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Chapter 4 Rewiring the brain: orthodenticle loss-of-function results in

neuroblast lineage transformation in postembryonic brain

development of Drosophila melanogaster.

Introduction

During embryogenesis, identifiable neuroblast lineages develop at highly stereotypical

locations in the ventral neuroectoderm. This stereotypy in the formation and specification of

embryonic neuroblasts is, in part, controlled by key developmental patterning genes that initially

define the body axes during early embryogenesis (Urbach and Technau, 2004). Cephalic gap genes,

such as orthodenticle (otd) and empty spiracles (ems) are one such class of genes. Cephalic gap genes

are among the earliest genes to be zygotically transcribed by the Drosophila embryo. They encode

transcription factors and are under the control of maternally contributed genes such as bicoid and

torso (Dalton et al., 1989; Finkelstein and Perrimon, 1990; Walldorf and Gehring, 1992; Gao and

Finkelstein, 1998). Their expression is first seen in the early blastoderm where they are expressed in

broad, overlapping stripe-like patterns, localized in the anterior region of the embryo (Jurgens and

Hartenstein, 1993). These transcription factor encoding genes are essential for establishing the

segmental identity of the cephalic region of the embryo, and their absence results in specific gaps in

the cephalic anlagen due to defects in specification of particular head segments (Dalton et al., 1989;

Cohen and Jürgens, 1990; Finkelstein and Perrimon, 1990; Finkelstein et al., 1990; Walldorf and

Gehring, 1992; Schmidt-Ott et al., 1994). As a result, all embryonic structures that derive from these

defective head segments are deleted upon the loss of the corresponding cephalic gap genes. For

example, loss-of-function of the cephalic gap gene otd, which is expressed in the ocular segment of

the cephalic region, or the loss-of-function of the cephalic gap gene ems, which is expressed in the

adjacent antennal segment, results in gap-like phenotypes in the embryonic head ectoderm and in the

anterior regions of the embryonic brain that derive from these corresponding head segments [for

recent reviews see (Lichtneckert and Reichert, 2008)].

In recent years it has emerged that these “early embryonic patterning genes” are also required

later in postembryonic development of Drosophila. Prominent examples of this are the multiple roles

that the cephalic gap genes otd and ems play in the formation of sensory systems. As described

earlier, nervous system development in Drosophila occurs during two phases; the larval nervous

system is generated during embryogenesis and the adult nervous system is generated primarily during

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postembryonic development [reviewed in (Hartenstein et al., 2008)]. Correspondingly, complex

sensory systems, such as those involved in olfaction and vision, are essentially created twice during

development; once during embryogenesis for the larva and once again during postembryonic

development for the adult fly [reviewed in (Morante et al., 2007; Rodrigues and Hummel, 2008;

Brochtrup and Hummel, 2011; Hadjieconomou et al., 2011)].

The ems gene has multiple roles in the postembryonic development of the olfactory sense

organs. It is required for the specification of olfactory sense organs on the antenna, and is also

essential for the targeting of the axons of olfactory sensory neurons to their appropriate central brain

targets in the glomeruli of the antennal lobe (Sen et al., 2010). The ems gene is also required for the

postembryonic development of olfactory interneurons in the central brain. It acts in two of the four

neuroblasts lineages that give rise to the adult-specific olfactory projection neurons and local neurons.

(A fifth neuroblast lineage gives rise to olfactory/gustatory interneurons; Das et al., in preparation).

In the absence of ems, interneurons in the lateral olfactory neuroblast lineage fail to survive, while

interneurons of the anterodorsal olfactory neuroblast lineage manifest targeting defects in the antennal

lobe (Das et al., 2008; Lichtneckert et al., 2008). The otd gene has also been shown to have multiple

roles in the postembryonic development of the adult visual sense organs. otd is required in the

development of photoreceptors of the compound eye for proper rhabdomere formation and maturation

as well as for the correct expression of appropriate rhodopsin subtypes; otd is also required for the

development of ocelli in the eye-antennal disc (Royet and Finkelstein, 1995; Vandendries et al., 1996;

Tahayato et al., 2003; Sprecher et al., 2007; Blanco et al., 2009; Fichelson et al., 2012). However, in

contrast to ems, there is currently little information about the role of otd in postembryonic

development of the central brain.

The central brain in Drosophila is organised in a modular fashion. Each of the brain

hemispheres develops from about 100 stem cell like precursors called neuroblasts (Urbach and

Technau, 2003, 2004). Neuroblasts divide in an asymmetric manner to give rise to two daughter cells;

one neuroblast and another terminally differentiated ‘ganglion mother cell’ (GMC), which has the

ability to divide only once more to generate two neural cells (Doe, 1992). During embryogenesis,

neuroblasts can generate up to 20 neural cells. Uniquely, in insects, the neurons of a single lineage,

that is, those that are born from the same neuroblast, remain tightly clustered and their axons

fasciculate together into a common bundle, the primary axon tract (PAT) (Therianos et al., 1995;

Nassif et al., 1998, 2003). By the end of embryogenesis, the embryonically born neurons form

appropriate pre- and post-synaptic connections, thus creating the neuropilar compartments of the

larval brain. These compartments form hubs of information processing, as they are dense in synapses,

usually exclude neuronal cell bodies, and are ensheathed by glial membranes (Younossi-Hartenstein

et al., 2006). Having generated the embryonic neurons, neuroblasts undergo a period of mitotic

quiescence that lasts until the early larval stage, after which they re-initiate the process of asymmetric

75

cell division to give rise to adult-specific, postembryonic secondary neurons (Ito and Hotta, 1992;

Pereanu and Hartenstein, 2006). This phase of proliferative cell division lasts up to early pupal life.

Up until then, the postembryonic neurons remain arrested in an immature state, with no terminal

arborisations in the neuropile compartments. It is only during pupal life, after the second phase of cell

division is over, that these neurons attain their mature, elaborate innervations patterns that invade the

neuropile compartments. However, much like the primary neurons of the embryo, the arrested,

postembryonic neurons of the larval brain differentiate in a lineage-specific manner, organising their

axons into a common bundle called the secondary axon tract (SAT) and their cell bodies into a tight

cluster (Dumstrei et al., 2003; Pereanu and Hartenstein, 2006). Furthermore, in a lineage-specific

manner, cell bodies of the secondary neurons remain in close proximity to the primary neurons of the

same lineage, and the axonal trajectories taken by the SATs utilise the framework laid down by the

PATs. Thus, within a lineage, the SATs of the secondary neurons fasciculate with the PATs of the

primary neurons and eventually contribute to the same neuropilar compartment as the primaries

(Truman, 1990; Larsen et al., 2009). Moreover, Larsen and colleagues have followed identified

neuroblast lineages through embryonic and postembryonic development and have shown that the

PATs and SATs, which are developmental intermediates, actually correspond to the trajectories that

are seen in the fully mature adult brain (Larsen et al., 2009). Therefore, the compartments of the

adult brain can be traced back during development, and are identifiable even as early as larval life

(Pereanu et al., 2010). Thus, in insects, a neuroblast lineage seems to represent a developmental unit

that underlies the projection circuitry of the functional compartments in the brain.

The best studied examples of neuroblast lineages and their contribution as developmental

units for brain compartments are the lineages that make up the mushroom body, the antennal lobe and,

more recently, the central complex. The mushroom body is a protocerebral brain compartment

associated with olfactory learning and memory (Heisenberg, 2003; Davis, 2011). It consists of

approximately 2000 Kenyon cells whose projections form a stalk called the peduncle and arborize in

two distinctly identifiable lobes, the medial and the vertical lobes that lie at mutually orthogonal

planes with respect to each other (Technau and Heisenberg, 1982). The entire set of Kenyon cells that

make up the mushroom body derive from four neuroblasts that have been traced back in the embryo

and can be identified in the procephalic neurectoderm (Kunz et al., 2012). These neuroblasts begin to

divide during embryogenesis and continue until late into pupal life without going into quiescence (Ito

and Hotta, 1992; Ito et al., 1997; Noveen et al., 2000; Kunz et al., 2012). Studies that have looked at

the mature and immature neurons from these lineages at both the embryonic and the postembryonic

phases find that neurons from these lineages innervate the mushroom body compartment at all times

during development (Ito et al., 1997; Kunz et al., 2012). Thus, PATs and mature primary neurons

innervate the mushroom body compartment in the embryo (Nassif et al., 1998; Noveen et al., 2000;

Younossi-Hartenstein et al., 2006); the SATs in the larva and the mature secondary neurons also

76

innervate the mushroom body compartment at later stages (Ito and Hotta, 1992; Ito et al., 1997).

Therefore, it can be said that the mushroom body is constructed during embryonic and postembryonic

development from the progeny of four neuroblast lineages.

Another well studied compartment is the deutocerebral antennal lobe, which is sub-divided

into about 60 synapse dense regions called glomeruli (Laissue et al., 1999). The neuronal

composition of this compartment is complex, with about 550 diverse kinds of olfactory interneurons.

These can be subdivided into uniglomerular and multiglomerular projection neurons that project to the

protocerebrum via different tracts, and the oligoglomerular and multiglomerular local interneurons

that do not leave the antennal lobe (Jefferis et al., 2001; Das et al., 2008; Lai et al., 2008; Chou et al.,

2010). All of these diverse interneuron types are generated from five neuroblasts that are non-

identical. These are the anterodorsal [BAmv3,(Jefferis et al., 2001)], the lateral [BAlc; (Das et al.,

2008; Lai et al., 2008; Chou et al., 2010)], the ventral [BAla1; (Lai et al., 2008)] and the ventral-LN

[BAla2; (Lai et al., 2008; Das et al., 2011)]. Additionally, there are reports of a fifth antennal lobe

lineage whose cell types have yet to be described [BAlp4; Das et al, submitted, (Pereanu and

Hartenstein, 2006)]. The direct relationship between these lineages and the antennal lobe

compartment is exemplified by ablation experiments where either chemical or genetic ablation of the

lateral lineage results in a drastic reduction in the size of the antennal lobe compartment (Stocker et

al., 1997; Das et al., 2008). For all of these lineages, both embryonic and postembryonic neuronal

progeny that contribute to the antennal lobe compartment have been documented, and their respective

PATs and SATs have been shown to resemble the trajectories seen in the adult brain (Jefferis et al.,

2001, 2004; Dumstrei et al., 2003; Marin et al., 2005; Pereanu and Hartenstein, 2006; Das et al., 2008,

2011; Larsen et al., 2009). Thus, the antennal lobe compartment develops from five distinct

neuroblast lineages that contribute specific cell types to this compartment throughout development.

A third example for this lineage-to-compartment relationship that has only recently been

described, is the central complex and its lineages. The central complex, situated at the midline of the

protocerebral region of the brain, is an apparently unpaired composite of neuropile compartments - the

central body, ellipsoid body, protocerebral bridge, noduli, and the associated lateral accessory lobe

(Hanesch et al., 1989). Behavioural studies have shown that this structure is required for many

complex behaviours such as visual memory and orientation, locomotor control and courtship

behaviour (Strauss, 2002; Sakai and Kitamoto, 2006; Ilius et al., 2007; Neuser et al., 2008; Ofstad et

al., 2011). As many as 50 different kinds of neurons have been described that innervate this neuropile

(Young and Armstrong, 2010). Most of these neurons derive from 10 known neuroblast lineages, all

of which contribute their neurons to different neuropile compartments of the central complex (Pereanu

and Hartenstein, 2006; Pereanu et al., 2010; Boyan and Reichert, 2011). Although embryonic born

neurons do contribute to the central complex, the role of these primary neurons to central complex

development has not been fully investigated as the entire structure develops only during pupal life.

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The lineage compartment relationships documented in the mushroom body, the antennal lobe

and the central complex demonstrate that the fly brain is a strikingly modular structure, with

neuroblast lineages representing the ‘modules’ that underlie the basic macro-architecture of the

brain’s circuitry. A further extension of this idea relates neuroblast lineages to behavioural functions.

Neuropile compartments are functionally segregated in flies; the antennal lobe neuropile is a site for

olfactory processing, the mushroom body neuropile is a site for olfactory learning and memory and

the central complex is a site for visual memory and locomotor co-ordination. Thus, the neuroblast

lineages that construct these neuropiles might also be seen as ‘functional modules’ of the fly brain.

This developmental and functional modularity presents itself as a convenient situation in the analysis

of complex behaviours from a genetic or circuit perspective, since we have some knowledge

regarding the combinatorial genetic codes involved in the specification of individual neuroblast

lineages (Urbach and Technau, 2003).

While this lineage principle underlies the macro-architecture of the fly brain, the micro-

architecture of the neural circuitry depends on the individual neurons of the lineage. For example,

within a lineage, individual neurons may differ both in terms of their neurotransmitter identity and

their finer targeting within a neuropile at both the axonal and dendritic terminals (Jefferis et al., 2001;

Marin et al., 2002; Wong et al., 2002; Das et al., 2008; Lai et al., 2008). While many studies that

have investigated the molecular basis of central brain development have identified genes involved in

neuroblast specification, asymmetric division pattern, cell division and local dendritic or axonal

targeting of the neurons; however, little is known about the molecular basis of the underlying macro-

architecture of the brain circuitry.

In this chapter, I investigate the role of otd in postembryonic development of the adult brain.

MARCM based clonal mutational inactivation of otd during postembryonic brain development

revealed an unexpected role for otd in the formation of adult-specific interneuronal wiring. Clonal

inactivation of otd in postembryonic brains resulted in the striking appearance of an ectopic

neuroblast lineage innervating the antennal lobe. This ectopic lineage innervated the antennal lobe

and higher brain centres in a manner similar to wild type olfactory projection neurons. Further

experiments identified an Otd-expressing lineage, ventral to the antennal lobe, to be the lineage that is

respecified into an antennal lobe lineage upon the loss of otd function. Under normal circumstances,

this identified lineage (BAmv1) contributes wide-field neurons to the central complex (Pereanu et al.,

2011). Strikingly, the respecified lineage not only showed a morphological transformation from a

central complex lineage to an antennal lobe, projection neuron lineage, but this transformation was

also accompanied by changes in the neurotransmitter identity of this lineage. As assayed by the

reporter lines OK371-Gal4 and Cha7.4-Gal4, it was determined that the wild type BAmv1 lineage is

primarily a glutamatergic lineage, while upon transformation to an antennal lobe lineage, this

glutamatergic identity was suppressed in BAmv1, and a novel cholinergic identity was assumed by

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the otd null BAmv1 lineage. The respecification of this lineage occurred only if otd function was

removed from the neuroblast, while removal of otd function from the postmitotic neurons failed to

result in a transformation. Taken together, this chapter documents the action of otd in postembryonic

development of the Drosophila central brain. Moreover, it describes the respecification of an entire

neural lineage upon the mutation of a single gene.

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Results

Clonal inactivation of otd during postembryonic brain development results in an ectopic antennal

lobe lineage

otd loss of function results in embryonic lethality. To determine if otd acts later in

postembryonic brain development, otd loss-of-function clones were generated in the neuroblast

lineages of the larval brain in an otherwise heterozygous animal, using the MARCM method (Mosaic

Analysis with a Repressible Cell Marker). The ubiquitously expressed tubulin-Gal4 driver was used

together with a UAS-mCD8::GFP reporter to label randomly targeted mutant MARCM neuroblast

clones that were induced 0-4h after larval hatching and recovered in the adult central brain. Two

different otd loss-of-function alleles were used, and similar results were obtained with both alleles.

Both alleles used were amorphic; the otdYH13 allele used was generated by EMS mutagenesis, and the

oc2 allele was an X-ray induced mutation resulting in a small deletion within the otd gene.

In control wild type (WT) clonal experiments, five different types of neuroblast clones

innervating the antennal lobe were recovered that corresponded to the adNB, lNB, vNB, vLN, and the

fifth, BAlp4 lineages (Figure 1; Das et al, submitted). In the corresponding otd-/- experiments, a

striking lineage-specific mutant phenotype was observed in about 13.6% of the brains. Mutant

neuroblast clones of this type consisted of a cluster of neural cells located ventromedially to the adult

antennal lobe, a position that does not correspond to any of the five WT antennal lobe neuroblast

clones; neurites from these cell bodies projected into the antennal lobe from the medial aspect of the

lobe, a point of entry that also does not correspond to any of the five known lineages (Figure 2 A-F).

Within the antennal lobe, this ventromedial, otd-/- neuroblast clone projected dendrite-like processes

that formed multiglomerular arborisations in more posterior regions and also innervated

interglomerular regions (yellow asterisk in Figure 2A,D,G). An axon-like tract continued from the

antennal lobe towards the protocerebrum via the inner antennocerebral tract (iACT), which is

normally utilised by the projection neurons of the adNB, lNB, and the fifth lineages (yellow arrows in

Figure 2G-O). In the protocerebrum, the axons targeted the mushroom body and the lateral horn in a

manner similar to WT projection neurons from the adNB and lNB lineages (yellow arrows in figure

2M and O). In addition to the dendrite-like processes to the antennal lobe and the axonal processes

via iACT in the mushroom body and lateral horns, the mutant lineages sometimes manifested

‘misprojections’ to the contralateral antennal lobe and sub oesophageal ganglion (SOG, not shown).

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Figure 1. Four known neuroblast lineages that make up the antennal lobe. Neuroblast clones of four of the five neuroblast lineages that innervate the antennal lobe – the anterodorsal lineage (adNB; A-C), the lateral lineage (lNB; D-F), the ventral lineage (vNB; G-I), and ventral-LN (vLN NB; J-L) lineage. Magenta dotted lines encircle the cell bodies of the respective lineages, and the white dotted lines encircle the antennal lobe. Note that none of the lineages enter the antennal lobe (yellow arrows) from near the midline (yellow lines). These neuroblast clones were generated at in the late embryo or 0-4 hours after larval hatching (ALH). Genotype - FRT19A,hsFLP,TubGAL80/FRT19A; Tub>GFP/+.

While the dendritic and axonal projection patterns of the cells of this ventromedial otd-/-

neuroblast lineage were comparable to that of wild type olfactory projection neurons, other

neuroanatomical features, such as position of cell bodies and point of entry into the antennal lobe,

were distinctly different from any of the wild type neuroblast lineages that innervate the antennal

lobe. Furthermore, in the different brain preparations, this mutant lineage was recovered alongside

neuroblast clones of the other known antennal lobe lineages, confirming that this was indeed a lineage

different from the five known antennal lobe lineages (not shown). In further support for this being an

additional antennal lobe lineage, none of the corresponding WT MARCM clonal brains contained

neuroblast clones having these features in the corresponding brain region. These findings indicate

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that the otd mutant neuroblast lineage is an additional antennal lobe lineage, to be called the 'ectopic

antennal lobe lineage' henceforth.

Figure 2. Random inactivation of otd in the postembryonic brain results in an ectopic antennal lobe lineage. Two examples of brains containing an ectopic antennal lobe lineage in one hemisphere (A-C right hemisphere, and D-F left hemisphere). Note the novel ventromedial position of the cell bodies (encircled in magenta, A-F) and the point at which the axonal bundle of this lineage enters the antennal lobe (magenta arrows in A-F) near the midline (yellow line), which is different from the known antennal lobe lineages shown in Figure 1. Stars in A, D, and G indicate the multiglomerular innervations within the antennal lobe. In G-O, the higher centre projections of the ectopic lineage shown in the D-F panel has been traced. The yellow arrows in G and I show the axonal bundle leaving the antennal lobe towards the protocerebrum. The yellow arrows in J and L show the axon bundle in the protocerebrum, and the yellow arrows in M and O show the innervations in the mushroom body and lateral horn. These neuroblast clones were generated at in the late embryo or 0-4 hours after larval hatching (ALH). Genotype - FRT19A,hsFLP,TubGAL80/FRT19A otd[YH13]; Tub>GFP/+.

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The ectopic antennal lobe lineage does not result from the survival of a lineage normally fated to

die in an Otd dependent manner

The presence of the ectopic otd mutant antennal lobe lineage could be due to the abnormal

survival of a neuroblast lineage that undergoes Otd-dependent programmed cell death during

postembryonic development in the wild type brain. Programmed cell death (PCD) of neuroblasts that

is dependent on the expression of other homeodomain-containing transcription factors has been

shown to occur during normal postembryonic CNS development in Drosophila (Bello et al., 2003). If

Otd indeed initiated PCD in a WT neuroblast to eliminate it from innervating the antennal lobe, we

hypothesised that inhibiting PCD, independent of Otd function, should result in the appearance of the

ectopic lineage. Blocking of cell death was achieved by two independent methods. Either the pan-

caspase inhibitor, p35, was overexpressed (UAS-p35) in neuroblast MARCM clones using the

ubiquitously expressed tubulin-Gal4, or the three pro-apoptotic genes, grim, reaper and hid were

removed from neuroblasts by making MARCM mutant clones of the deletion Df(3L)H99. Among a

total of 45 brains (90 hemispheres) with labelled clones recovered in the UAS-p35 experiments and 75

brains (150 hemispheres) with labelled clones recovered in the Df(3L)H99 experiments, none had the

neuroanatomical features characteristic of the otd mutant ectopic lineage (Table 1). This indicates

that Otd-dependent programmed cell death does not normally eliminate this lineage in the wild type,

and implies that the presence of the ectopic otd mutant lineage is not a consequence of an abnormal

lineage-specific cell death block. An alternative explanation for the presence of the ectopic otd

mutant antennal lobe lineage is that a non-olfactory lineage that expresses Otd in the wild type is

transformed into the ectopic, olfactory lineage in the absence of Otd.

N=X

Hemi-

segments

ad

NB

clones

lateral

NB

clones

ventral

NB

clones

ventral-

LN NB

clones

Ectopic

NB

clones

Tub p35 90

10 3 16 5 0

Tub

Df(3L)H99

150

14 9 12 20 0

Table 1: The ectopic lineage does not result from the survival of a lineage normally fated to die in an Otd dependent manner. While neuroblast clones of the four olfactory interneuron lineages were recovered with normal frequencies, the ectopic lineage innervating the antennal lobe was not uncovered. Clones were generated at L1-L2 larval instar stages. Numbers in red indicate number of brain hemispheres. Genotypes - FRT19A,hsFLP,TubGAL80/FRT19A UAS-p35; Tub>GFP/+ or yhsFLP/+ or Y; Tub>GFP/+; FRT2A, Df(3L)H99/FRT2A Tub Gal80.

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An Otd-positive neuroblast lineage, ventral to the antennal lobe, is transformed into the ectopic

antennal lobe lineage upon the loss of otd function

To investigate the possibility that a normally non-antennal lobe, Otd-positive lineage becomes

mis-specified into an antennal lobe lineage upon the loss of otd function, we first carried out

immunolabelling studies on the adult wild type brain to determine if any Otd-expressing cells are

located ventral to the antennal lobe, in a position that corresponds to that of cells of the ectopic otd

mutant lineage. Anti-Otd immunolabelling reveals five prominent groups of Otd-positive cell bodies

adjacent the antennal lobe of the adult brain (Figure. 3). One large and two smaller groups of Otd

immunoreactive cell bodies are located lateral to the antennal lobe; the position of these cells does not

correspond to that of the cells of the ectopic otd mutant lineage (yellow dotted lines in Figure 3A-C).

Another large cluster of Otd immunoreactive cell bodies is located ventral to the antennal lobe, in a

position that does correspond to that of the cell bodies of the ectopic otd mutant lineage (magenta

dotted lines in Figure 3). (Yet another cluster of otd immunoreactive cells is located more ventro-

anteriorly in the suboesophageal ganglion; the position of the cell bodies in this cluster does not

correspond to that of the cells in the ectopic lineage. White dotted lines in Figure 3.)

Figure 3: Otd expression in the adult brain. At least five clusters of Otd-positive cells lie in the vicinity of the antennal lobe, circled in white, yellow and magenta in A-C. These clusters may, or may not correspond to lineally related cells. The ventromedial cell cluster circled in magenta dotted lines correspond to the location of the ectopic lineage (A-C). This cluster of cells corresponds to a single neuroblast lineage (D-G). Genotype in A-C: CS. Genotype in D-G: FRT19A,hsFLP,TubGAL80/FRT19A; Tub>GFP/+.

In order to determine if this ventral cluster of Otd-positive cells in the wild type brain

corresponds to a single neuroblast lineage or to several neuroblast lineages, MARCM-labelling of

individual neuroblast clones was carried out together with Otd immunolabelling. Clones were

induced at larval hatching and recovered in the adult brain. These double labelling experiments

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demonstrate that the ventral cluster of Otd-expressing cells represents the lineage of a single

neuroblast (figure 3D-G).

The location of these cell bodies in the WT brains coincided with the location of the cells of

the ectopic lineage in the otd mutant brains. To determine if this Otd-positive lineage in the WT brain

indeed corresponded to the lineage, which, when mutant for otd function, is transformed into the

ectopic lineage, otd null MARCM experiments were carried out and the brains were counter-stained

with the anti-Otd antibody. If the WT Otd-positive lineage was indeed the one that was transformed

into the ectopic lineage (upon the loss of otd function), brains in which the ectopic lineage was

present, should correspondingly lack the ventrally placed Otd-positive cell cluster. In these

experiments, a total of 7 brains with an ectopic lineage in one hemisphere were analysed. In none of

these brains, did the hemisphere that contained the ectopic lineage also manifest the ventral, Otd-

positive cell body cluster. In contrast, all of the contralateral brain hemispheres, lacking ectopic

lineages, manifested the ventral, Otd-positive cell body cluster (figure 4).

Figure 4: An Otd-positive neuroblast lineage, ventral to the antennal lobe, is transformed into the ectopic lineage upon the loss of otd function. The presence of the ectopic lineage always corresponds to a loss of Otd expression from the Otd expressing cells ventral to the antennal lobe. The magenta arrows show the ectopic lineage in the right hemisphere (A and D), and the magenta asterisk shows the innervations from the ectopic lineage in the right antennal lobe. Correspondingly, the magenta dotted lines in the left hemisphere show the wild type Otd-expressing cell cluster, and the yellow dotted lines indicate the loss of Otd expression from this cluster. Genotype - FRT19A,hsFLP,TubGAL80/FRT19A otdYH13; Tub>GFP.

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In figure 4, which documents one such brain preparation, the left hemisphere does not contain

the ectopic lineage, while the right hemisphere does (magenta arrows in Figure 4A and D).

Correspondingly, the ventral cluster of Otd-positive cells is present in the left hemisphere (magenta

dotted lines), while this cluster is absent in the right hemisphere (yellow dotted lines).

These data imply that an Otd-positive neuroblast lineage, ventral to the antennal lobe is

indeed the WT lineage, which when mutant for otd function, is transformed into the ectopic lineage

innervating the antennal lobe.

The Otd positive neuroblast lineage, ventral to the antennal lobe, is the BAmv1 lineage, which

normally innervates the central complex

Having established that a neuroblast lineage is transformed into the ectopic lineage upon the

loss of otd function, we investigated the identity of this neuroblast lineage and its corresponding wild

type neuropilar compartment. To do this, we generated wild type MARCM clones using the

ubiquitously expressed tubulin-Gal4 driver and counter-stained the brains with the anti-Otd antibody.

We then recovered brain hemispheres that contained positively labelled neuroblast clones of the

ventral Otd-positive lineage, and followed their axonal trajectories in the brain. Although MARCM

experiments with the tubulin-Gal4 driver usually also resulted in simultaneous labelling of other

neuroblast clones in the brain, it was possible to establish that the axonal tract of the ventral Otd-

positive lineage in the wild type projected around the lateral aspect of the antennal lobe in the more

posterior sections and innervated a specific neuropile in the protocerebrum called the lateral accessory

lobe, which is known to be a compartment associated with the central complex (Figure 5). As the neuroblast composition of the central complex had recently been described, we

compared the trajectory of the Otd-positive lineage to the 10 published lineages that construct the

central complex (Pereanu et al., 2011). Amongst these 10 known lineages, only the BAmv1 lineage

corresponded to the ventral, Otd-positive lineage of interest, both because of the location of the cell

bodies and the axonal tract projections of the lineage. In their study, Pereanu and co-workers, utilised

the Per-Gal4 driver to label the BAmV1 lineage. Preliminary work in our lab suggested that the

OK371-Gal4 driver might also be used to label the BAmv1 lineage and the ventral, Otd-positive

lineage of our interest.

To establish definitively whether the ventral Otd-positive lineage, the Per-Gal4 labelled BAmv1

lineage, and the OK371-Gal4 labelled lineage all correspond to the same neuroblast lineage, we

carried out WT MARCM analyses using the OK371-Gal4 and the Per-Gal4 driver lines and counter-

stained the brains with an anti-Otd antibody. In all of these experiments, when clones with cell body

positions and axonal trajectories similar to the ventral Otd positive lineage were recovered, they were

invariably also positive for Otd expression (Figure 6A-D and K-N). This result confirmed that the

ventral, Otd-positive lineage did, in fact, correspond to the BAmv1 lineage, labelled by the OK371-

Gal4 and the Per-Gal4 driver lines. Importantly, in these experiments, it was possible to trace the

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axonal trajectory and innervation patterns of the BAmV1 lineage more precisely than in experiments

using the tubulin-Gal4 driver. Neuroblast clones of BAmv1 labelled by OK371-Gal4 and Per-Gal4

appear to have wide field innervations in the entire lateral accessory lobe, the central body, the

ellipsoid body and the nodule of the central complex (Figure 6 E-J and O-T). Unlike the reports in

Pereanu and co-workers, we did not recover cells that innervated the optic tubercle (Pereanu et al.,

2011). This may be due to timing of clone induction.

Taken together, these data suggest that the Otd-expressing BAmv1 lineage, which normally

innervates the central complex, is transformed or rewired into an antennal lobe innervating lineage

upon the loss of otd function.

Figure 5. The Otd positive neuroblast lineage, ventral to the antennal lobe, is the BAmv1 lineage, which normally innervates the lateral accessory lobe of the central complex. The Otd-positive lineage, ventral to the antennal lobe is shown with magenta dotted lines and asterisk. The axonal tract of this lineage (magenta dotted lines) skirts around the antennal lobe (yellow dotted lines) and innervates a central complex associated neuropile called the lateral accessory lobe (LAL), which lies between the antennal lobe and the mushroom body (yellow dotted lines labelled ‘MB’). Clones were generated at L1-L2 larval instar stages. Genotype - FRT19A,hsFLP,TubGAL80/FRT19A; Tub>GFP/+.

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Figure 6: The BAmv1 lineage is labeled by OK371-Gal4 and Per-Gal4 and contributes wide field neurons to most of the central complex. A-J shows Per-Gal4 WT MARCM clones, and K-T shows OK371-Gal4 WT MARCM clones. A-D shows the Otd-positive BAmv1 lineage, labelled by the Per-Gal4 line, which innervates the lateral accessory lobe (LAL) and the central complex (CC). Innervation in the central complex ellipsoid body (‘EB’ in E-G) and the central body and nodule (‘CB’ and ‘N’ in H-J) have been shown in the E-J. These magnifications are not from the preparation shown in A-D. K-N shows the OK371-Gal4 labelled BAmv1 lineage, labelled by the OK371-Gal4 line, also innervating the lateral accessory lobe (LAL) and the central complex (CC). O-Q are magnified images of the innervations patters of the OK371-Gal4 labelled BAmv1 lineage the central complex ellipsoid body (‘EB’) and R-T are the innervations in the central body and noduli (‘CB’ and ‘N’). Clones were generated in the late embryo or early first larval instar. Genotype in (A-J) - FRT19A,hsFLP,TubGAL80/FRT19A; Per>GFP/+, Genotype in (K-T) - FRT19A,hsFLP,TubGAL80/FRT19A; OK371>GFP/+.

Antennal lobe transformation of central complex neurons does not occur in postmitotic neurons

The identification of the WT Otd-expressing lineage of the central complex and the

availability of Gal4 lines with restricted expression patterns that label the WT lineage allowed us to

investigate whether the ability to rewire this central complex lineage into an antennal lobe lineage is

restricted to the neuroblast of BAmv1, or is also present in the postmitotic neurons. To address this

issue, we used the OK371-Gal4 and the Per-Gal4 drivers to generate single cell MARCM clones that

were either WT or otd null and counter-stained the brains with an anti-Otd antibody. (Single cell

MARCM clones in neuroblast lineages represent postmitotic neural cells.) In wild type experiments,

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we recovered a total of 7 single cell MARCM clones in the BAmv1 lineage, all of which innervated

the central complex, regardless of which of the two GAL4 driver lines were used (Figure 7A-D). In

the corresponding otd null clones (as determined by the anti-otd antibody; insets in Figure 7E-L), we

recovered a total of 11 single cell MARCM clones, all of which also manifested targets in the central

complex that corresponded to those of WT BAmv1 lineage neurons. For example, single cell otd null

clones innervating the lateral aspect of the lateral accessory lobe and projecting onto a single nodule

were recovered in both the OK371-Gal4 and Per-Gal4 experiments (Figure 7E-L). While otd

function in postmitotic neurons could be required for the targeting of these neurons within the central

complex, with respect to the observed rewiring phenotype, these results suggest that postmitotic

neurons are unable to make the transformation from central complex lineage cells into antennal lobe

lineage cells upon the loss of otd.

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Figure 7: Antennal lobe transformation of central complex neurons does not occur in post-mitotic neurons. A-D shows a single cell WT MARCM clone of the BAmv1 lineage innervating the lateral accessory lobe and one of the noduli labelled by OK371-Gal4 (yellow arrows). The inset shows Otd staining in the cell body. E-H and I-L show otd null single cell clones from the BAmv1 lineage labelled by OK371-Gal4 and Per-Gal4. Insets in E-L show loss of Otd staining in the otd null cells. Both otd null cells retain their targeting to the nodulus and lateral accessory lobe of the central complex (yellow arrow). Clones were generated in the early first instar larva. Genotype in (A-D) - FRT19A,hsFLP,TubGAL80/FRT19A; OK371>GFP/+, Genotype in (E-H) - FRT19A,hsFLP,TubGAL80/FRT19Aoc2; OK371>GFP/+, Genotype in (I-L) - FRT19A,hsFLP,TubGAL80/FRT19Aoc2; Per>GFP/+.

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The morphological transformation of BAmv1 from a central complex lineage to an antennal lobe

lineage is accompanied by molecular transformations

As mentioned above, both the Per-Gal4 driver line and the OK371-Gal4 driver line resulted

in reliable MARCM-based clonal labelling of the BAmv1 lineage in the wild type. However, these

two driver lines never resulted in MARCM-labelled neuroblast clones of the corresponding otd null

transformed lineage. For example, in the MARCM labelling experiments using OK371-Gal4 to drive

reporter gene expression, we recovered 6 neuroblast clones and 6 single cell clones of the wild type

BAmv1 lineage projecting to the central complex. We calculated the frequency of obtaining

neuroblast clones in BAmv1 with the OK371-Gal4 driver was 10 percent. In the corresponding otd

null experiments using the OK371-Gal4 driver, we recovered 6 single cell clones innervating the

central complex and no neuroblast clones innervating either the central complex or the antennal lobe.

Interestingly, we occasionally recovered 1-5 cell clones of the ectopic morphology in these otd null

experiments (Figure 8A-D and E-H). Accompanying these kinds of clones was a complete loss of

Otd staining from the Otd-positive cell cluster ventral to the antennal lobe, which corresponds to the

BAmv1 lineage (yellow dotted lines in Figure 8). Indeed, we sometimes even recovered brain

hemispheres in which the entire Otd positive cell cluster corresponding to the BAmv1 lineage was

lacking but no cells of the ectopic morphology were labelled (figure 8 I-L and M-P). This implies that

these brain hemispheres contain otd null MARCM neuroblast clones of the BAmv1 lineage and

therefore suggests that the OK371-Gal4 driver is suppressed in the transformed otd null BAmv1

lineage. (Comparable findings were obtained for the Per-Gal4 driver; data not shown.) Thus, both

the Per-Gal4 and the OK371-Gal4 driver lines, which are normally active in the WT BAmv1 lineage,

are inactive in the otd null BAmv1 lineage.

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Figure 8. The OK371-Gal4 enhancer is downregulated in the transformed, otd null BAmv1 lineage. A-D and E-H show two examples of brains which have unilateral otd null neuroblast clones. The left hemisphere in A-D and the right hemisphere in E-H are null for otd as seen by loss of Otd staining in that hemisphere (yellow dotted lines). The contralateral lobe, which is not null for otd function, retains expression of Otd (magenta dotted lines). 5 and 3 cells of the transformed morphology are visible in these preparations (yellow arrows in A and E). The innervations from these transformed cells in the antennal lobe has been indicated by the filled, yellow asterisks in A-H. I-L and M-P represent two examples of brains that have unilateral otd null clones as seen by loss of Otd staining in that hemisphere (yellow dotted lines). However, in these prepaprations, no cells of the transformed morphology were visible, and the antennal lobe contained no innervations from the otd null, BAmv1 cells (empty asterisks in I-P). Clones were generated in the early first instar larva. Genotype - FRT19A,hsFLP,TubGAL80/FRT19Aoc2; OK371>GFP/+.

The above results suggest a change in the profile of enhancers that are normally active in this

lineage following the lineage transformation that occurs upon the loss of otd function. This prompted

us to ask if the corollary was also true; might enhancers that are normally silent in the WT BAmV1

lineage, become activated in the transformed, otd null Bamv1 lineage? To investigate this, we

assayed the GH146-Gal4 driver line, which is normally not active in the WT BAmv1 lineage. We

generated WT and otd null MARCM clones utilizing the GH146-Gal4 driver to visualise the clones.

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In the adult brain, GH146-Gal4 drives reporter gene expression in a subset of the olfactory projection

neurons, namely in typical projection neurons of the adNB and lNb lineages and in ~7-10 cells of the

vNB lineage (Stocker et al., 1997; Jefferis et al., 2001; Das et al., 2008). Correspondingly, when this

driver was used to visualise WT clones, only adNB, lNB and vNB clones innervating the antennal

lobe and projecting up to the mushroom body and lateral horn via the iACT or the mACT were

recovered (Figure 9A-F). However, when GH146-Gal4 was used to label cells in otd mutant clones, a

fourth kind of neuroblast clone, distinct from the three neuroblast clones seen in the WT experiments,

was also recovered. The neuroanatomical features of this novel clone corresponded to those of the

ectopic antennal lobe lineage (otd null BAmv1 lineage, figure 9G-L). Thus, its cell bodies were

located ventro-medial to the antennal lobe (magenta arrows in figure 9G,I,J and L), and they projected

dendrite-like processes into the antennal lobe (magenta asterisks in figure 9G,I,J and L) as well as an

axon bundle (via the iACT) towards the protocerebrum where it innervated the mushroom body and

the lateral horn, similar to wild type antennal lobe projection neurons (compare yellow arrows in

figure 9D to those in figure 9G). This result suggests that the GH146 driver, which is normally

inactive in the WT BAmv1 lineage, is activated in the otd null, transformed BAmv1 lineage.

Furthermore, the ectopic activation (in the transformed lineage), of a driver line that is normally active

only in olfactory projection neurons, suggests that not only does the otd null BAmv1 lineage resemble

olfactory interneurons morphologically, but it also takes on molecular characteristics that are typical

of olfactory interneurons.

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Figure 9. The transformed, otd null BAmv1 lineage, and not the wild type BAmv1 lineage, expresses the enhancer GH146-Gal4, which is specific to olfactory projection neurons. A-C show WT clones of the adNB and vNB antennal lobe lineages and D-F show WT clones of the lNB antennal lobe lineage as labelled by GH146-Gal4 enhancer. In the wild type adult brain, the expression pattern of this Gal4 line is restricted to these olfactory projection neurons. In otd null clones an additional antennal lobe lineage, i.e. the otd null BAmv1 lineage, is seen. Two examples of this are shown in G-I and J-L where the cell bodies of this ectopic antennal lobe lineage is indicated by a magenta arrowhead, and the antennal lobe innervations are indicated by magenta asterisk. G-I shows a brain preparation where the higher centre projections of the ectopic lineage are clearly visible and can be compared to the wild type projection neuron innervations (yellow arrows). Clones were generated either in the late embryo, or in the early first instar larva. Genotype in A-F - FRT19A,hsFLP,TubGAL80/FRT19A; GH146>GFP/+, Genotype in G-L - FRT19A,hsFLP,TubGAL80/FRT19Aoc2or otdYH13; GH146>GFP/+.

Loss of otd function in the BAmv1 lineage transforms the glutamatergic central complex lineage,

into a cholinergic antennal lobe lineage

The OK371-Gal4 is an enhancer trap in the dvGlut gene, and has been shown to be a reliable

reporter for glutamatergic neurons (Mahr and Aberle, 2006). Therefore, the finding that OK371-Gal4

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is normally expressed in the WT BAmv1 lineage suggests that the neurons of this lineage are

normally glutamatergic (Figure 6 K-N). Moreover, since this driver was suppressed in the

transformed, otd null BAmv1 lineage (Figure 8 I-P), the glutamatergic identity of this lineage might

also be suppressed upon the loss of otd function and the subsequent transformation into an antennal

lobe lineage. If this were the case, we hypothesised that the transformed lineage might be taking on a

different neurotransmitter identity. WT olfactory projection neurons are not known to be

glutamatergic; their typical neurotransmitter identity is cholinergic. To test whether the transformed

lineage was also taking on the neurotransmitter identity typical of antennal projection neurons, we

generated WT and otd null MARCM clones using Cha7.4-Gal4 to drive reporter gene expression.

The Cha7.4-Gal4 is a promoter fusion line that has been used to label cholinergic neurons; in this line,

Gal4 is driven by a 7.4Kb DNA fragment that corresponds to the upstream region of the dvChat gene,

known to be expressed in cholinergic neurons (Salvaterra and Kitamoto, 2001). In WT adult brains,

this reporter line does not label the BAmv1 lineage (data not shown). Consequently, in WT clonal

brains, we never recovered the BAmv1 clone (72 hemispheres examined). However, in the

corresponding otd null MARCM experiments, we often recovered neuroblast clones with the

neuroanatomy of the ectopic lineage (figure 10). Thus, the cell bodies of these clones were

ventromedial to the antennal lobe (magenta arrows in figure 10 A and E), and they innervated the

antennal lobe (magenta asterisks), as well as projected into the protocerebrum via the iACT (not

shown). In these brain hemispheres, the Otd positive cell cluster corresponding to BAmv1 was

invariably absent, indicating that these brains did, in fact, contain the transformed, otd null BAmv1

lineage (magenta dotted lines in figure 10 B, D, F and H). Thus, in addition to the inactivation of the

reporter for glutamatergic neurons OK371-Gal4, the transformed, otd null lineage also activates the

reporter for cholinergic neurons Cha7.4-Gal4, suggesting that the transformation of these neurons

extends to their neurotransmitter identity as well.

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Figure 10. Loss of otd function from the BAmv1 lineage transforms the glutamatergic, central complex lineage, into a cholinergic, antennal lobe lineage. A-D and E-F are two examples of brain preparations containing the otd null, transformed BAmv1 lineage unilaterally, which express the Cha7.4-Gal4 reporter. In A-D the transformed lineage is in the right hemisphere and in E-H, the transformed lineage is in the left hemisphere. In both cases, the cell bodies of the lineage are indicated by magenta arrows, and the innervations in the antennal lobe are indicated by magenta asterisks. Magenta dotted lines in B, F, D and H show the loss of otd from the BAmv1 lineage (compare to wild type Otd staining in the contralateral hemisphere highlighted by yellow dotted lines). Note that in both cases where the BAmv1 lineage is null for otd function, the Cha7.4-Gal4 reporter labels the transformed lineage. Clones were generated either in the early first instar larva or late embryo. Genotype - FRT19A,hsFLP,TubGAL80/FRT19A otdYH13; Cha7.4>GFP/+.

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Discussion

In this report we show that the cephalic gap gene otd has a key role in lineage specification

during postembryonic brain development. In the wild type, otd is expressed in the interneurons of the

BAmv1 lineage that innervate the central complex; in the absence of otd, the interneurons in this

lineage are transformed and innervate the antennal lobe. This otd-dependent, lineage-specific

respecification of interneurons has implications for our understanding of the development,

organization and evolution of olfactory circuitry in the insect brain.

The specification of olfactory interneurons occurs through complex interactions between cell-

autonomous intrinsic genetic programmes and non cell-autonomous context dependent interactions

(Jefferis and Hummel, 2006; Brochtrup and Hummel, 2011). Among the previously characterized

cell-intrinsic control elements that operate in developing olfactory interneuron lineages are the

homeodomain transcription factors Ems and Cut, the POU transcription factors Acj6, Pdm3, Drifter,

the LIM homeodomain transcription factors Lim1 and Islet, and the BTB-Zn-finger transcription

factor Lola (Komiyama et al., 2003; Komiyama and Luo, 2007; Spletter et al., 2007; Das et al., 2008;

Lichtneckert et al., 2008). Mutations in these genes lead to a wide spectrum of differentiation and

targeting defects, including reduction of interneuron number and defects in dendritic/axonal targeting.

For example, Acj6 and Drifter are expressed in mutually exclusive sets of lineage-specific projection

interneurons, and misexpression of Acj6 in the lNB lineage or Drifter in the adNB lineage leads to

inappropriate innervation of glomeruli normally targeted by the other lineage (Komiyama et al.,

2003). However, mutation of these two transcription factors never resulted in a complete

transformation of the projection interneurons from either lineage.

Here we report a complete transformation of all of the projection neurons in a central complex

lineage into projections neurons that innervate the antennal lobe. All of the neurons in the lineage are

transformed and all of the transformed neurons manifest dendritic and axonal targeting that is typical

for antennal lobe projection neurons. This morphological transformation is accompanied by changes

in the molecular profile of the lineage, which also includes the neurotransmitter identity of the

lineage. This lineage-specific transformation occurs during postembryonic development if the Otd

transcription factor, which is normally expressed in the BAmv1 lineage, is mutated. The

transformation occurs only if Otd is mutated in the neuroblast, and not if it is mutated in the

postmitotic neurons. This indicates that Otd acts in the neuroblast to specify the normal lineage-

specific identity of these neurons, and that respecification of their identity in the absence of Otd does

not occur in the GMC. This implies that the identity of the postmitotic neurons is determined by the

‘combinatorial code of transcription factors’ in the lineage-specific progenitors and operates in the

neuroblast to translate lineage-specific information into appropriate dendritic and axonal targeting.

The role of Otd in the BAmv1 lineage specification is a striking example of the extent to which a

single member of this putative transcription factor code can dictate neural cell fate.

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The Otd protein has not been detected in any of the known olfactory interneuron lineages.

However, given its role in preventing the formation of an ectopic antennal lobe lineage, it can be

viewed as a key molecular control element in the normal development of antennal lobe circuitry.

From this point of view, the development of the olfactory system would depend both on genes that act

in antennal lobe lineages to promote the development of appropriate circuitry and on genes that act in

non-antennal lobe lineages to prevent the formation of inappropriate circuitry. It is noteworthy that

the BAmv1 lineage, like the four known antennal lobe lineages, derive from neuroblasts located in the

same basoanterior region of the developing deutocerebrum (Pereanu and Hartenstein, 2006). Thus, in

neuromeric terms, the transformation of the BAmv1 lineage into an ectopic antennal lobe lineage

represents the transformation of one deutocerebral lineage identity into another deutocerebral lineage

identity. The fact that the absence or presence of a single gene can result in complete transformation

of one lineage into another suggests that many of the other elements of a potential neural identity code

might be shared among basoanterior lineages in the deutocerebral neuromere.

In terms of their cellular morphology, the interneurons of the ectopic antennal lobe are

remarkably similar to typical olfactory projection neurons. They form dendritic arbours in the

antennal lobe that target specific glomeruli, project their axons through the iACT, and form axonal

arborisations in the mushroom bodies and in the lateral horn. This suggests that these ectopic

projection neurons are functionally integrated into antennal lobe circuitry and, hence, might

participate in olfactory information processing. It will be interesting to combine targeted genetic otd

mutation with neurophysiological and behavioural studies in order to investigate this. If this is the

case, then otd mutation in the BAmv1 lineage would result in a functional rewiring of the olfactory

circuitry due to the addition of an entire neuroblast lineage to the normal population of projection

interneurons. In more general terms, this type of lineage-specific rewiring might fuel the evolutionary

modification of neural circuitry in the brain. In this context, it is noteworthy that most neuroblast

lineages in the central brain target their proximal dendritic arbors to the neuropile of their neuromere

of origin (Kumar et al., 2009). Thus, genetically controlled retargeting of dendritic arborisation to the

neuropile of adjacent neuromeres or to different neuropiles of a given neuromere, could be a simple

and effective evolutionary strategy for rewiring brain circuitry.

Acknowledgements

This work was supported by grants from the TIFR, and the Indo Swiss Joint Research

Program. The Otd antibody was a kind gift from Henry Sun.

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