bonemorphogenetic protein (bmp)signaling in the...

Bone Morphogenetic Protein (BMP) Signaling in the Neuroectoderm 273

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BoneMorphogenetic Protein (BMP) Signaling in the Neuroectoderm

C M Mizutani and E Bier, University of California, atSan Diego, La Jolla, CA, USA

ã 2009 Elsevier Ltd. All rights reserved.

Introduction

The nervous system is a highly heterogeneous tissuecomprising a great diversity of cell types that inter-connect in complex patterns to control a myriad ofconscious and unconscious behaviors. Not surpris-ingly, creating such an intricate system requires aseries of many cellular interactions during develop-ment. Because various organisms have a wide rangeof different life strategies and needs, there is also agreat diversity in the function and development ofnervous systems across species. Notwithstanding theinherent complexity and diversity of nervous systemfunction and development, there are remarkable par-allels between the formation and function of the ner-vous system in organisms ranging from fruit flies andnematodes to vertebrates. In several cases, homolo-gous gene sets play critical roles in processes such asneural induction, neurite pathfinding, synaptogen-esis, action potential propagation, transmitter secre-tion and reception, and behavior. This high degree ofconservation of basic cellular and molecular func-tions suggests that the common ancestor of currentliving metazoans had a well-formed nervous systemwith many of the core properties shared by diversepresent-day organisms.One of the best characterized examples of con-

served pathway function in neural development isthe role of bone morphogenetic protein (BMP) signal-ing during neural induction. During this early phaseof embryonic development, BMP signaling activelyrepresses neural cell fates in epidermal regions of theembryo. In neuroectodermal regions, BMP signalingis blocked by various BMP antagonists, which per-mits the default program of neural development toprevail. Because many of the pathway componentsrequired for neural induction are similarly deployedin vertebrates and invertebrates, it seems highly likelythat this similarity reflects the conservation of anancestral mechanism for specifying neural versus epi-dermal cell fates. BMPs also play important roles inthe subsequent patterning of the nervous systemalong the dorsal–ventral (DV) axis. It is less clear,however, whether this latter phase of neural pattern-ing is accomplished by homologous or convergentmechanisms. In this article, we briefly review theevidence for a conserved function of BMP signaling

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during neural induction and then focus on how BMPsare believed to act during neural patterning in differ-ent organisms. We propose that a unifying theme mayunderlie the apparent diversity of these patterningmechanisms, wherein BMPs act by a common mech-anism to repress the expression of neural genes in adose-dependent fashion. We also consider how con-served and diverse elements of neural patterning mayhave evolved.

Evolutionary Conservation of BMPInhibition during Neural Induction

Nearly a century ago, Hans Spemann and HildeMangold showed that ventral transplantation of thedorsal lip of an amphibian embryo into a recipientembryo led to the production of a secondary neuralaxis. Using distinguishable host and donor embryosthey demonstrated further that the dorsal lip, orSpemann organizer as it is now often called, wasthe source of secreted neural-inducing signals whichcould redirect the development of surrounding cellsfated otherwise to give rise to epidermis. Since theseseminal experiments, there has been great interest inisolating and understanding the function of neuraliz-ing factors. Several neural inducers have been identi-fied from Xenopus in recent years, including Noggin,Chordin (Chd), and proteins in the DAN family,which are expressed in dorsal mesodermal cellsmaking up the Spemann organizer during late blas-tula and early gastrula stages. These structurallydiverse neural inducers function via a common dou-ble negative mechanism by antagonizing the functionof BMP signaling (Figure 1(a)). They bind to BMPs(BMP2/BMP4) with high affinity, preventing themfrom activating BMP receptors. In the nonneuralectoderm, where BMP4 is expressed at high levels,BMP signaling functions to promote epidermal fatesand to repress the expression of neural genes. Simi-larly, in Drosophila, the Chd homolog known asShort Gastrulation (Sog) is expressed in the lateralneuroectoderm and blocks BMP signaling in thedorsal ectoderm. It is likely that the DVaxes in verte-brate and invertebrate embryos were inverted duringevolution, such that the epidermis forms ventrally invertebrates but dorsally in invertebrates. In flies, as invertebrates, BMP signaling represses the expressionof neural genes and activates the expression of non-neural genes. It is noteworthy that in Drosophilasignificantly less BMP signaling is required to repressthe expression of neural genes than to activate expres-sion of epidermal genes. One of the genes activated by

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BMP

Sog/Chd

Autoactivates

Skin

Neurogenesis

Blocks Dpp autoactivation

Permits neurogenesis

Nonneuralectoderm

a b

Neuroectoderm(default state)

Figure 1 BMPs suppress neuronal fates in the ectoderm of vertebrates and arthropods: (a) BMP signaling in the nonneural ectoderm

represses the expression of all neural genes and activates the expression of epidermal genes, including the BMP4/Dpp genes, thereby

creating a positive feedback loop, referred to as autoactivation; (b) an example of the conserved nature of BMP inhibition and neural

induction in Xenopus embryos. In (a), neural-inducing factors, such as the extracellular BMP antagonists Sog/Chd, are supplied to the

neural ectoderm, where they bind to BMPs and prevent them from triggering BMP autoactivation, thereby preventing the invasive spread

of BMP signaling into the neuroectoderm. This double-negative mechanism allows cells in the neuroectoderm to follow the default neural

development pathway. In (b), the injection of sogmRNA from Drosophila into ventral blastomeres of early Xenopus embryos leads to the

formation of secondary neural axes (top embryo; compare to wild-type embryo on bottom) similar to those observed in the original

embryo-grafting experiments of Hilde Mangold and Hans Spemann. BMP, bone morphogenetic protein; Dpp, Decapentaplegic (the

ortholog of vertebrate BMP4/2); Sog, Short gastrulation (the ortholog of vertebrate Chordin (Chd)). Adapted from Biehs B, Francois V, and

Bier E (1996) The Drosophila short gastrulation gene prevents Dpp signaling from autoactivating and suppressing neurogenesis in the

neuroectoderm.Genes & Development 10: 2922–2934; and Schmidt J, Francois V, Bier E, and Kimelman D (1995) The Drosophila short

gastrulation gene induces an ectopic axis in Xenopus: Evidence for conserved mechanisms of dorsal-ventral patterning. Development

121: 4319–4328.

274 Bone Morphogenetic Protein (BMP) Signaling in the Neuroectoderm


BMP signaling in Drosophila and vertebrates is theDpp/BMP4 gene itself, which results in a positivefeedback loop, referred to as autoactivation. If unop-posed by BMP antagonists, BMP autoactivation canresult in the invasive spread of BMP signaling intothe neuroectoderm. This ectopic expression of BMPligands leads to the repression of neural gene expres-sion and to the activation of epidermal genes.Conservation of the Chd/BMP signaling system

extends to the functional level, as revealed in cross-species experiments. For example, injection ofDrosophila sog mRNA into the ventral blastomeresofXenopus embryos generates duplicated neural axes(Figure 1(b)) similar to those induced by injection ofvertebrate Chd or by transplantation of Spemannorganizer tissue. Similarly, vertebrate BMPs andBMP antagonists have the same activities inDrosoph-ila as they do in vertebrate embryos. Other extracel-lular components of the BMP pathway identified inDrosophila have also been shown to play similar rolesin early vertebrate embryos (Figure 2). For instance,embryos lacking Tolloid (Tld) and Twisted gastrula-tion (Tsg) activity have defects in BMP signaling. Tldis a metalloprotease that can cleave and inactivateSog, whereas Tsg forms a trimeric complex withSog/Chd and BMPs and modifies the BMP inhibitoryfunction of Sog by binding to it and by generatingalternative Tld cleavage products. Similarly, in verte-brates, the Xenopus counterpart of Tld, Xolloid(Xld), cleaves Chd in positions corresponding totwo of the four sites in Sog that are cut by Tld,

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thereby reducing Chd activity. In addition, Xenopusand zebra fish homologs of Tsg also can form a ter-nary complex with Chd and BMPs to modulate BMPsignaling.

Opposing Graded BMP and Hh SignalsPattern the Vertebrate Neuroectoderm

A wealth of embryological and genetic evidence invertebrates indicates that, following their role inneural induction, BMPs play an important role asmorphogens in organizing gene expression along thedorsal–ventral axis of the developing nervous system(note that morphogens are molecules distributed ina graded fashion that function in a dose-dependentfashion to activate or repress gene expression). Oncethe dorsal-most ectodermal region of the vertebrateembryo is specified as neuroectoderm (often referredto as the neural plate), these cells undergo a concertedset of bilaterally symmetric apical constrictions, caus-ing them to fold inside the embryo by the process ofinvagination (also referred to more specifically asneurulation). BMP-expressing epidermal cells border-ing the neural plate are thereby brought into juxtapo-sition to form a single coherent dorsal epidermal mass(Figure 3(a)). The invaginated neural plate forms alongitudinal cylinder, which then closes on itself andseparates from the overlying epidermis to form theneural tube. The dorsal-most cells of the neural tubelie immediately below the BMP-expressing epidermisand are subsequently induced to express BMPs. This

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Sog

Supersog

Tsg+

Tld

Tld

Endocytosis

Dpp Scw, Gbb

Tkv

Put

Sax

Put

Outsidecell

Insidecell

MAD/Medea

pMAD/Medea

(Epidermal genes:e.g., dpp, zen)

(Neural genes: e.g., AS-C, msh)

Nucleus

Degraded

Figure 2 Extracellular regulation of BMP signaling. Diagram of BMP signaling pathway from Drosophila, highlighting elements that are

conserved in vertebrates. BMP homodimers (e.g., Dpp-Dpp) or heterodimers (e.g., Dpp-Scw) induce the dimerization and then tetra-

merization of type I and type II BMP receptors. Following receptor dimerization, the type I receptor chain (e.g., Sax or Tkv receptors)

phosphorylates the type II chain (e.g., the Put receptor), leading to phosphorylation of the cytoplasmic signal transducer SMAD (or MAD/

Medea in Drosophila). Phosphorylated MAD (pMAD) then enters the nucleus, where it acts as a transcriptional cofactor to either activate

gene expression (e.g., epidermal genes, including dpp and zen) or repress it (e.g., neural genes, including those of the Achaete-Scute

complex or msh). Extracellular modulators of BMP signaling include Sog/Chd, which binds to Scw and inhibits peak BMP signaling

mediated by the Dpp-Scw heterodimer. Tolloid is a metalloprotease that cleaves and inactivates Sog. Dpp is required as a cofactor of Tld

in this cleavage reaction, both in vitro and in vivo. Tsg binds to Sog and Dpp and has been proposed to act in one of two possible ways.

First, by forming a trimeric complex with Sog and Dpp-Scw heterodimers, as well as Dpp-Dpp homodimers, it broadens the BMP inhibitory

range of Sog. The trimeric complex may also act as a carrier that protects BMPs from receptor-mediated endocytosis and thereby helps

concentrate BMP heterodimers along the dorsal midline. The second role of Tsg is to alter the cleavage of Sog by Tld such that alternative,

more broadly active forms of Sog (called Supersog) are formed. These truncated forms of Sog can bind directly to either Dpp-Dpp

homodimers or Dpp-Scw heterodimers and inhibit their activity. AS-C, achaete-scute gene complex; BMP, bone morphogenetic protein;

Dpp, Decapentaplegic (the ortholog of vertebrate BMP4/2); Gbb, Glass bottom boat; msh, muscle specific homeobox; Sax, Saxophone;

Scw, Screw; Sog, Short gastrulation (the ortholog of vertebrate Chordin (Chd)); Tkv, Thick veins; Tld, Tolloid (the ortholog of vertebrate

Xolloid (Xld)); Tsg, Twisted gastrulation; zen, zerknullt.



dorsally restricted BMP expression is thought to leadto the formation of a BMP activity gradient, which ishigh dorsally and low ventrally. High levels of BMPsignaling in the dorsal regions of the neural tuberesult in the expression of genes such as Msx1/2 andPax7 in cells giving rise to migratory neural crest cellsand sensory cells, whereas lower BMP levels result inthe expression of lateral markers such as Gsh, Pax6,and Dbx1/2 in cells generating various interneurons(Figure 4(a)). It is not known whether BMPs actdirectly or indirectly to activate dorsal markers. Incurrent models, BMPs are typically portrayed as hav-ing a direct positive role inducing gene expression, inpart because a BMP-responsive enhancer region of theMsx1 gene has been shown to have binding sites forSMADs that are required for the activation of this cis-regulatory element. However, it is not clear that thiselement is responsible for Msx1 expression in dorsalcells of the neural tube becauseMsx1 is also expressedin ventral cells of the embryo during this same period.In addition to the gradient of dorsally produc-

ed BMPs, the neural tube also receives ventral induc-tive cues provided by the Sonic Hedgehog (SHh)morphogen. As a consequence of the prior invagination


of themesoderm, cells derived from the Spemann orga-nizer form a stiff longitudinal structure known as thenotochord, which underlies the neural tube. Thesenotochord cells secrete SHh and induce the neighbor-ing ventral neural tube cells (called the floorplate) toacquire notochord-like properties, such as expressionof the transcription factor HNF3b and SHh itself,which maintains its expression by a positive feedbackmechanism (Figure 4(a)). Notochord cells also con-tinue to express BMP inhibitors such as Noggin andChd. SHh produced in the notochord and floorplate ofthe neural tube is distributed in a concentration gradi-ent reciprocal to that of the BMP gradient (i.e., SHh ishigh ventrally and low dorsally). High levels of SHhresult in the expression of ventral genes, such asNkx2.2 and Nkx6.1, in cells that ultimately give riseto motor neurons, whereas lower levels of SHh lead tothe expression of lateral markers.

In addition to organizing gene expression in thedorsal and ventral regions of the neural tube, BMPsand SHh also antagonize one another. For example, co-expression of BMP antagonists with limiting amountsof SHh greatly increases the ventralizing activity ofSHh. Reciprocally, when BMPs are provided at levels

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Neuraltube

Dorsald.m.l of embryo

NeuroectodermNonneuralectoderm

Mesoderm

Ventral Notochord

Epidermis

Somitesv.m.l.of NT

Notochord

Dorsal

Epi

Neuro/epi

Meso

Ventralv.f.

Meso

Epi

Neuro/epi

v.m.l.

Dorsalepi

Neuro

Denticles Ventralepi

v.m.l.

b

a

(1) (2) (3) (4)

Figure 3 Early neural development in vertebrate and fly embryos, cross-sectional views of embryos: (a) neurulation in vertebrates;

(b) delamination of neuroblasts in Drosophila. In (a), (1) is a cross section of the dorsal region of the embryo indicating the neuroectoderm

(blue), or neural plate, which later invaginates along the dorsal midline. The adjacent more ventrally located nonneural ectodermal cells

(orange) express high levels of BMPs and repress the expression of neural genes. Mesodermal cells (red) have already entered the

interior of the embryo by this stage, by the process of involution, which begins dorsally at the blastopore and then expands ventrally. In

(2) and (3), as invagination of the neural plate proceeds, its two ventral borders are brought into contact and the adjacent epithelial cells

fuse into a single coherent domain as the neural ectoderm detaches to form the neural tube. In (4), The invagination of the neural plate

leads to a reversal of dorsal-ventral (DV) polarity of the nervous system with respect to the primary embryonic DV axis because cells

located originally at the dorsal midline of the embryo (white triangle) assume the most ventral position in the internalized neural tube. The

ventral-most cells of the vertebrate neural tube, which are referred to as the floorplate, come into direct contact with a specialized

mesodermal derivative known as the notochord, a rigid rodlike structure that provides support to the tadpole. Cells from the Spemann

organizer give rise to the notochord. Flanking the neural tube laterally are the somites, a mesodermal tissue that gives rise to the adult

bony skeleton and to muscle. In (b), (1) is a cross-section view of the early Drosophila embryo indicating the three germ layers along the

DVaxis. In (2) and (3), invagination of the mesoderm brings the left and right halves of the neural ectoderm into contact to form the ventral

midline of the embryo and nervous system. In (4), neural precursor cells (neuroblasts) individually delaminate from the ectodermal

epithelium and reside between the overlying epithelium and the more internal mesoderm. An important comparative point is that, due to

the double inversion of the DV axis in vertebrates relative to flies, the final orientation ends up being the same. For example, the ventral

midlines of vertebrates and flies are both formed by cells that were originally furthest from the source of BMPs in the nonneural ectoderm.

BMP, bone morphogenetic protein; d.m.l., dorsal midline; NT, neural tube; v.f., ventral front; v.m.l., ventral midline. (See also Figure 4.)Adapted from Bier E (2000) The Coiled Spring: How Life Begins. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.



typical of the dorsal or lateral regions of the neuraltube, they can override the induction of ventralmarkersby SHh.

Graded BMP-Mediated Repression ofNeural Genes in Drosophila

Following their respective resident roles in consoli-dating cell fate choices within the epidermal and neu-ral regions of the fly embryo during neural induction,Dpp and Sog play nonautonomous roles in the furthersubdivision of these two regions. Cells in the dorsalregion of the embryo express uniform levels ofdpp RNA and are initially equivalent because they


are defined by the absence of the maternally derivedDorsal (Dl) morphogen. (Dl is a transcription factorrelated to mammalian nuclear factor (NF)-kB that setsup the initial DV polarity of the embryo. High levelsof Dl ventrally specify the mesoderm, graded lowlevels of Dl define the neuroectoderm, and theabsence of Dl in dorsal cells permits expression ofdpp; see Figure 4(b).) Polarity in the dorsal region iscreated by Sog diffusing dorsally from the lateralneuroectoderm, where it is cleaved and inactivatedby the Tld protease, which is co-expressed with Dppin dorsal cells. The adjacent ventral source of Sog anddorsal Tld sink result in the formation of a Sog pro-tein gradient in the dorsal region, which is high

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BMP4Msx1

Pax7

Pax6Gsh

Nkx6.1Nkx2.2

SHh

Notochord

Chd HNF3b

Dbx1

NE

EPI

MES Vnd

Ind

Msh

DI

Sog

Dpp

a b

Figure 4 BMPs pattern the vertebrate and Drosophila neuro-

ectoderm, cross-sectional diagrams: (a) the vertebrate neural

tube, indicating the opposing gradients of BMPs and Sonic

Hedgehog (SHh); (b) a blastoderm-stage Drosophila embryo,

indicating the opposing Dpp and Dl morphogen gradients. In (a),

high levels of BMPs dorsally result in the expression of the neural

identity gene Msx1, and lower levels specify cells expressing

lateral genes such as Gsh. High levels of SHh ventrally lead to

the expression of the ventral neural identity gene Nkx2.2, whereas

lower levels result in the expression of more lateral markers. The

expression patterns of some other transcription factors along the

dorsal-ventral (DV) axis are also indicated, including Pax7, Dbx1

(and, but not shown, Dbx2, which extends more ventrally than

Dbx1), Pax6 (which also plays a conserved role in eye formation),

and Nkx6.1. In (b), threshold-dependent repression mediated by

the Dpp gradient helps pattern the ind and msh domains of

expression, whereas concentration-dependent activation by the

Dl gradient helps defining the vnd/ind border. An important mech-

anism contributing to the sharp, mutually exclusive neural domains

is the ventral dominant cross-inhibition among the neural identity

genes, wherein Vnd represses the expression of ind and msh and

Ind inhibits expression ofmsh. Adapted fromMizutaniCM,MeyerN,

Roelink H, and Bier E (2006) Threshold-dependent BMP-mediated

repression: A model for a conserved mechanism that patterns the

neuroectoderm. PLoS Biology 4: e313 (online).



ventrally and low dorsally (Figure 5(a)). It has beenproposed that this Sog gradient creates an inverseBMP activity gradient with peak levels in dorsal-most cells and lower levels in more ventral cells,which can be visualized by the in situ activationof the signal transducer phospho-MAD (pMAD).Sog may also carry Dpp dorsally and concentrate italong the dorsal midline. This BMP gradient results inthe nested activation of a series of genes, includingthe transcription factors zen, pannier, and ush. Theprimary consequence of the graded activation ofDpp target genes is the subdivision of the dorsalregion into two parts: a dorsal-most extra-embryonicdomain (amnioserosa) and a more ventral epidermaldomain. Note, however, that even the lower relativelevels of BMP signaling present in the epidermalportion of the dorsal region are sufficient to repressthe expression of all neural genes in those cells.There is also evidence for a reciprocal influence of

the dorsal ectoderm on patterning the lateral neuro-ectoderm mediated by Dpp diffusing ventrally(although diffusion of small amounts of Dpp ventrally


remains to be demonstrated directly). Because Sogand a transcriptional repressor of BMP signalingknown as Brinker (Brk) are expressed in the neuro-ectoderm, the levels of BMP signaling in neuroecto-dermal cells would be expected to be much lowerthan those in the dorsal region, where Dpp isexpressed and only low levels of graded Sog are pres-ent. As a consequence of Dpp being present in limit-ing amounts within the neuroectoderm, its abilityto repress neural gene expression becomes dosagedependent. This dosage-sensitive repression has beenmost conclusively studied with regard to the expres-sion of the neural identity genes vnd, ind, and msh,which are required to specify the fates of the threeprimary rows of neuroblasts in the embryonic centralnervous system (C NS) ( Figure s 4(b) and 5(b )). vnd,the homolog of vertebrateNkx2.2, is expressed in theventral-most row of the neuroblasts; ind, the homo-log of Gsh, is expressed in the middle row of neuro-blasts; andmsh, the homolog of Msx1/2, is expressedin the dorsal row of neuroblasts. The fact that ortho-logous sets of neural identity genes are expressed inthe same relative ventral-to-dorsal order with regardto BMP-expressing cells in vertebrates and flies sug-gests that this configuration reflects an ancestral statethat has been conserved during evolution (Figure 4).(Note that, despite the fact that the primary DV axesare inverted in vertebrates and Drosophila embryos,the final relative order of neural identity genes endsup being the same as a consequence of the neural DVpattern being reversed with respect to the remainderof the embryo following invagination of the neuralplate. Such a secondary reversal does not take place inDrosophila, in which neuroblasts delaminate isotopi-cally from the epidermis to form a subepithelial layer,as indicated in Figure 3(b).)

An important regulatory feature of neural identitygenes in Drosophila is that they cross-inhibit oneanother in a ventral-dominant fashion in which Vndrepresses expression of both ind and msh, and Indinhibits the expression of msh (Figure 4(b)). As Dppdiffuses ventrally, it represses expression of the inter-mediate neural identity gene indmore effectively thanmsh. This results in ind, but not msh, being repressedby BMP signaling in dorsal cells of the neuroecto-derm, which are closest to the Dpp source. BMP-mediated repression of ind expression, in turn,relieves ventral-dominant repression of msh by Ind,resulting inmsh expression in the dorsal-most domainof the neuroectoderm. Thus, as a consequence of thecross-inhibitory interactions among neural identitygenes, sharp boundaries of neural gene expressiondomains are established in response to graded Dppsignaling along the neuroectoderm. Similar cross-regulation of neural identity genes has also been

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Dorsal

a

b

High BMP

Low BMPTId

Amnioserosa

Ventral

(1)

(1)(2)

(2) (3)

Dpp

Dpp

Nonneural genesNeural genes

Neuronal genes

vnd

msh

Sog

ind

Nonneuralectoderm

Epi

Neuro

Meso

Sog proteinSog proteinsog RNA/

dpp

mshind

vnd

Figure 5 BMP-mediated patterning of the fly neuroectoderm: (a) reciprocal BMP gradient created by diffusion of Sog into dorsal regions

and its cleavage by Tld protease in the dorsal-most cells; (b) ventral diffusion of Dpp from the dorsal ectoderm into the lateral

neuroectoderm. In (a), double staining of Sog RNA and Sog protein reveals that Sog protein can be detected further dorsally forming a

gradient, beyond its domain of expression within the lateral neuroectoderm ((1) and (2) show a lateral view and (3) shows a transversal

view of the embryo). In (b) the ventral diffusion of Dpp forms a concentration gradient to pattern the expression domains of neural identity

genes msh (red), ind (green), and vnd (blue) mRNA. BMP, bone morphogenetic protein; Dpp, Decapentaplegic; Sog, Short gastrulation;

Tld, Tolloid. (a), (1) and (2), from Srinivasan S, Rashka KE, and Bier E (2002) Creation of a Sog morphogen gradient in the Drosophila

embryo. Developmental Cell 2: 91–101. (b), (2), From Kosman D, Mizutani CM, Lemons D, Cox WG, McGinnis W, and Bier E (2004)

Multiplex detection of RNA expression in Drosophila embryos. Science 305: 846.



observed in vertebrates, athough it remains to bedetermined whether they follow a ventral-dominantpattern as has been shown in Drosophila.In ventral regions, it appears that the primary system

involved in patterning the neuroectoderm is the oppos-ing ventral-to-dorsal Dl gradient, which is providedmaternally as previously described. Moderate levelsof Dl in ventral cells activate vnd,whereas lower levelsactivate ind. Because Vnd represses the expression ofind and msh, the graded action of Dl results in vndbeing expressed exclusively in ventral-most cells ofthe neuroectoderm and ind expression in the adjacentintermediate domain where the levels of Dl are too lowto activate vnd. Although Dpp signaling can alsorepress the expression of vnd and can regulate thedorsal borders of all three neural identity genes, theborder between the vnd and ind domains is establishedprimarily by graded activation of these genes by Dl,whereas the border between ind andmsh is determinedprimarily by the threshold-dependent repression ofthese genes by Dpp signaling emanating from dorsalepidermal cells.


Neural Patterning in Other Groups ofOrganisms

Primary insights into the mechanisms of neural induc-tion have been provided by classical model systemssuch as flies, frogs, zebra fish, and mice, however, it isimportant to complement these studies with analysesof organisms from other phylogenetic groups. Suchevo-devo studies provide two important types ofinformation. First, cross-genome comparisons haverevealed a striking degree of gene loss during theevolution of lineages that include the model systemsDrosophila and Caenorhabditis elegans. Thus, findinga vertebrate gene not present in flies or other insectsdoes not necessarily imply that the gene evolved withinthe vertebrate lineage following its divergence frominvertebrates but, rather, that it may simply have beenlost in the insect lineage. Second, one of the mostinteresting features of evolution is the appearanceof novel structures within specific lineages whichcan only be understood through comparative studiesusing diverse organisms.

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Insecta Nematoda

Ecdysozoa

Lophotrochozoa

Bilateralia

Vertebrata

Chordata

Tunicata

Hemichordata

Echinodermata

Figure 6 Simplified phylogeny of the bilateralia. Evolutionary tree indicating the relationships of the three major groups of bilateral

animals. This tree is based on cladistic analysis of morphological characters and on 18S ribosomal RNA sequence-divergence data.

Because of the rapid diversification of animal forms during and preceding the Cambrian radiation, the relationships at the base of the tree

are not certain. The current view depicted in this tree is that the bilateralia consist of three major groups: (1) chordates (which include

vertebrates and tunicates), hemichordates, and echinoderms; (2) the ecdysozoa, which includes arthropods such as insects and

nematode worms such as C. elegans, and (3) the lophotrochozoa, which includes mollusks, flatworms, and annelid worms. The great

majority of developmental studies have focused on the first two branches of the tree (chordates and ecdysozoa); there has been much

less analysis of lophotrochozoan development. Further comparisons of developmental strategies and genetic pathways among these

three groups will provide a much improved view of the common ancestor of the bilateralia.



Current phylogenies tentatively group metazoa inone of three major groups: deuterostomes, whichinclude chordates (vertebrates and ascidians), hemi-chordates, and echinoderms, ecdysozoa (which includearthropods such as Drosophila and nematodes suchas C. elegans), and lophotrochozoa (which includeflatworms, annelid worms, mollusks, and other shell-enclosed organisms) (Figure 6). Conspicuously missingamong the model organisms, which have been used todefine developmental paradigms, are those in the largediverse group of lophotrochozoa. Studies from addi-tional members of the ecdysozoa and chordate lineageswould also provide more generality to our currentviews of development and should shed light on whichfeatures are truly conserved versus which indepen-dently evolved in different lineages.We briefly summa-rize some current evo-devo studies in other organismsthat bear on themechanistic origins of neural inductionand patterning. It is important to bear in mind, how-ever, that each of these species is also likely to have lostgenes that were present in the common ancestor ofbilateral animals.In spider embryos, the DV axis is established in a

very different way than in Drosophila or vertebrates.A small group of Dpp-expressing mesodermal cellsmigrates under the epidermis, leaving a linear track of


overlying epidermal cells in which BMP signalingpersists and which ultimately forms the dorsal mid-line. The spider sog gene is expressed in the ventralectoderm which gives rise to the nervous system, as inother arthropods. sog function is required for ventralcell-fate specification, including the nervous system,because the reduction of sog activity by RNA inter-ference (RNAi) results in the spread of high BMPsignaling into ventral cells and the subsequent lossof ventral structures. The invasion of BMP signalinginto the neuroectoderm of sog RNAi spiders and itssuppression of neuroectodermal fates parallel the roleof BMP signaling in Drosophila and vertebrates.Despite the difference in how BMP signaling is estab-lished in the spider embryo, the way it is employedsupports the view that an ancestral role of neuralinducers was to prevent BMP from spreading intothe neuroectoderm and suppressing neurogenesis.

Hemichordates, which are thought to be mostclosely related to echinoderms, include marine wormsand other sessile marine organisms that retain only amoderate degree of DVorganization as a consequenceof their nearly rotationally symmetric body plans.Early during development, BMP4 and Chd/Sog areexpressed in opposing domains and define a DV axisin hemichordate embryos consisting of three germ

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layers and distinct domains of gene expression, exceptfor the nervous system. Unlike vertebrates and arthro-pods, the nervous system in hemichordates is notcondensed into either dorsal or ventral ganglia but,rather, consists of dispersed neurons which send theiraxons into one of two major axonal bundles, onerunning dorsally and the other ventrally. Because neu-rons form around the entire circumference of theseembryos, BMP signaling does not inhibit the forma-tion of neurons in the dorsal region, nor does BMPoverexpression inhibit neuron formation elsewhere.In addition, although hemichordates have recogniz-able counterparts of at least vnd/Nkx2.2 and msh/Msx, the homologs examined so far do not displayany obvious restriction in their expression along theDV axis. One possible explanation for these observa-tions is that the neural repressive function of BMPs invertebrates, arthropods, and spiders arose followingthe separation of these lineages and that the originalfunction of BMP signaling in bilateral ancestors mayhave been to establish DV polarity. Alternatively, theneural suppressive function of BMP signaling mayhave been lost during the course of hemichordateevolution as specialization along the DV axis becamegreatly simplified and the animals assumed a nearlyrotationally symmetric body plan. This latter viewaccounts for the common pattern of neural identitygene expression in vertebrates and arthropods, aswell as its potential common dosage-sensitive regula-tion by BMP signaling. Future experiments shouldresolve this question, particularly by examining theexpression of neural identity genes in other chordatebranches and in various lineages of the lophotrocho-zoa, the third major branch of the metazoan evolution-ary tree. When compared to the other groups, thelophotrochozoa appear to be one of the slowest evol-ving groups, having lost far fewer genes present in thecommon bilateral ancestor and typically having ventralnerve cords similar to those in arthropods, althoughprimitive flat worms (platyhelminths) have eitherdiffuse nervous systems or only anterior nerve nets.In sum, the current knowledge of neural induction

in diverse bilateral embryos suggests that the role ofBMPs in neural induction reflects the conservation ofa mechanism that evolved from a common bilateralancestor, although it is formally possible that this mayhave arisen independently in several different lineages.Clearly there are species-specific aspects that have beendescribed, but it is not clear whether this is evidenceagainst a common origin rather than an indicationthat the mechanism has been lost or highly modifiedin various lineages. Further analysis of additionalgroups should resolve these issues.A second important evolutionary question is whether

the role of BMPs in patterning neural identity also


originated in a common ancestor. Indeed, becausevertebrates and flies share a common set of neuralidentity genes expressed in the same relative orderwith regard to a source of BMPs and because BMPsplay a prominent role in patterning the dorsal regionof the nervous system in both flies and vertebrates,it seems likely that neural patterning by the BMPswas a common feature of the bilateral ancestor.Clearly, other species-specific signaling pathways arealso important in DV patterning of the nervous sys-tem; for example, primary morphogens involved inventral neural patterning appear to be different inflies (i.e., Dl) and vertebrates (i.e., Hedgehog (Hh)).Nonetheless, it is tempting to speculate that BMPsonce were sufficient to pattern the entire neural DVaxis. According to this hypothesis, additional signal-ing systems were then added to buttress patterningat the low end of the BMP gradient during the diver-gence of the vertebrate and invertebrate lineages.Consistent with this view are experiments on DVpatterning of the mouse spinal cord. When the func-tion of the Hh signaling pathway is completely abol-ished (i.e., by removing both SHh and the defaultrepressor of the Hh pathway, known as Gli3), muchof the ventral pattern is restored relative to what islost in SHh-single mutants. In addition, under condi-tions of low-level Hh signaling, the gene-expressionprofile in neural-plate explants can be adjusted toventral, lateral, or dorsal levels by adding increasingdoses of BMPs, indicating again that BMPs alone areable to pattern the full DV span of neural cell fates.Thus, in the early bilateral ancestors,which are believedto have been very small (less than 2-mm long), a singleBMP morphogen gradient may have been sufficient tocreate a pattern along the entire DVaxis.

DV Inversion in Vertebrates?

The fact that DV polarity of the nervous systemand the circulatory system appears to be reversedin vertebrates relative to invertebrates was notedby the renowned French comparative anatomistGeoffroy St.-Hilaire, who proposed that verte-brates were essentially upside-down invertebrates(Figure 7(a–c)). The patterns of gene expression invertebrates and invertebrates summarized here haveled many modern evo-devo enthusiasts to supportGeoffroy St.-Hilaire’s hypothesis. One possible excep-tion to the axis inversion model, however, is the headregion. Comparison of gene-expression markers foreyes such as Pax6/eyeless (which are thought tohave played an ancestral role in specifying some prop-erties of light-sensitive organs in metazoa), as well asgenes expressed in the vertebrate hypothalamus anda potentially homologous neuroendocrine organ in

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Arthropod

ab

dc

e

Chordate

Trunk rotated 180�relative to the head

Whole-body inversion →

Head nervous system only in ancestor →Independent evolution of trunk CNS onopposite sides in arthropods vs. chordates

Mouth moves ventrally

Figure 7 Possible origins of dorsal–ventral (DV) axis inversion in vertebrates: (a) invertebrate DV axis; (b) vertebrate DV axis;

(c) Geoffroy St.-Hilaire model; (d) and (e), alternative models. In the early nineteenth century, Geoffroy St.-Hilaire proposed that the

DVaxis of vertebrates was inverted with respect to that of invertebrates, based on the opposite positions of the nervous system and heart

(dorsal in flies pumping anteriorly vs. ventral in vertebrates pumping posteriorly). Several scenarios have been proposed to account for the

apparent axis inversion in light of recently obtained molecular data. In the original Geoffroy St.-Hilaire model (c), the entire DV axis was

inverted, followed by a ventral migration of the mouth orifice. Alternatively (d), only the trunk region was rotated by 180� with respect to the

head, followed by migration of the mouth opening as well. An attractive feature of this hypothesis is that it also explains why the left and

right sides of the vertebrate sensory nervous system map primarily to the opposite side of the brain. Another possibility (e) is that the last

common bilateral ancestor had only a condensed anterior nervous system (or brain) and that the condensed central nervous system

(CNS) trunk later evolved separately (and with opposite DV polarity). This hypothesis does not account for the similar BMP-mediated

mechanisms for establishing the conserved neural patterning along the DV axis.



Drosophila, suggests that the order of DV patterningin the brain might be the same in flies and inverte-brates. Thus, the relative DV patterns in the head andtrunk appear to be opposite. One explanation for theapparent differences in head and trunk patterning isthat the anterior brain may have evolved first from ananterior net of cells and then condensed trunk nervoussystems developed later and in opposite DV orienta-tions following the split of vertebrates and arthropods(Figure 7(e)). One argument against this model is theshared DV pattern of neural identity gene expressionand the dosage-sensitive regulation of these genesby BMPs, which seems difficult to imagine havingevolved twice by chance. Another possible explana-tion is that the inversion of neural pattern wasconfined to the trunk and that the body was rotatedby 180� with respect to the head, which remained ina fixed DV orientation (Figure 7(d)). This hypothe-sis could also offer a potential explanation for an


otherwise puzzling feature of the vertebrate nervoussystem – that the primary sensory axonal projectionscross from left to right (or decussate, in the jargon).In other words, the right hand maps primarily to theleft sensory cortex, as does the right eye to the left visualcortex. There is no evidence for an analogous primarycross-representation in invertebrates. For example,eyes project primarily ipsolaterally in all invertebratesexamined. It is also possible that apparent differencesbetween the head and trunk reflect a sampling bias andthat further analysis of additional conserved gene setsexpressed in the headwill support the originalGeoffroySt.-Hilaire model for the full-body axis inversion. Oneinteresting testable prediction of the head–trunk rota-tion model is that genes expressed along the entireanterior–posterior (AP) axis of the nervous system ina restrictedDVpattern in arthropodsmight have oppo-site split DV expression domains in the head versustrunk regions of vertebrates.

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Conclusion

BMPs play a similar all-or-none role in repressing theexpression of neural genes in epidermal regions invertebrates and arthropods and then play a dosage-sensitive role in establishing a conserved pattern ofneural identity gene expression during early pattern-ing of the neuroectoderm. An important question toresolve is whether BMPs function in vertebrates asthey do in flies, by using threshold-dependent repres-sion of neural identity genes in conjunction with ven-tral-dominant cross-inhibition among neural identitygenes. Analysis of the role on BMPs in patterning thenervous systems of other organisms will provide addi-tional information for reconstructing the elements ofneural induction present in the common bilateralancestor. Such broadened evo-devo studies will alsoreveal how evolutionary novelties arise in specificlineages to give rise to the rich array of neural devel-opment and function in diverse organisms.

See also: Basal Ganglia: Internal Organization; Genetic

Regulation of Circadian Rhythms in Drosophila; Learning

and Memory in Invertebrates: Drosophila; NeuralInduction in Chicks; Olfaction in Invertebrates:

Drosophila; Sleep and Waking in Drosophila.

Further Reading

Arendt D (2005) Genes and homology in nervous system evolution:

Comparing gene functions, expression patterns, and cell type

molecular fingerprints. Theory in Biosciences 124: 185–197.Arendt D and Nubler-Jung K (1999) Comparison of early nerve

cord development in insects and vertebrates.Development 126:2309–2325.

Biehs B, Francois V, and Bier E (1996). The Drosophila shortgastrulation gene prevents Dpp signaling from autoactivating

and suppressing neurogenesis in the neuroectoderm. Genes &Development 10: 2922–2934.

Bier E (2000) The Coiled Spring: How Life Begins. Cold SpringHarbor, NY: Cold Spring Harbor Laboratory Press.

De Robertis EM, Larrain J, Oelgeschlager M, and Wessely O

(2000) The establishment of Spemann’s organizer and pat-terning of the vertebrate embryo. Nature Reviews Genetics 1:171–181.

Geoffroy St.-Hilaire E (1822) Considerations generales sur la

vertebre. Memoirs du Museum National d’Histoire Naturelle9: 89–119.

Gilbert SF (2006) Developmental Biology, 8th edn. Sunderland,

MA: Sinauer Associates.


Harland R (2000) Neural induction. Current Opinion in Geneticsand Development 10: 357–362.

Kosman D, Mizutani CM, Lemons D, Cox WG, McGinnis W, and

Bier E (2004) Multiplex detection of RNA expression in Dro-sophila embryos. Science 305: 846.

Lee KJ and Jessell TM (1999) The specification of dorsal cell fates

in the vertebrate central nervous system. Annual Review ofNeuroscience 22: 261–294.

Lowe CJ, Terasaki M,WuM, et al. (2006) Dorsoventral patterning

in hemichordates: Insights into early chordate evolution. PLoSBiology 4: e291 (online).

Mizutani CM, Meyer N, Roelink H, and Bier E (2006) Threshold-

dependent BMP-mediated repression: A model for a conserved

mechanism that patterns the neuroectoderm. PLoS Biology 4:

e313 (online).Raible F and Arendt D (2004) Metazoan evolution: Some animals

are more equal than others. Current Biology 14: R106–R108.

Sanes DH, Reh TA, and Harris WA (2006) Development of theNervous System, 2nd edn. Amsterdam: Elsevier Academic Press.

Schmidt J, Francois V, Bier E, and Kimelman D (1995) The Dro-sophila short gastrulation gene induces an ectopic axis inXeno-pus: Evidence for conserved mechanisms of dorsal-ventralpatterning. Development 121: 4319–4328.

Srinivasan S, Rashka KE, and Bier E (2002) Creation of a Sog

morphogen gradient in theDrosophila embryo.DevelopmentalCell 2: 91–101.

Spemann H and Mangold H (1924) Uber Induction von Embryo-

nanlagen durch Implantation artfremderOrganisatoren.WilhelmRoux’ Archiv fiir Entwicklungsmechanik der Organismen 100:

599–638.Stathopoulos A and Levine M (2002) Dorsal gradient networks in

the Drosophila embryo. Developmental Biology 246: 57–67.

Tessmar-Raible K and Arendt D (2003) Emerging systems: Betweenvertebrates and arthropods, the Lophotrochozoa. CurrentOpinion in Genetics and Development 13: 331–340.

Wolpert L, Beddington RS, Brockes J, Jessell TM, Lawrence P, and

Meyerowitz E (2001) Principles of Development, 3rd edn.New York: Oxford University Press.

Relevant Websites

http://www.sdbonline.org – Atlas of Drosophila Development andSociety for Developmental Biology.

http://www.expasy.ch – ExPASy, Swiss Institute of Bioinformatics.

Caenorhabditis elegans worm enzymes.

http://flybase.net/ – Flybase.http://flybrain.neurobio.arizona.edu – FlyBrain.

http://www.informatics.jax.org – Informatics. Mouse genome.

http://www.ncbi.nlm.nih.gov – National Institutes of Health.

Zebra fish genome.http://www3.ncbi.nlm.nih.gov – National Institutes of Health.

OnlineMendelian Inheritance inMan (OMIM). Human disease

genes.

http://sdb.bio.purdue.edu – Purdue University Interactive Fly.http://genome-www.stanford.edu – Stanford University. Saccharo-

myces yeast genome.

e (2009), vol. 2, pp. 273-282

http://www.sdbonline.org

http://www.expasy.ch

http://flybase.net/

http://flybrain.neurobio.arizona.edu

http://www.informatics.jax.org

http://www.ncbi.nlm.nih.gov

http://www3.ncbi.nlm.nih.gov

http://sdb.bio.purdue.edu

http://genome-www.stanford.edu

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