domains at the neurula

stage, individualized placode identity will become more defined as neurulation

proceeds. For example, Six1 and Eya2 expressed throughout the pre-placode region

at the early neurula stage are down-regulated in the lens region but are maintained in

the adenohypophseal and olfactory as neurulation progresses (Bailey and Streit

2005; Schlosser 2006). Moreover, Pax6 expressed in the entire rostral pre-placode

region is strongly up-regulated in the lens and olfactory ectoderm and reduced from

stage E4.5 of the chick adenohypophyseal region (Li, Yang et al. 1994;

Bhattacharyya, Bailey et al. 2004). On the other hand, Pax2 becomes restricted to

the otic and epibranchial placodes, and Pax3 to the prospective trigeminal ectoderm

(Groves and Bronner-Fraser 2000; Bailey and Streit 2005).

Placode precursors are dispersed in such a way that is comparatively colinear with

their final location (Kozlowski, Murakami et al. 1997; Streit 2002; Bhattacharyya,

Bailey et al. 2004; Xu, Dude et al. 2008). Developmental studies on rostral placodes

and caudal placodes using focal dyes and fate maps suggest that adjacent ectodermal

placodal precursors can contribute to diverse placodes (Streit 2002; Bhattacharyya,

Bailey et al. 2004; Bhattacharyya and Bronner-Fraser 2004; Xu, Dude et al. 2008).

Thus, placodal precursors have been shown to overlap in fate map experiments. For

example, around stage 6 prospective olfactory and lens precursors intermingle in the

anterior border region (Bhattacharyya, Bailey et al. 2004; Sjodal, Edlund et al.

2007). In a similar manner, the trigeminal, otic and epibranchial precursors

intermingle in the posterior border region at stage 5 (Streit 2002; Freter, Muta et al.

2008; Xu, Dude et al. 2008).

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Individual placode identity

Lens placode

The vertebrate eye develops with synchronized communications between the

neuroectoderm in the forebrain and the surface ectoderm (lens ectoderm). The

neuroectoderm associated with the ventral forebrain evaginates, which further

results in the formation of bilateral optic vesicles. The distal part of the optic vesicle

will make contact with the overlying lens ectoderm, which is then thickened to form

the lens placode (Fig. 3B) (Fuhrmann 2010). The lens placode later invaginates to

form the lens pit (Fig. 3C). Successively the lens pit further deepens and the contact

with the overlying surface ectoderm or the prospective cornea is abolished and

ensues in the formation of the lens vesicle (Fig. 3D) (Gunhaga 2011). Until the

formation of the lens vesicle the majority of the ectodermal lens cells proliferate.

After this stage, the posterior-most cells in the vesicle exit the cell cycle and

elongate to become primary lens fibre cells, whereas anteriorly situated cells form

the lens epithelium and retain their high proliferative capacity. Eventually the rate of

cell proliferation declines and becomes restricted to the equatorial zone, a small

region of epithelial cells above the lens equator, which will remain mitotically active

throughout life (Jarrin, Pandit et al. 2012).

Figure 3: Morphological changes occur during the early development of lens and retina during

chick embryonic development. (A) Around neural tube stage (stage 11) prospective lens

ectoderm (LE) in green comes in contact with evaginating optic vesicle in light red color. Once

the optic vesicle comes in contact with the lens ectoderm, the ectoderm thickens to form the

lens placode (LP) around stage 13. (B, C) From stage 13, the lens placode starts to invaginate

8

simultaneously with the underlying optic vesicle. (D) By stage 16, the lens ectoderm forms into a

vesicle and the underlying optic vesicle forms into a bilayered optic cup. The inner layer of the

optic cup becomes the neural retina and the outer layer forms the RPE. Lens cells in the

posterior part close to the optic cup start elongating and form the primary fibre cells. MC,

mesenchymal cells; RPE, retinal pigmented epithelium; NR, neural retina; LV, lens vesicle.

Specification of lens placodal cells

Rostral neural plate border cells are specified as rostral placodal progenitor cells,

and they maintain the ability to acquire either lens or olfactory placodal fate (Fig. 1).

Using explant assays it was shown that lens placodal cells are initially specified at

the late gastrula stage, and that BMP signals play a key role in this process at least

partly by inhibiting neural fate (Sjodal, Edlund et al. 2007). These results also

provided evidence that beyond this stage lens placodal cells do not require any

interactions with adjacent epidermal or neural ectoderm or underlying mesoderm;

this proposes that maintenance and maturation of the lens placodal cells is dependent

on signals from the prospective placode ectoderm itself (Sjodal, Edlund et al. 2007;

Patthey, Gunhaga et al. 2008). At gastrula stages, Wnt activity in the caudal region

inhibits lens and olfactory placodal specification while promoting generation of

neural crest cells (Litsiou, Hanson et al. 2005; Patthey, Gunhaga et al. 2008;

Gunhaga 2011). Slightly later at the neural fold stage prospective lens placodal

explants cultured with premigratory or migratory neural crest cells fail to acquire

lens character. Moreover, partial ablation of prospective neural crest cells from the

forebrain to midbrain levels at the neural fold stage resulted in occasional ectopic

lens formation, suggesting a lens-inhibitory activity in the neural crest that restricts

lens formation to forebrain levels (Bailey, Bhattacharyya et al. 2006).

At the neural fold stage, pSmad1/5/8, a downstream transducer of BMP signaling is

detected in the prospective lens ectoderm. During this stage prospective lens cells

can switch between olfactory and lens placodal fate upon modulation of the levels of

BMP signaling, suggesting that at the neural fold stage BMP signals promote

generation of lens cells at the expense of olfactory placodal cells (Fig. 4) (Sjodal,

Edlund et al. 2007). At later stages, Bmp7 is expressed in the lens ectoderm and

optic vesicle, while Bmp4 is expressed in the optic vesicle (Dudley and Robertson

1997; Wawersik, Purcell et al. 1999). Bmp4 mouse mutant embryos display failed

9

lens placode formation, but the formation of olfactory placode appears to be normal.

Although, Bmp4 knockout mice embryos lack a lens placode, the expression of Pax6

and Six3 is still maintained in prospective lens ectoderm, which is indicative of the

presence of lens precursor cells (Furuta and Hogan 1998). Similar to Bmp4 mouse

mutant, Bmp7 mutant embryos show disturbed lens placode invagination.

Furthermore, deletion of genes encoding the type I BMP

receptors (Bmpr1a and Acvr1) in mouse embryos prevented lens formation. But the

deletion of BMP canonical transducers (Smad4, Smad1 and Smad5) have shown no

effect on lens specific markers and lens development (Rajagopal, Huang et al.

2009). This demonstrates that both Bmp4, Bmp7 together with their type I receptors

are required for the development of the lens (Dudley and Robertson 1997;

Wawersik, Purcell et al. 1999). Further studies are needed to determine the

downstream signaling of the BMP pathway for lens development.

Other signaling molecules like FGF have also been shown to play an important role

during the development of the lens. In support, several FGF family members are

expressed in the eye region and all four FGF receptors are expressed in the lens

(Robinson 2006). As mentioned earlier, at the late gastrula stage FGF activity

prevents prospective lens/olfactory cells in the rostral neural plate border from

acquiring epidermal fate (Sjodal, Edlund et al. 2007). In addition, mis-expression of

Fgf8 in the competent surface ectoderm at the neural tube stage inhibited the

generation of epidermal cells, thereby enabling BMP signals to ectopically induce

lens character as indicated by L-Maf expression (Kurose, Okamoto et al. 2005).

Moreover, mouse eye explants from E8.5 to 9.5 cultured with an FGF inhibitor

exhibited reduced Pax6 expression with significant size reduction in the lens placode

and delayed placode invagination. In the mouse, conditional deletion of FGFR1/2/3

during the lens pit stage exhibited normal invagination with the expression of lens

fiber cell maker α- and β-crystallins (Faber, Dimanlig et al. 2001; Zhao, Yang et al.

2008). A similar phenotype is also observed in mice mutants lacking Frs2α, an

anchor protein mediating FGF signaling via the ERK pathway (Gotoh, Ito et al.

2004). Despite the fact that initial differentiation of the primary lens fiber cell and

expression of the differentiation marker δ-crystallin are independent of FGF activity,

further differentiation of lens fiber cells appears to require FGF activity (Lorén,

10

Schrader et al. 2009). Together, these results indicate that BMP signals together with

FGF activity regulate the specification and development of lens placodal cells.

Olfactory placode

The olfactory placode lies in proximity to the ventral telencephalon, and gives rise to

both sensory neural and respiratory non-neural epithelium (Maier, von Hofsten et al.

2010). As mentioned in the previous sections, the prospective olfactory and lens

placodal cells are intermingled at the rostral border (Figure 1) (Bhattacharyya,

Bailey et al. 2004). Consistently, explants cultured from this rostral border region

generate cells of both lens and olfactory character in distinct domains (Sjodal,

Edlund et al. 2007). At the neural fold stages, rostral placodal cells can change

between lens and olfactory identities in response to alterations in BMP activity, in

which BMP activity promotes lens character at the expense of olfactory placodal

cells (Sjodal, Edlund et al. 2007). However, at this stage FGF activity is not

sufficient to induce olfactory identity in lens progenitor cells, and inhibition of FGF

activity does not prevent generation of olfactory cells (Bailey, Bhattacharyya et al.

2006; Sjodal, Edlund et al. 2007). In spite of this, FGF signaling is necessary for

generation of olfactory placode derived neurons, in part by inhibiting the generation

of epidermal cells (Sjodal, Edlund et al. 2007).

At stage 14, Fgf8 expression is detected in the anterior part of the olfactory placode

and Bmp4 expression is identified in the posterior part of the placode. At around

Figure 4: Schematic diagram of a St

8 (neural fold stage) chick embryo. At

this stage prospective olfactory and

lens cells are spatially segregated

within the rostral placodal region, as

indicated in green and blue,

respectively. Within the caudal

placodal region, the otic placode is

indicated in orange. ANR, anterior

neural ridge.

11

stage 20, cells in the anterior portion of the olfactory pit express Fgf8, whereas weak

Bmp4 expression can be detected at the olfactory rim. In the center part of the

olfactory pit, cells at the rim express Fgf8 and Bmp4, and in the posterior part of the

olfactory pit cells express Bmp4 with weak Fgf8 expression. The activated BMP

transducer, pSmad1/5/8, is detected in more or less every cell in the posterior part

and in scattered cells in the anteromedial part of the olfactory pit (Maier, von

Hofsten et al. 2010).

The olfactory placode is first morphologically evident in chick around stage 14

(Bhattacharyya, Bailey et al. 2004; Maier and Gunhaga 2009). During olfactory

placode patterning the specification of neurogenic versus non-neurogenic domains is

a critical step that involves many signaling molecules and gene networks (Maier,

Saxena et al. 2014). In chick and mouse, BMP signals promote respiratory epithelial

cells, and FGF signaling is required for the generation of sensory epithelial cells

(Maier, von Hofsten et al. 2010). Previous studies reveal that the spatiotemporal

expressions of several transcription factors like Hes5, Dlx3, Dlx5, Sox2, Pax6,

Msx1/2, and Id3 are important for olfactory placode formation (Cau, Gradwohl et al.

2000; Bhattacharyya, Bailey et al. 2004; Maier and Gunhaga 2009; Maier, von

Hofsten et al. 2010). Hes5 and HuC/D are expressed in the sensory part of the

epithelium, and Id3 and Msx1/2 are expressed in future respiratory epithelial cells.

Together these genes determine the neurogenic and non-neurogenic regions of the

olfactory placode (Maier, von Hofsten et al. 2010).

Neurogenesis in the olfactory epithelium

In the olfactory epithelium neurogenesis occurs throughout life and it is further

categorized into two phases: 1) Primary neurogenesis occurs during embryonic

development and 2) secondary neurogenesis is the homeostatic replacement of

neurons in the adult to maintain the matured pseudostratified epithelium (Maier,

Saxena et al. 2014). Primary neurogenesis begins with placode formation at around

stage 14 in chick (Maier and Gunhaga 2009). As mentioned, primary neurogenesis

forms the essential structure of the olfactory epithelium from which the axons of

olfactory sensory neurons extend to the most rostral part of the telencephalon where

12

the future olfactory bulb forms (Treloar, Miller et al. 2010; Maier, Saxena et al.

2014)). During initial establishment of the olfactory placode, Hes5 is expressed in a

cluster of neural progenitor cells and it is implicated in defining the neurogenic

domain (Cau, Gradwohl et al. 2000). Ngn1 is expressed in immediate neuronal

precursors in the basal region of the placode and it is also detected in the first early

migrating neurons. In mice lacking Ngn1 (Ngn1−/−), basal progenitors fail to express

several neuronal differentiation-marker genes including NeuroD. Furthermore, Ngn1

is required in the basal progenitors for the activation of the differentiation program

for the olfactory sensory neurons (Cau, Casarosa et al. 2002; Maier and Gunhaga

2009).

Otic placode

Adjacent to the hindbrain region at the 4-6 somites stage, otic placode induction

takes place (Jidigam and Gunhaga 2013). The otic placode gives rise to the inner ear

and the eighth cranial nerve. The inner ear is a complex structure that is composed

of several cell types including ciliated mechanoreceptors that detect sound. The

signals are transduced to the central nervous system via the neurons of the eighth

cranial nerve, the cochleovestibular nerve.

Several FGF family members are expressed in the developing inner ear and in

adjacent tissues, and they regulate otic induction, neuronal production, and

morphogenesis (Ladher, Anakwe et al. 2000; Alsina, Abelló et al. 2004). From the 8

somites stage on, Fgf10 is expressed in the anterior region, while Bmp7 expression

is detected throughout the posterior region of the otic epithelium (Abello, Khatri et

al. 2010).

Pax2 is one of the earliest known markers expressed during otic induction, with an

onset at the neural fold stage (Christophorou, Mende et al. 2010). The Pax2

expressing domain of the surface ectoderm contains progenitors for both otic and

epibranchial placodal cells (Groves and Bronner-Fraser 2000; Streit 2002; Streit

2004). This Pax2 positive domain has been referred to as the otic and epibranchial

placode domain (OEPD) (Fig.1), the posterior placodal area (PPA), and the pre otic

field.

13

Several studies have shown that members of the FGF family are required for the

induction of the otic and epibranchial placode domains (Schimmang 2007). In chick,

around stage 5, Fgf19 and Fgf3 are expressed in the mesoderm underlying the otic

pre-placodal region, together with Fgf8 expressed in the endoderm (Ladher, Anakwe

et al. 2000; Schimmang 2007). At the neural fold stage, this expression is expanded

to the hindbrain adjacent to the developing otic placode. Although knockdown of

Fgf19 did not show any significant effect, OEPD induction was blocked by

inhibiting both Fgf19 and Fgf3 signals (Freter, Muta et al. 2008). Expression of

Fgf10 (in mouse) and Fgf19 (in chick) seems to be under the control of Fgf8. That is

why in the chick Fgf8 alone seems to be sufficient for otic induction (Ladher,

Wright et al. 2005). By the neural tube stage (st10/11), Fgf10 and Fgf8 are

expressed within the chick otic placode (Ladher, Anakwe et al. 2000; Karabagli,

Karabagli et al. 2002; Schimmang 2007). During this stage, FGF family members

are expressed in and around the developing otic placode; this suggests that FGF

signals play a significant role in otic placode formation (Schimmang 2007). Cranial

paraxial mesoderm positive for Fgf19 expression is essential for avian otic placode

induction; by ablating the paraxial mesoderm the otic placode fails to develop (Kil,

Streit et al. 2005). Conversely, overexpression of FGF signaling molecules like Fgf3

near the otic vesicle induced ectopic vesicle-like structures expressing otic related

markers (Vendrell, Carnicero et al. 2000). In addition, placing FGF2 and FGF8

beads close to the otic region induces ectopic expression of otic placode markers

like SOHo1 and Pax2. Taken together, it is clear that FGF signaling plays an

important role for otic induction and further for otic placode formation.

Wnt signaling is also known to play an important role in otic induction (Ladher,

Anakwe et al. 2000). Wnt activity in combination with FGF signals induce otic

character, and excludes the epibranchial identity (Freter, Muta et al. 2008). In an

explant assay, exposing the competent non-neural ectoderm to mesodermal signal

FGF19 together with Wnt8 is sufficient to induce otic markers (Ladher, Anakwe et

al. 2000). Consistent with sequential steps for otic induction, inhibiting Wnt

signaling with Dkk1 blocked the expression of SOHo1 (inner ear marker), but did

not inhibit the early otic marker Pax2 (Freter, Muta et al. 2008; Sai and Ladher

2015).

14

Placode Invagination

Ectodermal thickening (placode)

Thickening of the placodes occurs at different stages for different cranial placodes.

Before the invagination process, cells in the placode elongate along the apical basal

axis. In chick, the olfactory placode is shaped at stage 14, the lens at stage 13, and

the otic placode is formed at stage 8/9. However, molecular mechanisms regulating

the thickening of the placode epithelium have received less attention. It has been

shown in the otic placode that Pax2 is a potential candidate for placode thickening

(Christophorou, Mende et al. 2010), but detailed mechanisms for the formation of

placodes are still unclear. However, a study in the mouse paves the path for

investigating the members of the small GTPases family as potential candidates,

which suggests that Rac1 is a main effector for cell elongation in the lens placode

(Chauhan, Lou et al. 2011). Further studies are needed to determine if this small

GTPases is essential for the thickening in the otic and olfactory placodes. Cells

within the placode undergo cell division, through a process called interkinetic

nuclear migration (IKNM), which permits the epithelial cells within the placode to

be tightly packed for a higher number of cells (Sauer 1936). The strong association

of placode formation and morphogenesis supports the idea that epithelial thickening

might be essential for epithelial bending or placode invagination (Chauhan,

Plageman et al. 2015).

Following thickening, placodes invaginate to form structures called pits or sacs. In

chick, the initial invagination of the olfactory placode starts at stage 17, the lens at

stage 14, and the otic placode at stage 10/11 (Jidigam and Gunhaga 2013). During

these early stages of development, the morphological process of the lens epithelium

is different when compared to olfactory and otic placode morphogenesis. Once the

lens pit deepens, the lens epithelium fuses with itself, and the connection with the

overlying surface ectoderm is broken. In all three placodes, transition from the

placode to pit structure involves the cells in the epithelium to undergo a biphasic

process – phase one being basal cell expansion and phase two apical constriction

(reduced apical cell surface) (Sai and Ladher 2008; Christophorou, Mende et al.

2010; Plageman, Chung et al. 2010). Numerous well known transcription factors,

15

cell adhesion molecules and cytoskeleton elements have been connected with the

regulation of placodal invagination (Hogan 1999; Jamora and Fuchs 2002), but the

signaling molecules regulating them is still not known.

One of the driving forces behind morphogenesis is change of cell shape via

rearrangements in the cytoskeleton, i.e. the network of actin, intermediate filaments

and microtubules, which are the backbone for the structure of the cell (Lecuit and

Lenne 2007). Both apical constriction and placodal invagination are known to be

mediated by networks of F-actin, which contract by interaction with myosin II. At

stage 10, F-actin is detected in both the apical and basal side of the otic placode, and

by stage 11 F-actin is depleted basally and enhanced apically (Sai and Ladher 2008).

Blocking myosin II and F-actin polymerization using blebbistatin and cytochalasin

D, respectively, results in the failure of placode invagination (Sai and Ladher 2008;

Borges, Lamers et al. 2011; Plageman, Chauhan et al. 2011). In addition,

accumulation of phosphorylated Myosin and apical constriction requires apical

localization of RhoA (Borges, Lamers et al. 2011; Plageman, Chauhan et al. 2011).

Rho GTPases are known to act as molecular switches for transfer of information

within the cell by cycling between a GTP bound “active state” and GDP bound

“inactive state”. Rho GAP and Rho GEF are identified to promote the switching to

the active and inactive states of Rho proteins, respectively (Hall 2012). Myosin is

directly phosphorylated by ROCK, a downstream molecule of RhoA, and ROCK

inhibition disrupts placode invagination (Amano, Ito et al. 1996; Borges, Lamers et

al. 2011). Moreover, apical accumulation of RhoA is sufficient to induce apical

constriction, and RhoA plays a key role in apical localization of Shroom3 (an actin

scaffold protein). Blocking Shroom3 causes reduction in the apical distribution of

both F-actin and myosin in the lens placode (Plageman, Chung et al. 2010). In cell

culture experiments it is shown that Trio, a RhoA GEF molecule, is required for

Shroom3 dependent apical constriction. Together this points to the idea that a Trio,

RhoA, Shroom3 and ROCK pathway is required for apical constriction (Plageman,

Chauhan et al. 2011). Molecular signals regulating this pathway are still not known.

Several transcription factors have been shown to play substantial roles in the

formation and invagination of placodes. Pax2 plays a key role in otic placode

16

morphogenesis; in the absence of Pax2 cells fail to elongate due to the loss of

apically localized N-cadherin and N-CAM. In addition, ectopic Pax2 in surface

ectoderm did not promote cell elongation but induced N-Cadherin and N-CAM

expression (Christophorou, Mende et al. 2010). On the other hand, Spalt4, a Zinc

finger transcription factor that is strongly expressed in the olfactory and otic and

weakly expressed in the lens placode, was shown to be involved in the early stages

of otic placode development and to initiate cranial ectodermal invagination.

Overexpressing Spalt4 in the non-placodal surface ectoderm from stages 8-11

induced ectopic invaginations. On the other hand, it was also shown that

implantation of FGF2 beads into the area opaca (extra embryonic ectoderm in chick)

induced Spalt4 expression (Barembaum and Bronner-Fraser 2007).

Retina

Once the neural plate is established, the anterior neural domain progressively

becomes regionalized into different regions that later generate the telencephalon, eye

field (optic), hypothalamus and diencephalon (Puelles and Rubenstein 2003). The

eye-field gives rise to most structures of the eye, such as the neural retina, the retinal

pigment epithelium (RPE) and the optic stalk. The cellular and molecular

mechanisms required for the differential specification of these regions are not well

characterized. In chick embryos, fate map studies at the neural plate stages show that

prospective eye-field cells are located in a well-defined region in the anterior neural

plate (Fernández-Garre, Rodríguez-Gallardo et al. 2002; Sánchez-Arrones, Ferrán et

al. 2009). One of the initial markers expressed within the eye field region are the

members of Rax/Rx family. At the time of initial specification of the eye field,

prospective lens ectoderm and retina are not in close contact, and the initial contact

is observed at the neural tube stage. The initial sign of morphogenesis in the eye

region is the evagination of the optic vesicle from the ventral forebrain region at the

neural tube stage. The neural retina is formed from the distal portion of the optic

vesicle and the retinal pigment epithelium from the dorsal part (Kagiyama, Gotouda

et al. 2005; Hirashima, Kobayashi et al. 2008).

17

Eye (retina) formation includes a complex series of morphogenetic events during

vertebrate development. The basic morphology of the eye is visible when the optic

vesicle evaginates and forms a bilayered optic cup in which the inner epithelium is

neural and the outer is pigmented epithelium (Fig. 3C). Eventually, and consistent

with a morphogenetic wave of differentiation, the sensory neural retina, ciliary body

and iris are committed from this simple structure along the central to peripheral axis.

Rax/Rx (homeodomain transcription factor) genes are shown to play vital roles in

optic vesicle evagination. In zebrafish embryos lacking Rx3 (an ortholog of Rax1),

the evaginated distal portion of the optic vesicle moving outwards is completely

disturbed, which indicates the importance of Rx3 in extensive cell movements

(Winkler, Loosli et al. 2000; Rembold, Loosli et al. 2006; Stigloher, Ninkovic et al.

2006). In Xenopus, the Rx gene is shown to regulate cell proliferation, and Optx2,

which controls proliferation in the eye-field, is known to be dependent on Rx

function (Zuber, Perron et al. 1999; Zuber, Gestri et al. 2003).

At the optic vesicle stage, neuroepithelial cells are bipotent, i.e. the presumptive

retinal cells are competent to develop into either RPE or neural retinal cells (Rowan,

Chen et al. 2004; Horsford, Nguyen et al. 2005; Westenskow, McKean et al. 2010).

The earliest genes that are expressed in the retina and RPE domains are the

homeobox gene Vsx2 (Chx10) and the bHLH transcription factor Mitf, respectively

(Fuhrmann 2010). In the mouse it has been shown that Mitf is expressed in the

whole optic vesicle and consequently reduced after Vsx2 expression is up-regulated

(Nguyen and Arnheiter 2000). Both genes are needed for the maintenance of retinal

cell identity, and in addition Vsx2 controls other aspects of the development like cell

proliferation (Sigulinsky, Green et al. 2008). Lhx2 (LIM box transcription factor) is

a known retinal patterning gene expressed in the eye-field that is known to be

essential for Mitf and other retinal markers (Zuber, Gestri et al. 2003; Yun, Saijoh et

al. 2009). It has been suggested that the neuroretinal-inducing signal comes from the

surface ectoderm (Hyer, Kuhlman et al. 2003), suggesting a model whereby, signals

from the ectoderm promotes the cells in the optic vesicle to become NR rather than

RPE when the close contact is established between two tissues. It has been shown

that Vsx2 might act directly by suppressing transactivation of the Mitf gene in the

distal optic vesicle (Bharti, Liu et al. 2008). Another study has demonstrated that

18

Chx10 repression of Mitf is required for the maintenance of mammalian neural retina

identity (Horsford, Nguyen et al. 2005). However, the signals by which the spatio-

temporal expression of these transcription factors is regulated during the early stages

of development remain to be described.

Although neural retina and telencephalon both arise within the forebrain, how

these two domains are differentially specified is not well defined. Signaling

pathways like FGF and BMP were shown to play important roles in several phases

of early eye development. Studies in the mouse have shown that FGF signaling is

necessary for the maintenance of neural retina characteristics, rather than the

specification (Horsford, Nguyen et al. 2005). However, the signals required for the

early specification of neural retinal cells are not known. Other Bmp mutant studies in

the mouse have shown severe defects in early eye development (Furuta and Hogan

1998; Wawersik, Purcell et al. 1999). The spatio-temporal requirements of BMP

signals during the early stages of the eye development are not known. Moreover, the

molecular mechanisms that regulate the induction and maintenance of eye-field cells

and the specification of neural retina cells are poorly understood.

19

Aims

The studies described in this thesis are about the requirement of BMP signals for

induction and specification of lens and retina cells as well as its importance in the

morphogenesis of sensory and non-sensory placode invagination.

Specific aims of this thesis are:

1. To determine at what stages BMP signals are required for initial identity of

lens cells.

2. To examine when lens fibre cell differentiation becomes independent of

BMP signalling.

3. To analyse the molecular signals involved in the specification of retinal

cells, and at what stage retinal cells are specified

4. To determine what signalling molecules are regulating placode

invagination.

5. To analyse if placode invagination is regulated by a common molecular

machinery, or is controlled by unique molecular codes for each individual

placode.

6. To examine whether placodes maintain their identity after the disruption of

invagination.

20

Materials and methods

Functional experiments:

Electroporation

In ovo electroporation: Lens and olfactory regions of the chick embryo were

electroporated in ovo at desired developmental stages to transfer a control green

fluorescent protein (GFP) vector, alone or together with a construct of interest.

DNA-constructs either to over-activate BMP or to inhibit the BMP signals were

used. DNA constructs were transferred using an Electro Square Porator ECM 830

(BTX) by applying 5 pulses (9-15 V, 25 ms duration) at 1-s intervals. The

electroporated embryos were allowed to grow to a desired developmental stage.

Embryos with GFP expression within the desired region were selected for further

analyses. The intact chick embryo assay allows to examine morphological processes

and is a valuable tool to strengthen the physiological relevance of in vitro results.

Chick explant assay

-minus +plus

Olfactory

Lens

21

Explant assay: Small pieces of tissue of interest (so called explants) are isolated

from chick embryos at different developmental stages using tungsten needles.

Thereafter the prospective placodal explants are cultured alone, or in the presence of

inhibitors or activators of specific signalling pathways. All explants are cultured in

vitro in collagen in serum-free conditions.

Whole embryo culture

Ex ovo culture of chick embryos: Stage 5-7 embryos were dissected and folded in

half along the midline into a semicircle, and cut along the outside edge. The

embryos were allowed to rest undisturbed in Ringer’s solution at room temperature

(RT) for approximately 45-60 min. Embryos were then transferred to a 4-well plate

containing 500µl of medium (a 2:1 mixture of thin albumen and Ringer’s solution

with penicillin and streptomycin 2 U/ml) alone or together with inhibitors. Each well

contained one to six embryos. Embryos were cultured in the presence of BMP

inhibitors Noggin, Dorsomorphin or ROCK inhibitor Y27632.

Evaluation of functional experiments: In both the explant and intact embryo

studies, the identity of the cells was analysed by immunohistochemistry and in situ

hybridization using specific antibodies and chick dioxigenin-labeled RNA probes,

respectively. Changes in cell proliferation and cell death were analysed by using

Caspase3, and phospho-HistoneH3. To obtain reliable statistics, explants and

embryos were sectioned on consecutive slides and stained in multiple ways. The

percentage of antigen-expressing cells was quantified by counting the number of

marker-stained cells, and compared to the total number of cells, determined by

counting DAPI-stained nuclei. Morphometric analysis: using confocal microscopy

Embryos in

+/- Inhibitors Stage 5/7

Incubate at 37 degrees

22

both control and BMP inhibited embryos were analysed. Apical cell width, cell

length, basal cell width were studied with the combination of confocal microscopy

and Volocity 6.0 software.

23

Results

Paper I: BMP-Induced L-Maf Regulates Subsequent BMP-Independent Differentiation of Primary Lens Fibre Cells

Localization of BMP signals and lens specific markers in the developing eye

region

To determine whether BMP signaling is active in the prospective lens from the optic

vesicle stage to the developing optic cup, we examined expression of the potential

BMP ligand, Bmp4, and pSmad1/5/8 (an active indicator of BMP signaling),

together with lens specific markers L-Maf and δ-crystallin. At stage 11, Bmp4 and

pSmad1/5/8 expression were found in the prospective lens ectoderm, but no L-Maf

and δ-crystallin expression was detected. By stage 13, initial L-Maf expression was

observed in the thickened epithelium (placode) with no δ-crystallin expression.

Initial δ-crystallin expression was observed first at stage 17, and by this time no

expression for Bmp4 was detected in the lens vesicle. This indicates that BMP

signals are initially detected before placode formation and lens specific markers are

detected after placode formation.

In in vitro conditions BMP activity is required for the initial expression of L-

Maf in lens placode cells

To test if BMP activity is required for the expression of L-Maf and/or δ-crystallin,

we dissected out explants containing the lens ectoderm together with optic vesicle

tissue at stage 11; at this time point, L-Maf and δ-crystallin were not expressed in

this region. These LR (lens/retina) explants were cultured in the presence or absence

of Noggin, a BMP antagonist (Lamb, Knecht et al. 1993) for 42h, which in intact

embryos corresponds to stage 21 chick embryos. Afterwards, explants were

processed for sectioning and analyzed by immunohistochemistry using L-Maf and δ-

crystallin markers. Stage 11 control explants generated L-Maf and δ-crystallin cells

in the lens domain, characteristic of primary lens fibre cells. Explants cultured with

24

Noggin failed to generate cells expressing L-Maf and δ-crystallin but instead

coexpressed HuC/D, Keratin and pan-Dlx, which was indicative of olfactory cell

identity.

In vivo, BMP activity is required for the up-regulation of L-Maf expression

Next we examined the requirement of BMP signaling for L-Maf expression in intact

chick embryos. To address this question, stage 11 prospective lens ectoderm was

electroporated with GFP alone or together with a BMP antagonist, Noggin (Timmer,

Wang et al. 2002), and cultured to stage 21. GFP positive cells within the lens

region were selected for future analysis. Control embryos electroporated with only

GFP exhibited normal lens morphology with expression of L-Maf and δ-crystallin.

In contrast, Noggin treated embryos displayed two phenotypes – severely affected

embryos showed complete loss of L-Maf and δ-crystallin with failure of optic cup

formation; slightly less affected embryos exhibited a disturbed smaller lens with

expression of L-Maf and δ-crystallin, while Keratin was detected in the surface

ectoderm in both phenotypes. The severity of the lens phenotype was associated

with the electroporation efficiency.

We wanted to better understand whether the mechanism behind the failure in lens

formation after BMP inhibition is due to reduced proliferation or increase in cell

death. We examined the cell proliferation marker pHH3 and the cell death marker

aCaspase3 in control and Noggin treated embryos that were cultured to different

time points. Embryos cultured to stage 13/14 did not show any significant changes

in cell proliferation in Noggin treated embryos compared to control embryos. In

embryos cultured to stage 15 and stage 17, a reduction in pHH3+ cells was observed.

At all the stages examined, we did not observe any changes in the levels of

aCaspase3+ in the Noggin and control treated embryos. These data indicate that the

failure in invagination at stage 15 and stage 17 is partly associated with reduced cell

proliferation. This indicates that BMP is required for the formation of the lens and

for the up-regulation of L-Maf and δ-crystallin expression.

25

BMP activity is sufficient to induce L-Maf expression

At gastrula stages, Bmp4 expression was detected in the rostral border ectoderm

region from where the lens cells arise. To investigate if BMP signals were sufficient

to induce L-Maf expression, stage 4 neural explants were dissected and cultured for

43-45h in the presence or absence of BMP4 (35ng/ml). Neural explants cultured

alone generated L5 and Sox1 positive cells, indicative of neural character. By

contrast, addition of BMP4 blocked the generation of L5 and Sox1 positive neural

cells and induced L-Maf positive lens cells. This suggests that BMP is sufficient to

induce L-Maf expression.

Initial elongation of primary fibre cells is independent of BMP activity

We wanted to understand if BMP signals are required directly for δ-crystallin

expression or indirectly required via L-Maf induction which in turn promotes δ-

crystallin expression. To examine this issue, we dissected out stage 13 LR explants

and cultured them alone or in the presence of Noggin to the time point equivalent to

stage 21. In control explants both L-Maf and δ-crystallin were detected within the

lens domain. Similar results were obtained when stage 13 LR explants were cultured

in the presence of Noggin; this is characteristic of primary lens fibre cells. Taken

together, after the specification of lens placodal cells or after the onset of L-Maf

expression, the up-regulation of δ-crystallin and initial elongation of primary fibre

cells was independent of BMP activity.

L-Maf expression is sufficient to induce lens fate independent of BMP signals

Our results provide evidence that after the onset of L-Maf, inhibition of BMP

signaling did not prevent early fiber cell differentiation; this suggests that this event

in primary fiber cell differentiation is dependent on L-Maf and not on BMP signals.

To analyze this further, an L-Maf over-expression vector (Ogino and Yasuda 1998)

was electroporated alone or together with Noggin in stage 9/10 olfactory epithelium

and surface ectoderm, and cultured to stage 18 and 21. In the olfactory epithelium

we detected δ-crystallin expression in the presence or absence of BMP signaling.

Also in the head ectoderm L-Maf induced δ-crystallin expression in the absence of

26

BMP signaling. Taken together these results provide evidence that the up-regulation

of δ-crystallin is independent of BMP activity; this implies that L-Maf acts as a

regulator of primary lens fibre differentiation.

27

Paper II: Neural retina identity is specified by lens-derived BMP signals

Characterization of optic vesicle and forebrain markers in the developing chick

head

To analyze when the eye-field cells acquire neural retina identity, we examined a set

of markers in the different regions of the forebrain during early chick embryonic

development from stage 9-21. At stage 9, Rax2 was expressed in cells of the

evaginating prospective retina and in the prospective hypothalamus; also FoxG1 was

expressed in prospective telencephalic cells. By stage 11, Rax2 expression was

limited to the prospective optic vesicle and FoxG1 was detected in the periphery of

the optic vesicle and a stronger expression in the telencephalon region. At stage 13,

when the lens placode is formed and makes a contact with the underlying optic

vesicle, Vsx2 was up-regulated in a region overlapping with that of Rax2, whereas

Mitf was only detected in the prospective RPE. At stage 21, Rax2 and Vsx2 were

expressed in the neural domain of the optic cup, with weaker expression of Rax2 in

the tuberal hypothalamus. Both FoxG1 and Emx2 were expressed in the dorsal

telencephalon, and no other regions of the forebrain area were shown to express

these markers. Based on these expression patterns, Rax2 and Vsx2 can be used as

retinal cell markers and FoxG1 and Emx2 together as dorsal telencephalic markers.

At blastula stages BMP inhibition is required for the generation of eye-field

cells

A previous study in zebrafish has shown that BMP signals in the anterior neural

ectoderm promote telencephalic identity at the expense of eye-field character

(Bielen and Houart 2012). To understand if a similar mechanism was followed in

the chick, we dissected out blastula stage medial (M) explants, and cultured them

alone or in the presence of Noggin to a time point equivalent to stage 10.

Subsequently, explants were then sectioned and processed for in situ hybridization

and expression levels of Rax2 and FoxG1 were examined.

28

Stage 2 medial explants cultured alone showed FoxG1 expression, but no Rax2

expression was detected. This is in agreement with the previous findings that medial

explants are specified as dorsal telencephalic character (Patthey, Edlund et al. 2009).

In contrast, medial explants cultured in the presence of Noggin generated Rax2 and

FoxG1 positive cells. This suggested that eye-field cells are not specified by the

blastula stage, and in addition, BMP inhibition at this stage is required for the

generation of eye-field cells within the forebrain region.

Prospective retinal cells acquire dorsal telencephalic identity during in vitro

culture

To understand if the initial specification of eye-field cells and neural retina cells

occur in single or distinct inductive events, we analyzed if the eye field cells can

differentiate into Rax2 and Vsx2 positive cells in culture. To address this issue, we

isolated stage 9, 10, 11 and 13optic vesicle (OV) and performed analysis at the onset

of culture (0h), or after 46-54h in culture, which corresponds to around stage 21 in

intact embryos. All explants isolated from stage 9, 10, 11, and 13 and cultured for 0h

expressed Rax2 with few cells positive for FoxG1 in the boundary of the explants.

But stage 9, 10 and 11 OV explants when cultured longer did not show any Rax2,

Vsx2 or Mitf expression but instead generated FoxG1 and Emx2 positive cells,

indicative of dorsal telencephalic character. This suggests that stage 9-11 OV

explants acquire dorsal telencephalic identity when cultured longer. In contrast,

stage 13 OV explants generated Rax2 and Vsx2 positive cells, but no Emx2 was

detected when cultured to 38-40h, suggesting neural retinal cells are specified at

stage 13. Thus prospective retinal cells require signals from adjacent tissue for

maintenance of eye-field character and acquisition of neural retina character at the

expense of telencephalic identity.

Stage 9-11 optic vesicle requires lens ectoderm and BMP signals

To determine if the signals emanating from the lens ectoderm are important for

specification of neural retina characteristics in the underlying optic vesicle, we

dissected out the optic vesicle together with lens (OVL) explants and cultured to a

29

time point equivalent to stage 21. Under these conditions, δ-crystallin positive lens

fiber cells and Rax2 and Vsx2 positive retinal cells were detected in non-overlapping

regions. No FoxG1, Emx2 and Mitf were detected in these explants. This suggests

that lens ectoderm is sufficient for the optic vesicle to maintain Rax2 expression and

further to acquire neural retina identity.

We have shown in our previous publications that Bmp4 is expressed in the

prospective lens ectoderm (Pandit, Jidigam et al. 2011), and phosphorylated smad1

is detected in the optic vesicle (Belecky-Adams, Adler et al. 2002). These data

suggest that BMP signals from the lens ectoderm is important for the maintenance of

Rax2 expression in the optic vesicle and for subsequent generation of neural retina

character. To further investigate this, we cultured stage 9/10 OVL explants in the

presence of Noggin. After BMP inhibition, the generation of neural retina markers

Rax2 and Vsx2 were suppressed and instead FoxG1 and Emx2 expression was

induced; this is indicative of telencephalic character. In addition, we have previously

shown that BMP inhibition blocked the generation of δ-crystallin positive lens cells

(Sjodal, Edlund et al. 2007; Pandit, Jidigam et al. 2011). These results suggest that

BMP signals emanating from prospective lens ectoderm are required for

specification of neural retinal identity by suppressing dorsal telencephalic identity.

Retinal identity is dependent on BMP signaling emanating from the lens

ectoderm

To examine if BMP signals are required for the induction of neural retina cells in

ovo, stage 9/10 optic vesicles were electroporated with GFP alone or together with

Noggin expressing vector (Timmer, Wang et al. 2002), and cultured to stage 15/16.

Embryos with GFP staining within the retina region were selected and further

sectioned and processed for in situ hybridization and immuno staining. All the non-

electroporated side, as well as control GFP electroporated embryos, displayed

normal retina morphology with normal expression of Rax2, Vsx2 and FoxG1. By

contrast, optic vesicles electroporated with Noggin exhibited failed invagination and

did not form bilayered optic cup. Moreover, expression of the neural retina marker

Vsx2 was lost, with down-regulation of Rax2 and expansion of FoxG1 expression.

30

In, addition, the lens failed to invaginate and did not form a vesicle. This suggests

that BMP activity is required for the early development of the neural retina.

To further analyze whether the BMP signals are derived from the lens or from other

adjacent tissues, we co-electroporated Noggin together with GFP in the following

various locations of the chick head: 1) in the lens ectoderm; 2) in the head ectoderm

lateral to the prospective lens ectoderm; and 3) in the ventral midline. When Noggin

was electroporated in the lateral surface ectoderm and ventral midline, no difference

was observed in the eye morphology or expression of Rax2, Vsx2 and FoxG1 when

compared to control embryos. In contrast, when Noggin was electroprated in the

prospective lens ectoderm, the optic vesicle failed to invaginate and did not form a

bilayered optic cup. In addition, Vsx2 expression was lost, and Rax2 expression was

downregulated with no obvious change in FoxG1 expression. Altogether these data

suggest that lens derived BMP signals are required for the induction of neural retina

cells.

Direct requirement of BMP activity for specification of neural retina

To determine whether neural retina specification requires direct involvement of

BMP signaling independent of lens ectoderm, we dissected out stage 13 OV

explants that in vitro generate Rax2 and Vsx2 in the absence of lens ectoderm. When

such explants were cultured in the presence of Noggin, all exhibited low levels of

Rax2 with weak or no Vsx2 expression in half of the explants; in the other half Rax2

and Vsx2 were completely abolished with FoxG1 up-regulated. No Emx2 was

detected in stage 13 OV explants when cultured in the presence of Noggin.

Altogether this suggests that half of the explants acquired tuberal hypothalamus

identity and the other half acquired FoxG1 positive forebrain cells, although distinct

from dorsal telencephalic identity. These results indicate that a direct BMP activity

is required for the specification of neural retina cells.

Next we analyzed if BMP is sufficient to suppress dorsal telencephalic identity and

induce retinal characteristics. To test this, stage 9/10 dorsal telencephalic (dT)

explants were dissected out and cultured alone or in the presence of BMP4. Stage

31

9/10 dT control explants generated FoxG1 and Emx2, indicative of dorsal

telencephalic identity, with no Rax2, Vsx2 neural retinal cells and Mitf positive RPE

cells. With addition of BMP4 protein to the cultured dT explants, dorsal

telencephalic identity was completely inhibited, and instead Rax2 and Vsx2 positive

neural retinal cells were induced, even with relatively low concentration of BMP4.

However, Mitf positive RPE cells were not detected with lower levels of BMP4.

Increased concentration of BMP4 did not induce RPE positive cells. These data

show that BMP signals can substitute for the missing lens ectoderm, and provide

evidence that low levels of BMP are sufficient to induce neural retina identity.

BMP induces Fgf8 expression in prospective retinal cells

FGF signals have been shown to play a role in promoting the identity of neural

retinal cells (Pittack, Grunwald et al. 1997; Hyer, Kuhlman et al. 2003). In chick

embryos Fgf8 expression is detected in the medial part of neural retina at stage 13-

14 and it is also detected at stage 21. To determine if the FGF signals act together

with BMP activity in specification of neural retinal cells, we dissected out stage 9/10

OVL explants and cultured them alone or in the presence of Noggin. Control

explants generated Fgf8 positive cells in the Rax2 positive domain of the explants.

By contrast, in the presence of Noggin, Fgf8 expression was inhibited in the

prospective retinal cells suggesting that BMP acts upstream of Fgf8.

To assess whether the loss of Rax2 and Vsx2 markers after BMP inhibition is due to

the secondary effect of loss of FGF activity, we cultured stage 9/10 OVL explants

together with Noggin and FGF8 (250 ng/ml). However, the addition of FGF8 could

not rescue retinal identity in the absence of BMP signaling. In addition, when OVL

explants were cultured with SU5402, a pharmacological inhibitor for FGF, Rax2 and

Vsx2 markers were still maintained in the explants. These results suggest that early

FGF signals are not required for the specification of neural retinal identity, and in

the absence of BMP signals eye-field cells acquire telencephalic identity.

Wnt alone is not sufficient to specify neural retinal cells, but Wnt together with

BMP signals induce RPE cells

32

Wnt signals have been suggested to be important for the specification of RPE cells

(Fujimura, Taketo et al. 2009; Steinfeld, Steinfeld et al. 2013). Whether Wnt signals

play a role in the specification of the neural retina has not been determined. To

address this, we cultured stage 10 OVL explants and stage 13 OV explants together

with a soluble frizzled receptor (Frizzled-conditioned medium) to inhibit Wnt

activity (Hsieh, Rattner et al. 1999; Gunhaga, Marklund et al. 2003). Stage 10 OVL

and stage 13 OV explants cultured alone or in the presence of Frizzled protein

generated Rax2 and Vsx2 positive cells with no FoxG1 expression in stage 10 OVL

explants, whereas there were few FoxG1 positive cells in stage 13 OV explants. This

suggests that Wnt signals are not required for the specification of neural retinal cells.

To further analyze if the combination of Wnt together with BMP is sufficient to

induce neural retina or RPE identity, Stage 9/10 OV explants were cultured with

Wnt3A condition medium alone or together with low levels of BMP4. Explants

cultured with Wnt3A alone generated FoxG1 and Emx2, indicative of dorsal

telencephalic cells, while retinal markers like Rax2 and Vsx2 were not detected. By

contrast, the combination of Wnt and BMP activity inhibited the generation of

FoxG1 and Emx2 positive dorsal telencephalic cells, but instead induced Mitf

positive RPE cells co-expressing with neural retinal markers Rax2 and Vsx2. Thus,

Wnt activity is not sufficient to induce neural retina identity, but a combination of

Wnt and BMP can induce RPE cells.

33

Paper III: Apical constriction and epithelial invagination are regulated by BMP activity

Characterization of Bmp expression in placode regions around the time of

invagination

To study invagination we first characterized the timing of initial invagination in the

olfactory, lens, and otic sensory placodes in the chick. The initial invagination of

otic, lens and olfactory begins at stage 10/11, stage 14 and stage 17, respectively. It

has been shown that BMP signals are required for the generation of placodes

(Brugmann, Pandur et al. 2004; Glavic, Honoré et al. 2004; Sjodal, Edlund et al.

2007; Patthey, Gunhaga et al. 2008; Patthey, Edlund et al. 2009). To further

investigate if BMP activity plays a role in placode invagination, we examined the

expression pattern of Bmp4, Bmp7, and its transducer pSmad1/5/8 prior to and

around the time of placode invagination. At stages 8-10 Bmp4, Bmp7, and

pSmad1/5/8 were detected in the otic placode region. At stages 11-14 and stage 14-

17, Bmp4, Bmp7 expression and pSmad1/5/8 were observed in the lens and olfactory

region, respectively. Thus, in the olfactory, lens and otic placodes BMP expression

together with pSmad1/5/8 is indicative of BMP activity at the time of initial

invagination of sensory placodes.

BMP signals are essential for morphogenesis of placode and placode

invagination

To investigate whether BMP signals are involved in sensory placode invagination,

we performed in vivo experiments by blocking BMP signals and analyzed for the

changes in the sensory placode morphology. This was achieved by electroporating a

Noggin expressing vector in the stage 10/11 olfactory and lens placode regions. A

GFP vector was transfected alone or together with Noggin in the prospective

ectodermal region before the onset of invagination. Electroporated embryos were

cultured to stage 19/20 for olfactory, and stage 15/16 for lens targeted regions. GFP

stainings within the olfactory and lens were further processed for sectioning and

analyzed by in situ hybridization and immuno staining.

34

All control lens and olfactory electroporated embryos showed normal morphology

of the lens and olfactory epithelium. By contrast, Noggin electroporated embryos

displayed failed thickening of the lens placode and further invagination was

suppressed in both lens and olfactory. Depending on the electroporation efficiency,

occasionally a small olfactory invagination was detected in the GFP negative region.

While the majority of the Noggin electroporated olfactory regions still possessed

thickened epithelium, occasionally a few embryos completely lacked thickened

epithelium. Using a whole embryo three dimensional OPT imaging technique, the

disrupted invagination of the olfactory placode and the lack of pit formation was

clearly visible after BMP inhibition as compared to the controls.

We next analyzed whether BMP signals played a similar role in the otic placode

invagination as for the olfactory and lens. Electroporating stage 6/7 embryos in the

otic region, which lies in close proximity of the heart region, resulted in heart

malformation and arrested development, thus preventing analysis of otic placode

invagination. To avoid this issue, we used a modified Cornish pasty method (Nagai,

Lin et al. 2011) which is another type of ex ovo whole embryo culture technique. For

the whole embryos culture, stage 6/7 embryos were dissected and cultured in the

control medium alone or together with either Noggin or Dorsomorphin, to

approximately stage 12/13. All the embryos cultured in the control medium

exhibited normal morphology and underwent placode invagination. When BMP

signaling was inhibited by either Noggin or Dorsomorphin, the majority of the

embryos displayed normal placode thickening but disrupted placode invagination,

while a few embryos displayed complete lack of otic placode formation. Altogether,

these data suggest that the process of placode formation in the olfactory and otic

placode is BMP independent, while lens placode formation requires BMP signals.

But the invagination process for olfactory, otic, and lens all require BMP activity.

This suggests that placode formation and invagination are two independent

processes, and BMP signals are essential for the invagination process.

Disrupted placode invagination after BMP inhibition is not due to the loss of

placode identity

35

Several studies have shown that BMP signals are important for the specification of

lens fibre cells, partly by regulating L-Maf (Sjodal, Edlund et al. 2007; Pandit,

Jidigam et al. 2011). This was also confirmed in the current studies. Therefore, we

examined if the disrupted placode invagination after BMP inhibition might be a

consequence of disturbed acquisition of placode cell identity in the olfactory and

otic placodes, or if BMP signals are specifically important for the invagination

process. To address this issue, we investigated whether cells in the placode still

generated differentiated cell types in the absence of BMP signals by using a set of

molecular markers that uniquely define specific placodes.

In general, the olfactory placode gives rise to both sensory and respiratory parts of

the olfactory epithelium (Maier, von Hofsten et al. 2010). The cells in the sensory

domain express stem-cell like Hes5 positive cells and post-mitotic HuC/D neurons,

while cells in the respiratory region express Id3 (Maier, von Hofsten et al. 2010).

Moreover, Id3 has been shown to be a direct target gene of BMP signaling, and can

be used as a readout of BMP activity (Hollnagel, Oehlmann et al. 1999). Embryos

electroporated with GFP alone in the olfactory placodal region expressed Hes5 and

HuC/D cells in the sensory domain and Id3 in the respiratory region. In contrast,

Noggin electroporation in the olfactory placodal region blocked generation of Id3

positive respiratory cell, which confirmed the suppression of BMP activity; this is in

agreement with previous published data (Maier, von Hofsten et al. 2010). Even

though BMP inhibition blocked the generation of the respiratory domain, expression

of Hes5 and HuC/D characteristic of olfactory sensory identity was maintained.

Control cultured embryos expressed the non-neurogenic markers Irx1 and Lmx1b in

the posterior otic placode as expected. In the BMP inhibited embryos, the otic

placode failed to invaginate with Lmx1b expression throughout the otic epithelium,

while Irx1 positive cells were detected in the posterior part of the otic region. This

suggests that at this stage specification of otic placodal cells occur independent of

BMP signals. Altogether, these results suggest that cells can differentiate beyond the

placode stage and mature into differentiated cell types in the absence of

invagination. This proposes that acquisition of placode specific cell types and

invagination are two independent processes.

36

Failure in placode invagination after BMP inhibition is not due to changes in

cell death or proliferation

We then examined whether disruption of placode invagination after BMP inhibition

is due to the reduction of cell proliferation or increase in cell death. To examine this,

cell proliferation marker pHH3 and cell death marker aCaspase3 were analyzed in

the control and BMP-inhibited embryos (Noggin electroporated in the lens and

olfactory, Dorsomorphin treated embryos for the otic region) that were cultured to

different time points. We did not observe any significant changes in the levels of

aCaspase3 positive cells in the Noggin electroporated lens and olfactory embryos

compared to control embryos. Likewise, no significant changes for aCaspase3

positive cells were observed when otic regions were cultured in the presence or

absence of Dorsomorphin. Regarding the proliferative studies, there was no

significant change in pHH3/GFP double positive cells in the Noggin electroporated

in the lens; similarly no changes in the levels of pHH3 were observed in the otic

region treated with Dorsomorphin when compared to controls. In contrast, reduction

in pHH3 positive cells was observed in the Noggin electroporated in olfactory

region compared to controls. This suggests that reduced cell proliferation in the

olfactory region might affect the invagination process, but in general reduced

proliferation cannot explain failure in placode invagination after BMP inhibition.

BMP is required for cell shape changes in the prospective placodal cells

Cell shape changes like cell elongation and apical constriction were required for the

invagination process. We next analyzed if BMP inhibition prevented any cell shape

changes. We measured cell length, apical cell width and basal cell width in control

and Noggin treated embryos. The olfactory placode was optimal for these studies,

since BMP inhibition disrupted the placode invagination but not the placode

formation, and the GFP electroporated cells were easily visualized in the failed

olfactory placode. GFP positive cells from the medial invaginated part of the control

olfactory were compared to Noggin treated embryos, with a total of 74 control cells

and 61 cells from Noggin treated embryos.

37

BMP inhibition in the olfactory placode significantly increased the cell apical width

(average of 2.8µm) compared to the controls (average of 1.2µm). Moreover, Noggin

treated cells showed reduction in cell length (33µm compared to control average of

50µm). But no significant difference was observed with cell basal width in the

control with an average of 5.5µm and Noggin treated cells with an average of 5µm.

This resulted in an apical basal ratio of 0.56 for Noggin electroporated cells

compared to 0.22 for the control treated cells. This suggests that variations in cell

shape changes after BMP inhibition is one of the reasons for disruption of olfactory

placode invagination.

Apical localization of RhoA and F-actin is disrupted after BMP inhibition

Apical constriction and placode invagination have been shown to be mediated by

apical localization of RhoA that enables apical accumulation of F-actin, which in

turn activates the acto-myosin network (Sai and Ladher 2008; Plageman, Chauhan et

al. 2011). Using confocal microscopy, we analyzed if the apical accumulation of

RhoA and F-actin were disturbed after the inhibition of BMP in the placode regions.

In all the control invaginated placodes for the olfactory, otic and lens, RhoA and F-

actin were apically localized. By blocking BMP signals in all the three sensory

placodes, apical localization of RhoA and F-actin were disrupted. In the BMP

inhibited embryos RhoA expression was completely lost and the cellular localization

of F-actin was changed to the boundary of the cells instead of being apically

localized as seen in the control embryos. These results provide evidence that BMP

signals are required for the apical accumulation of RhoA and F-actin, and disruption

of this accumulation is the reason for failed sensory placode invagination.

To determine if this mechanism is common to all placodes, we also analyzed the

hypophyseal non-sensory placode by electroporating Noggin construct at stage 12

and cultured to stage 15/16. All the control GFP electroporated embryos exhibited

normal placode invagination with apical localization of F-actin, while Noggin

treated embryos demonstrated failed placode invagination with apical disruption of

F-actin. RhoA was not expressed at the examined stages in the hypophyseal

38

epithelium. This suggests that BMP signals are required for the apical accumulation

of F-actin and epithelial invagination in both sensory and non-sensory placodes.

ROCK activity is required for placode invagination

Rho-associated protein kinase (ROCK), a downstream effector of small GTPase Rho

is known to be one of the major regulators of cytoskeleton elements. It is well

known that RhoA forms a complex together with Trio, Shroom3 and Rock1/2, and

blocking any of the activity disrupts apical constriction and placode invagination

(Borges, Lamers et al. 2011; Plageman, Chauhan et al. 2011; Sai, Yonemura et al.

2014). To assess the importance of ROCK activity in all three sensory placodes, we

used whole embryo culture together with Y27632, a ROCK inhibitor. During the

control conditions the lens, otic, and olfactory showed normal apical localization of

RhoA and F-actin. By contrast, embryos treated with ROCK inhibitor exhibited

failed placode invagination in a similar manner as the embryos treated with BMP

inhibition. Moreover, using confocal microscopy we were able to show that

accumulation of RhoA was reduced or lost, and F-actin was located at the periphery

of the cells instead of being apically localized as seen in the controls. This suggests

that blocking ROCK activity results in the failure of placode invagination, a

phenotype similar to that of BMP inhibited sensory placodes. Altogether these

results indicate that BMP signals act upstream of ROCK during apical constriction

and placode invagination.

39

Discussion

Paper I: Lens

Several studies in the chick and mouse have shown the BMP activity is required for

the normal development of lens. Bmp4 or Bmp7 knockout mice revealed that BMP

signaling is essential for lens induction, but the cellular mechanisms underlying this

process are not well studied (Furuta and Hogan 1998; Wawersik, Purcell et al.

1999). Another study in mice demonstrates, inactivation of two type I BMP

receptors (Bmpr1a and Acvr1) act redundantly in a smad independent pathway

during lens formation (Rajagopal, Huang et al. 2009). Most of the BMP mice mutant

studies have shown its requirement for the normal development of eye, but the

spatio-temporal requirement of BMP signaling during lens development is not

addressed. Our results now extend that knowledge and show that prior to lens

placode formation, BMP signals are important for the initial induction of L-Maf,

which acts as a key regulator for lens fiber cell identity and is also known to be a

marker for early specification of the lens placode. In the absence of BMP signals,

lens cells switch to olfactory fate characterized by co-expression of Keratin, HuC/D

and Dlx. Furthermore, our results show that once L-Maf is up-regulated around

stage 13 (lens placode stage), further lens development becomes independent of

BMP signaling. At these stages, BMP inhibition did not exhibit any loss of the lens

specific markers L-Maf and δ-crystallin. This defined time window of BMP

dependent and BMP independent mechanisms, before and after placode formation is

very crucial for the early development of the lens.

Previous studies have shown that L-Maf is required for the up-regulation of δ-

crystallin which regulates cell cycle exit and lens fiber cell differentiation by

activating p27kip1 (Reza, Urano et al. 2007). Moreover, it was shown that L-Maf

expression is capable of ectopically inducing Prox1 and Crystallin genes in ovo,

while dominant negative L-Maf represses these genes with complete loss of lens and

optic vesicle invagination (Ogino and Yasuda 1998; Reza, Ogino et al. 2002; Reza,

Urano et al. 2007). It was previously shown that L-Maf expression is dependent on

40

Pax6. Although Pax6 alone ectopically could not induce L-Maf expression, addition

of Sox2 is sufficient to induce L-Maf (Reza, Ogino et al. 2002). We show that in the

presence of L-Maf, δ-crystallin is upregulated with initial elongation of fiber cells.

In our study we show that the upregulation of L-Maf is BMP dependent, and

subsequent δ-crystallin upregulation with initial elongation of fiber cell is BMP

independent.

Using mouse fibroblast cells it was shown that c-Maf is a potential candidate target

of Pax6 (Sakai, Serria et al. 2001). c-Maf is a member of large Mafs, which were

shown to be involved in lens fiber cell differentiation in the mouse (Kawauchi,

Takahashi et al. 1999). Moreover, c-Maf knockout studies in mice have displayed

reduced crystallin expression (Kim, Li et al. 1999). Although c-Maf and L-Maf have

similar functions, they are not functionally identical since L-Maf plays an important

role in lens placode formation in the chick, whereas c-Maf does not play a similar

role in the mouse. Our study extends the knowledge that BMP directly or indirectly

regulates L-Maf expression.

Otx2 and Pax6 are shown to maintain the competence of the surface ectoderm to

respond to the signals from the optic vesicles (Zygar, Cook et al. 1998; Ashery-

Padan, Marquardt et al. 2000). Many studies have shown that Pax6 plays a cell-

autonomous role in surface ectoderm and it is required to respond to the lens

inductive signal. Conditional deletion of Pax6 in the prospective lens-forming

ectoderm prevents lens development (Ashery-Padan, Marquardt et al. 2000). One

study of Pax6 conditional deletion in surface ectoderm prevented placode formation

with substantial decrease in ECM (extra cellular matrix) between the optic vesicle

and ectoderm (Huang, Rajagopal et al. 2011). One more study with placodal

deletion of Sox2, another transcription factor involved in lens development, showed

milder phenotypic changes with reduced crystallin expression in the invaginated

lens placode (Smith, Miller et al. 2009). In mice lacking Bmp4 normal Pax6

expression was shown with reduced levels of Sox2. Moreover in the Bmp7 mutants,

the expression of Pax6 is reduced with loss of Sox2 expression (Furuta and Hogan

1998; Wawersik, Purcell et al. 1999). Somewhat similar to that, our studies show

that BMP inhibition has severe consequences for lens development in addition to the

41

loss of L-Maf and δ-crystallin expression, although expression of Pax6 and Sox2

was not affected after BMP inhibition. One possible explanation is that Sox2 and

Pax6 have become independent of BMP signals at the time BMP was inhibited in

the chicken experiments. Alternatively, Sox2 and Pax6 expression is not directly

dependent on BMP, but the observed reduction in the mouse mutants might be

because the germline deletion of Bmp gene also impairs the earlier development of

ectodermal tissues (Rajagopal, Huang et al. 2009).

From our in vitro explant assay, it is evident that in the absence of BMP signals

prospective lens ectodermal cells acquire olfactory cell fate. This is in agreement

with previous studies at the neural fold stage, showing that continuous exposure of

olfactory placodal cells to BMP signals promotes the generation of lens cells

(Sjodal, Edlund et al. 2007). Studies in the chick spinal cord have shown that ectopic

activation of Bmp signaling induced expression of MafB (Chizhikov and Millen

2004) indicating that large Maf genes are regulated by a common molecular

mechanism. Altogether it can be suggested that Pax6 might play a potential role in

the regulation of ECM deposition, as well as in the morphogenesis of lens ectoderm

to come in close contact with optic vesicle which is crucial for further induction of

lens placode, whereas BMP plays a potential role in cytoskeleton reorganization

together with induction of lens placodal specific genes (Fig. 5). This proposes that

both Pax6 and Bmp4 appear to be regulated and to function independently of each

other during lens induction and both are required for proper lens development.

Fig. 5: BMP signals are required for induction of L-Maf. At the optic vesicle stage when the lens ectoderm (purple) is located close to the optic vesicle (green), BMP signals within the lens ectoderm are required for induction of L-Maf in response to inductive signals from the optic vesicle. Furthermore, upregulation of δ-crystallin is dependent on L-Maf. BMP signals are required for apical accumulation of RhoA and F-actin. Either BMP within the lens ectoderm is directly required for L-Maf induction or the signals from the optic vesicle induce it.

42

Paper II: Retina

The mechanism by which the eye field is specified within the anterior neural plate in

early embryonic development is poorly described. Simultaneously, the mechanism

by which the neural retina becomes regionalized from the forebrain region is not

well defined. Our study extends the knowledge of the molecular mechanisms for the

early specification of retinal cells and at the same time we provide evidence that

signals from the lens ectoderm are necessary for the specification and maintenance

of retinal identity. Briefly, our results provide evidence that at blastula stages the

presence of BMP signals in the anterior neural ectoderm represses eye field

characteristics by promoting subsequent acquisition of forebrain character.

Conversely, at the optic vesicle stage lens derived BMP signals are important for the

differential specification of the retina within the forebrain region, by inhibiting

telencephalic identity. Furthermore, our results provide evidence that retinal cells are

specified at around stage 13.

Using chick embryos, it has been shown that removal of prospective lens ectoderm

at stage 12, resulted in optic vesicle formation but further failure to form an

invaginated optic cup. In contrast, surgical removal of the lens placode at stage 13

did not show any adverse effects on optic cup formation or any effects on neural

retinal identity. This indicates that prospective lens ectoderm is necessary for optic

vesicle invagination (Hyer, Kuhlman et al. 2003). Although this study defined the

time window for the optic vesicle invagination, specific signals or molecules

important for the specification and maintenance of neural retina were not mentioned.

In mouse, lens specific deletion of Pax6 was shown to allow induction of

prospective lens ectodermal character and neural retina identity, but further

development of the lens is arrested (Ashery-Padan, Marquardt et al. 2000). Another

study in the chick showed that blocking of L-Maf and Pax6 function using dominant

negative constructs in the lens ectoderm prevented both lens placode formation and

optic vesicle invagination (Reza, Ogino et al. 2002). These studies however, do not

provide evidence on the developmental windows at which these gene products are

required for the specification and/or maintenance of neural retina identity. Our study

provides evidence that prior to stage 13, BMP signals from the prospective lens

43

ectoderm are crucial for the specification of prospective retinal cells as indicated by

Rax2 and Vsx2 expression; after stage 13, retinal cells develop independent of

signals from the lens placode.

Our in vitro studies show that blocking BMP signals at stage 9/10 inhibited the

generation of retinal cells, and up-regulated telencephalic markers. But the cell fate

switching from retinal cell to telencephalic identity is only partially observed by

blocking BMP signals at stage 13. In vivo studies exhibited similar results. Upon

reduction of BMP signals cells express reduced levels of Rax2 with complete loss of

the neural retinal marker Vsx2, and further expansion of FoxG1 expression in the

optic vesicle. However, we do not see any switch in cell identity suggesting that in

intact embryos signals from surrounding tissues like ventral midline or cephalic

mesenchyme might compensate for the effect of reduced BMP signaling. This

indicates that BMP signals are required for the specification of Vsx2+ neural retinal

cells; in absence of BMP signals, cells acquire telencephalic character. From our

study we showed that BMP signals are required for specification of neural retina and

there have been studies in mouse indicating that BMP4 and BMP7 are essential for

dorso-ventral patterning of the retina (Morcillo, Martínez-Morales et al. 2006; Yun,

Saijoh et al. 2009). Our present studies as well as previous studies from the mouse

indicate that BMP signals are essential for specification and/or maintenance of

neural retinal cells along with patterning of the retina.

Another study in the mouse with conditional deletion of Lhx2 showed a severe

phenotype with failed optic vesicle invagination towards the lens ectoderm, and

further loss of retinal specific markers (Yun, Saijoh et al. 2009; Hägglund, Dahl et

al. 2011). Rescue experiments in Lhx2 mutants have proven that the treatment of

Lhx2 mutant explants with exogenously added BMP recovered some aspects of eye

phenotype (Yun, Saijoh et al. 2009). Although Pax6 and Lhx2 mutants show similar

morphology, molecularly they are distinct. In the absence of the Pax6, the eye field

is still maintained through the optic vesicle stage as indicated by Rx, Six3, Pax2 and

neural retinal marker Vsx2 and RPE marker Mitf (Bäumer, Marquardt et al. 2003). In

Lhx2 mutants the eye field is still maintained with complete loss of neural retinal

identity shown by Vsx2 marker. The mutants maintain optic vesicle formation

44

because of the Rx expression and this gene is known to be essential for the optic

vesicle morphogenesis (Mathers, Grinberg et al. 1997). Our results after BMP

inhibition show a similar kind of phenotype with failed optic cup formation. This is

in agreement with a study by Murali et al, which showed that homozygous

inactivation of two type I BMP receptors resulted in down-regulation of Vsx2

expression and failed optic cup formation. The morphological effect could be

because of loss of the Vsx2 (Chx10) marker which was shown to have important

functions for retinal development, such as regulation of proliferation (Green, Stubbs

et al. 2003). Reduced Rax2 expression with loss of proliferative Vsx2 positive cells

resulted in decreased dorsal ventral expansion which in turn had an impact on the

invagination of the optic cup. In addition, it was shown that BMP signaling is also

implicated in the regulation of apoptosis in chick and mouse eye development

(Trousse, Esteve et al. 2001; Morcillo, Martínez-Morales et al. 2006). Further

studies are needed to determine if reduction in cell death has any effect on the

morphology. In contrast, using bead assays it was shown that ectopic BMP4

expression inhibited invagination of optic vesicle but not lens development (Hyer,

Kuhlman et al. 2003). Our in vitro experiments with dorsal telencephalic and optic

vesicle explants demonstrate that cells switch identity when cultured in the presence

of low levels of BMP. However, Hyer et al, did not examine the requirement of

BMP signals for the optic vesicle. We show that modulating the levels of BMP

signals has adverse effects on the development of the retina, suggesting that a

balance of BMP signals is required for the proper optic vesicle invagination.

Another study in the mouse demonstrates that the cells within the optic vesicle

change shape intensely, which is further accompanied by the transient alteration of

the basal lamina composition (Svoboda and O'Shea 1987). Further studies are

needed to determine if BMP signals play a role in the deposition of laminin during

the optic vesicle to optic cup formation. As the main focus of our paper was on

specification and maintenance of the eye field, a brief background is given about the

possibilities of morphological difference caused after BMP inhibition.

Studies from 3D stem cell differentiation have shown that embryonic stem cells can

either spontaneously acquire self-directed organization of the complex optic-cup and

neural retina morphology (Eiraku, Takata et al. 2011) or dorsal telencephalic

45

identity (Lancaster, Renner et al. 2013). The difference between these behaviors has

remained mysterious. Our study provides evidence that the absence or presence of

low levels of BMP might switch cell identity between retina and telencephalon.

46

Paper III: Placode invagination

Our study provides mechanistic insight into cellular mechanisms that drive the

morphogenetic process of the flat ectodermal sheet into invaginated cup like

structures. We advocate that one essential module of morphogenetic cell behavior

called placode invagination is not coupled with the acquisition of placode specific

identity in three sensory (olfactory, otic and lens) placodes. Moreover, based on the

studies from both sensory and non-sensory ectodermal placodes, our data show that

the regulatory mechanisms involved in the placode bending are common for all the

ectodermal placodes. Also, BMP signaling plays a central role in the sensory and

non-sensory placode invagination. Our results provide evidence that disturbed

placode invagination after BMP inhibition is not due to loss of placode identity, cell

death or reduced proliferation, but mainly due to failed apical accumulation of RhoA

and F-actin. Furthermore, our study shows that inhibition of ROCK, a molecule

known to be essential for invagination of the otic placode, results in flat placode

ectoderm.

Our expression analysis in the chick shows that the BMP ligands and its transducer

pSmad1/5/8 are detected before placode formation and around the onset of placode

invagination. In the present studies we provide evidence that BMP is required for

the normal development of sensory placodes (olfactory, otic and lens) and one non-

sensory placode (hypophyseal). Embryos in which BMP signals are blocked exhibit

failure of placode invagination as a result of disrupted apical accumulation of RhoA

and F-actin, which leads to a lack of apical constriction and reduced cell length. This

is in agreement with previous observations in chick that defective BMP signaling

also results in changes in olfactory and lens placode invagination (Maier, von

Hofsten et al. 2010; Pandit, Jidigam et al. 2015). Consistently, both Bmp4 and Bmp7

germline mice mutants demonstrate failed lens placode formation and subsequent

invagination (Furuta and Hogan 1998; Wawersik, Purcell et al. 1999). In addition,

conditional deletion of Bmpr1a and Acvr1 receptors demonstrated defects in lens

placode formation (Rajagopal, Huang et al. 2009).

47

Besides BMP, FGF signaling was shown to be important for placode invagination.

Previous studies from chick and mouse embryos showed that both olfactory and otic

placodes fail to invaginate after blocking FGF signaling (Zelarayan, Vendrell et al.

2007; Sai and Ladher 2008; Maier, von Hofsten et al. 2010). For otic

morphogenesis, FGF signaling was shown to activate myosin II through PLCγ-

mediated cascade which is essential for apical actin enrichment (Sai and Ladher

2008). Invagination in lens, olfactory and otic appears to be driven by both intrinsic

and extrinsic forces; it is still controversial as to which force dominates. Conversely,

whether these two processes are coordinated together is still unclear. Extrinsic forces

originate not from the placode itself, but from the surrounding epidermis and

mesenchyme. Another known extrinsic factor is the Rho family GTPase Cdc42, the

activity of which is vital for the formation of filopodia (Nobes and Hall 1999;

Ridley 2006). Conditional deletion of Cdc42 mutant mice resulted in reduced lens

pit invagination (Chauhan, Disanza et al. 2009). In contrast, intrinsic forces generate

through the apical constriction and cell elongation, but it is still a question if apical

constriction and cell elongation rely only on intrinsic forces. Our studies from the

three sensory placodes show that BMP signals regulate the process called apical

constriction and this process is accompanied by actin-myosin cytoskeletal changes,

and is supposedly a process of intrinsic forces.

BMP is required for apical accumulation of RhoA and actin-myosin

contractility

Our results provide evidence that loss of BMP activity in prospective placodal

regions of the head ectoderm disrupts accumulation of RhoA and F-actin at the

apical side of the placodal cells as well as for subsequent apical constriction. It has

already been shown that RhoA plays a central role in apical constriction, whereas

another small GTPase, Rac, is essential for cell height. RhoA mouse mutant embryos

show reduced lens pit curvature with increased cell length. Rac1 mutants show

increased apical constriction reduced cell length in the lens placode (Chauhan, Lou

et al. 2011). Based on this, it has been suggested that the RhoA mutant is indicative

of Rac1 pathway gain of function, and the Rac1 mutant is indicative of RhoA

pathway gain of function (Chauhan, Plageman et al. 2015). Similar mechanisms

48

have been shown in multinucleated giant cells (MNGC) in that the level of

microtubule polymerization inversely regulates Rho and Rac activities.

Depolymerization of microtubules leads to Rho activation by blocking Rac activity,

whereas repolymerization of microtubule induces Rac activation by inhibiting Rho

activity (Ory, Destaing et al. 2002). From our present studies, BMP inhibition in the

lens and olfactory placodes cause defects of both apical constriction and cell height

which suggests that BMP signaling might be upstream of both Rho and Rac

signaling.

In one study, blocking MID (a microtubule associated protein) in neuroepithelial

cells of Xenopus embryos caused failure of apical constriction and cell elongation,

which involves both actin and microtubules (Suzuki, Hara et al. 2010). Similar to

the role of Rac1, microtubules have long been hypothesized to be involved in

maintaining cell height (Burnside 1973; Picone, Ren et al. 2010). In the amnioserosa

(a squamous epithelial tissue in the dorsal-most region of the cellular blastoderm) of

gastrulating Drosophila embryos, a cell shape conversion from columnar to

squamous is observed due to change in the microtubule orientation from the apical-

basal axis to the planar axis (Pope and Harris 2008). Moreover, it was shown that

microtubules are needed for polarized localization of many factors like RhoA

(Nakaya, Sukowati et al. 2008), polarity regulators (Siegrist and Doe 2007),

RhoGEF (Rogers, Wiedemann et al. 2004) and adhesion molecules (Harris and

Peifer 2007). Whether the microtubule requirement for localization of RhoA is

direct or indirect is not known. Our studies provide evidence that BMP signals are

required in the sensory placodes for apical accumulation of RhoA. However,

microtubules regulating cell height are still under discussion.

An additional well studied mechanism for cell length was shown in the Drosophila

ventral furrow formation, suggesting that apical constriction generates cytoplasmic

flow and results in cell elongation (He, Doubrovinski et al. 2014). Our study in the

olfactory placode showed that blocking BMP signals in the olfactory ectoderm led

to reduced apical constriction with decreased cell length. If the reduced cell height is

because of the increase in apical width then this indirectly means that the

mechanism of cytoplasmic flow is also regulated by BMP signaling.

49

Although small Rho GTPases (Rho and Rac) have been shown to be important for

the process of epithelial bending, invagination still occurs in absence of RhoA and

Rac1, which suggests that other unknown potential molecules are possibly involved

in the process of epithelial invagination (Chauhan, Lou et al. 2011; Sai, Yonemura

et al. 2014). On the other hand, cells in both RhoA and Rac1 mutants display no

significant changes in the basal surface area. Studies in chick embryos demonstrate

that the cells in the otic placode undergo basal expansion first and apical constriction

later. Furthermore, basally localized FGF signaling generates basal cell expansion in

the otic placode by depletion of actin filaments. Activation of a RhoA-ROCK

dependent pathway results in myosin II activation essential to direct the contraction

of apical actin networks and subsequent apical constriction (Sai and Ladher 2008;

Sai, Yonemura et al. 2014). Studies in mouse mutant embryos show that when FGF

signaling is blocked the thickening of the lens placode is significantly reduced

(Faber, Dimanlig et al. 2001). But it is not further analyzed, if the FGF signaling is

essential for cell morphology in the lens placode invagination. Based on these

studies, a possible reason might be that the apical constriction with elongated cells

causes only slight bending, and basal cell expansion is further needed for deeper

invagination. From the present studies, inhibition of BMP signaling in the lens

ectoderm shows defective placode formation and subsequent invagination, whereas

in the olfactory and the otic placode formation is normal but the invagination is

shown to be affected. A possible reason might be that both the olfactory and otic

placodes have two distinct domains and these regions are known to be regulated by

FGF and BMP, respectively (Abello, Khatri et al. 2010; Maier, von Hofsten et al.

2010). In the absence of one domain, the placode still maintains its identity which is

sufficient for thickening of the ectoderm, whereas for invagination both domains are

required. Alternatively, a balance of both FGF and BMP activities is required for

placode invagination (Fig. 6).

50

In addition, the basal lamina might play a prominent role in placode invagination.

Using pull down assays it was demonstrated that laminin 10/11 activates Rac; in

contrast activation of Rho is influenced by fibronectin (Gu, Sumida et al. 2001). In

zebrafish fin epithelium it was shown that the laminin regulates cell shape changes

via canonical Wnt signaling pathway (Nagendran, Arora et al. 2015). In Drosophila

eye morphogenesis, integrins were shown to regulate apical constriction through

microtubule stabilization (Fernandes, McCormack et al. 2014). There are several

studies showing that integrins are cellular receptors for laminins, and they mediate

anchoring to basal lamina (Zagris 2001; Zagris, Christopoulos et al. 2004). Integrins

influence multiple functions by anchoring cells to the extracellular matrix (ECM).

Integrins in the extracellular region act as a bridge between the laminin-containing

ECM and the cytoskeleton of the cell in the cytosol, resulting in changes in cell

polarity, shape and migration. Based on my personal unpublished observations,

laminins are expressed as a thicker band before placode formation and later this

lamina is refined to a thinner region as the placode invaginates (Fig.7). Apical

constriction, increase in cell length, basal expansion and thinning of basal lamina

altogether might play a potential role for complete placode invagination. Disparity in

any of the stated events might show mild to severe defects in placode invagination.

Further studies are needed regarding the role of laminin during placode invagination,

and additional experiments have to be done to determine if the requirement for BMP

Figure 6: (A) schematic image showing Hes5 positive cells in the invaginated olfactory

placode and Id3 in the rim of the olfactory placode. (B) In the absence of BMP signaling

Hes5 positive olfactory placode cell identity was maintained. (C) In the absence of FGF

signaling Id3 positive olfactory placode cell identity was retained (Maier et al., 2010). FGF,

fibroblast growth factor; BMP, bone morphogenetic protein.

51

during sensory placode invagination is due to its impact on graded levels of laminin

deposition.

Figure 7: At olfactory placode stage (st 14) Laminin was broadly expressed within the olfactory

placode region. By stage 17, when the placode starts to invaginate, Laminin was concentrated

in a thinner region at the basal side of the epithelium underneath the slightly invaginated

placode. Cell nuclei stained with DAPI appear in blue and red indicates Laminin.

BMP activity acts upstream of ROCK

Vertebrate neural tube development is a typical model of epithelial invagination,

since the neural tube is the first organ to develop. For neural tube closure, RhoA

activation by ArhGEF11 is essential through activation of pMLC (Nishimura,

Honda et al. 2012). Similarly, in the otic placode, RhoA is apically localized in the

same way as during neural tube closure which is also known to be activated by

ArhGEF11. ArhGEF11 and its putative downstream effector ROCK activate the

levels of pMLC and F-actin at the apical junctions. Inhibiting RhoA, ArhGEF11and

ROCK all reduced apical constriction with reduced levels of pMLC and F-actin in

the apical junctions (Sai, Yonemura et al. 2014). During otic placode invagination,

the PCP (planar cell polarity) marker Celsr1 is precisely expressed in the center of

the otic placode; by blocking the function of this gene placodal cell displayed a

significant increase in apical surface area. It has been suggested that Celsr1 lies

upstream of RhoA during otic placode invagination (Sai, Yonemura et al. 2014).

Although blocking Rho, ArhGEF, and Celsr1 shows reduced apical localization of

pMLC and F-actin, invagination still occurs. Since ROCK is the downstream target

of all the above mentioned genes, blocking of ROCK function using a

52

pharmacological inhibitor results in drastic effects on otic placode invagination

(Chauhan, Lou et al. 2011; Sai, Yonemura et al. 2014). Altogether it can be

suggested that Rho and GEF molecules are expressed specifically in placode cells,

whereas ROCK is expressed in the placode as well as in the lateral ectoderm. By

inhibiting ROCK, the force being generated from the lateral surface ectoderm is also

inhibited. This suggests that the lateral surface ectoderm might be playing an

essential role in sensory placode invagination. Our present study shows that ROCK

inhibition exhibits failure in all the three sensory placode invagination with

disrupted accumulation of F-actin. Moreover, our results provide evidence that

morphologically the ROCK-inhibited placodal epithelia were indistinguishable from

the BMP-inhibited flat placodal domain. This indicates that BMP activity acts up-

stream of the ROCK-regulatory molecular machinery to regulate epithelial

invagination.

Another known suggested mechanism for epithelial invagination is apoptosis.

Studies in fly embryos demonstrate that apoptotic cell extrusion accelerates dorsal

closure (Toyama, Peralta et al. 2008). Another study in Drosophila epithelial cells

displays that apoptotic cells actively influence their surrounding cells by

accumulation of acto-myosin along their apical-basal axes and pulls the apical

surface inward resulting in the transient depression of the apical surface (Monier,

Gettings et al. 2015). However, our current study shows that failure in placode

invagination after BMP inhibition is not caused by alterations in cell death or loss of

placodal identity.

In contrast to the mechanism of apoptosis in epithelial invagination, it was suggested

that a mitotic cell rounding and interkinetic nuclear migration (IKNM) play an

important role in apical constriction and epithelial invagination (Messier 1978;

Stewart, Helenius et al. 2011). In Drosophila tracheal placode invagination, mitotic

cell rounding is known to play an important role, and using microtubule inhibitor

colchicine it was shown that cell rounding, but not cell division, is responsible for

tracheal placode invagination (Kondo and Hayashi 2013). However, in our study we

do not see any significant difference in cell proliferation in lens and otic placodes. A

possible reason for lack of placode invagination might be due to delayed mitosis

53

after BMP inhibition, which would affect invagination but not the number of

proliferating cells. Since IKNM was to be required for neural tube closure, a primary

question about IKNM is how different or similar the process is between tissue types.

54

Conclusions

1. Prior to the lens placode stage (stage 13) BMP signals are essential for

acquisition of lens identity both in vitro and in vivo. Until stage 13,

continuous exposure of BMP signals is required for induction of L-Maf

(lens placode identity marker). In the absence of BMP activity, explanted

lens cells acquire olfactory characteristics.

2. After the onset of L-Maf primary lens fibre cell differentiation indicated by

δ-crystallin, becomes independent of BMP signals.

3. At optic vesicle stage BMP signals from the lens ectoderm are required for

promoting retina cell fate in part by suppressing telencephalic identity. By

stage 13, retinal cells are specified suggesting that prospective retina cells

do not require lens tissue to maintain retina characteristics.

4. BMP signals are required for placode invagination.

5. BMP signals regulating the placode invagination is shown to be required

for both sensory (olfactory, otic and lens) and non-sensory (hypophyseal)

placode invagination.

6. Disruption in placode invagination after BMP inhibition is not due to loss

of placode specific identity, but mainly to failure in apical localization of

RhoA and F-actin.

55

Acknowledgements

There have been many people who have guided me, showed me opportunities during

the last few years and I would like to thank each and every one of them. I would like

to thank all the people who helped me reach my goal and please forgive me if i

forget someone.

First of all, I wish to express my gratitude to my supervisor, Lena Gunhaga. Thank

you for giving me the opportunity to be in your lab. The biggest dream of my life

was not only possible but also a pleasure with your unlimited support and guidance.

You are an excellent supervisor; I never hesitated to step into your office to discuss

my projects and experimental problems. You gave me endless freedom to learn

many things about the research which helped me improve my critical thinking and

analyzing skills. It was not just at work, the support you have given when my health

was deteriorating was amazing and unforgettable. I would like to thank from the

bottom of my heart for everything. Thanks a lot for your help for my thesis writing.

Am sure I will miss working with you in future.

I also wish to sincerely thank my co supervisor, Jonas Von Hofsten: I am really

thankful for teaching me the electroporation technique and grateful for all the

support during my tenure.

I wish to acknowledge the support received from the past and present lab members.

Previous lab members; Tanushree: It was really nice working with you. I am really

thankful for your help in teaching me the lab techniques in my initial joining days. I

really like the way you discuss with interest in every scientific and nonscientific

topics. I wish you all the best for your future. Miguel Jarrin: I miss the joy of

working with you and still miss the fun talks we used to have daily! It was really fun

discussing Spanish words with you. Esther: You are the first person who

encouraged me to visit IKSU and now i realize the importance of it. Nevertheless,

lately started visiting IKSU and would be thankful for the rest of my life for a good

start. Walter: I always see you working without taking any breaks. I wonder where

you get that energy to work like that without taking any breaks. Hanna Nord: I still

remember the days when i was new to the lab and you responded to endless

questions of mine with patience. I would like to thank you for all the help and

support. I wish you all the best for your future. Helena Alstermark: It was really

nice working with you during the initial days of work in this lab. I have tried my

best to learn from you in organizing the stuff. All the masters and project students

Nils, Harsha, Raghu, Manoj, Margaret, Ashleigh, Shori, Dåg, Emma, Miriam,

Elsa, Parham it was fun getting to know all of you.

Current lab members: Cedric Patthey, It was a pleasure to work with you. You

are very inspiring in several aspects of my PhD life. How to think rationally, clearly

and reasonably when it comes to research is something i have learnt from you. You

56

have not only given your time generously, but also provided responses and questions

that were valuable to my research. Though i have doubts, i hesitated to ask questions

during several lab meetings and it was from you i have learnt to open up about my

agreements or disagreements. Whenever i come to the lab during the late nights i

always see you in the lab or in your office. Your dedication, swiftness and

promptness inspired me a lot. I am really thankful for your help in correcting my

thesis. Tami, Soufien, Alice, and Raj: I am really thankful to you all and it was

really a great experience to know all of you. Raj i would really thank you for your

help when i was in need. Alice and Shori it was really fun working with both of you.

Tami and Soufien I really enjoyed nice discussions on th scientific and non scientific

topics.

Lars Nilsson: You are one of the wonderful persons i met during my PhD. I really

enjoyed discussions about the religious stuff and the atheism committee. You are

never tired of discussing scientific and nonscientific subject anytime anywhere.

Saba and Nawaz: It was really nice to know you guys. I felt really comfortable

discussing with you when i am more stressed. I wish you all the best for your future.

Agnes: Good luck with your Postdoc. It was really a nice dinner definitely i would

like to have one more of it. Hope you are having nice time with cute little Sara.

Christoffer Nord: As we were sharing the same office for thesis writing in the final

stages it was really helpful from you in getting the advice in using several software

programs including Volocity. Thank you for everything. Gowtham: I would like to

thank for your help. P-A, Mirella, Lina: i would like to thank you all for making

things easy for me in ordering and in the administrative documentation.

Jonathan Gilthorpe: I will never forget your help in suggesting some scientific

ideas despite your busy schedule. I would like to thank you for everything. Iwan

Jones: I am not sure if you remember you suggested me the tips for cloning. I still

follow those tips with lots of successful cloning results. Thank you so much! Åke

Renmen: It was you who made me more stable when my health was deteriorating

and i can’t thank enough for that. Annelisa Wallius: My swimming pool friend. We

had several nice discussions which was really helpful for me. Thank you. Anne &

Ash: Thank you for inviting us to the get togethers.

Saveer: You are one of my childhood friends whom i will never forget. I was really

happy when you came to Sweden for PhD. I will never forget those days whenever i

feel low i use to call you share my feelings with you. Thanks alot for your support.

Sudhakar: You are the person who suggested me to come to Sweden for PhD. This

day would not come if you have not suggested me to come to Sweden. I am really

thankful to you from the bottom of my heart. Srinivas N: I am glad we have done

masters together and we still share each other's problems.

Dozens of people have helped me make my home away from home. I would like to

thank Kiran & Sirisha for helping me put those first steps in the university and get

accustomed to the new place. Very special thanks to Pramod & Deepthi for the

57

friendly chats, informal dinners, long walks and all the help during their stay in

Umeå.

I would not have enjoyed little things in life if i hadn’t found friends outside work.

Thank you so much for happily talking about things other than just papers. Heartful

thanks to Ravi, Vidya; I am really thankful to Ravi for giving me the moral support

during my tough times. Karthik, Gowthami & Shyam, Reshma for making our

parenting journey easier. I would like to thank Karthik for his support during my

house shifting. I would like to thank you for everything.

My time in Umeå was made enjoyable in large part due to many friends that became

part of my life. I am grateful for the time spent with my roommate Edwin, sharing

weekend lunches with Manoj, Atiq and for the fun talks we had. I would also like

to acknowledge all friends who made my PhD journey memorable in Umeå. Thank

you Uma, Bala, Philge, Nisha, Aaron, Madhavi, Mohan, Munindar, Sharvani.

Especially thank Pramod for helping me in shifting my apartment. I would like to

also thank Harsha, Chaitanya for helping in shifting. I would like to thank all the

Indian friends Yugender, Venky, Srikanth Kolan, Shashank, and Rammurthy.

Amma, Nanna & Family: At every step of the way, they were with me even

though they were physically thousands of miles away. Whatever i have achieved so

far it is because of your support. I still remember how i was struggling to find a Phd

position and the support you gave me was incredible, because of your support i have

reached this far.

Kamla my wonderful wife... Her intelligence, honesty, and love have many times

been the reason why I did not turn around and run away. I cannot imagine a more

special soul-mate; I cannot imagine a better mother for my son. Support you gave

me when i have to come to the lab during late nights was fantastic. Vaikan my cute

little support system... There is nothing like the cute innocent face of a sleeping

child to bring out the strongest determination to succeed. It was his magic that

pulled me out of many intervals of depression. I love you both. Nothing would be

possible without you.

58

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