wingless

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Wingless targets in neural development

How is neuroblast-specific gene expression established? This paper's focus was on the huckebein gene, because it is expressed in a subset of neuroblasts and is required for aspects of neuronal and glial determination. hkb is required within the neuroblast 1-1, 2-2 and 4-2 lineages for proper axon pathfinding of interneurons and motoneurons and for proper muscle target recognition by motoneurons. The secreted Wingless and Hedgehog proteins activate huckebein expression in distinct but overlapping clusters of neuroectodermal cells and neuroblasts, whereas the nuclear Engrailed and Gooseberry proteins repress huckebein expression in specific regions of neuroectoderm or neuroblasts. Hedgehog activates hkb in cells that give rise to the 5HT expressing lineage), while Wingless activates hkb in cells that give rise to an eve expressing motorneuron lineage). Wingless and Hedgehog activate hkb in the neuroectoderm of hemisegment row 5 neuroblast precursors. Early-forming neuroblasts of rows 5 and 6 never express hkb even though they develop from Hkb+ neuroectoderm (row 5). Gooseberry functions to repress hkb expression in row 5 neuroblasts while Engrailed represses hkb expression in rows 6/7 neuroectoderm. Integration of these activation and repression inputs is required to establish the precise neuroectodermal pattern of huckebein, which is subsequently required for the development of specific neuroblast cell lineages (McDonald, 1997).

The nuclear proteins Spalt and Spalt-related belong to a conserved family of transcriptional regulators characterized by the presence of double zinc-finger domains. In the wing, they are regulated by the secreted protein Decapentaplegic and participate in the positioning of the wing veins. Regulatory regions in the spalt/spalt-related gene complex have been identifed that direct expression in the wing disc. The regulatory sequences are organized in independent modules, each of them responsible for expression in particular domains of the wing imaginal disc. In the thorax, spalt and spalt-related are expressed in a restricted domain that includes most proneural clusters of the developing sensory organs in the notum, and are regulated by the signaling molecules Wingless, Decapentaplegic and Hedgehog. spalt/spalt-related are found to participate in the development of sensory organs in the thorax, mainly in the positioning of specific proneural clusters. Later, the expression of at least spalt is eliminated from the sensory organ precursor cells and this is a requisite for the differentiation of these cells. It is postulated that spalt and spalt-related belong to a category of transcriptional regulators that subdivide the thorax into expression domains (prepattern) required for the localized activation of proneural genes (de Celis, 1999).

A prominent stripe of wg expression is seen in the thorax, within the sal non-expressing region 4 and close to the border of the sal-expressing region 3. This observation raises the possibility that wg may act as a repressor of sal expression in the thorax, and possibly in other regions of the wing disc. Indeed, in thoracic region 3, sal expression is repressed in and around clones of cells that overexpress wg. Furthermore, wg overexpression in the hinge region results in a reduction of sal expression, whereas a reduction of wg expression in the hinge of imaginal discs as a result of the regulatory mutation spade flag (wg^spd) results in a consistent increase in sal expression. Reduction of wg expression in the thorax in a heteroallelic wingless combination results in the expansion of Sal expression. However, Sal is not expressed in all region 4, indicating that a repressor other than wg is responsible for the exclusion of Sal in this region. It is likely that both sal activation by hh/dpp and its repression by wg are mediated by the regulatory regions that have been identified as responsible for direct sal/salr expression in the thorax. The repressive action of wg is mediated by sequences contained within a fragment called the LA fragment. In these experiments, the inhibitory action of wg can be counteracted by sequences of the endogenous regulatory region located outside the LA fragment. Consistent with a requirement for both hh and dpp signaling to activate sal/salr in the thorax, the expression of beta-gal in the line LA is not modified when dpp is expressed ectopically (de Celis, 1999).

A stepwise morphogenetic program of cell division and cell fate determination generates the precise neuronal architecture of the visual centers of the Drosophila brain. The assembly of the target structure for ingrowing retinal axons involves cell-cell interactions mediated by the secreted product of the wg gene. Expressed in two symmetrical domains of the developing brain optic lobe, wg is required to induce and maintain the expression of the secreted decapentaplegic gene product in adjacent domains (Kaphingst, 1994).

The secreted protein Hedgehog has been identified as the signal transmitted along retinal axons which serves as the inductive signal triggering neurogenesis in the lamina. The target of HH in the developing visual system is wingless, which in turn targets decapentaplegic and Distal-less. The lamina neurons and the cortical neurons that contribute axons to the medulla neuropil derive from a neuroblast population (OPC or outer proliferation center) that divides throughout most of larval development. Although cells expressing wg constitute only a small fraction of the OPC, the inactivation of wg at early times results in the later absence of nearly the entire target structure. Inactivation of wg at later times results in the formation of pregressively more complete central target structures. The expression of wg in the developing visual system is consistent with a role in organizing the dorsoventral axis. wg expression begins in a single ventral cell at approximately 10 hr of larval develpment. About 6 hr later, wg expression begins in a single dorsal cell. For the remaining stages of larval development wg is expressed in two OPC domains that define the dorsal and ventral termini of the developing target regions. These observations suggest that wg has a nonautonomous role in organizing the target region of retinal axons. The fact that early wg expression occurs prior to axon ingrowth, indicates that HH is required for the maintenance of wg expression and not for its initiation (Kaphingst, 1994).

Wingless regulates the onset and maintenance of dpp expression. Approximately 14 hr after the onset of wg expression, dpp expression begins in single cell domains immediately adjacent to the wg-expressing cells, and is maintained throughout larval development as these cell populations divide up to and including the period of retinal axon ingrowth. In dpp mutants many OPC progeny fail to down-regulate the expression of the cell adhesion molecule fasII, fail to express neuron markers, and fail to contribute axons to the medulla neuropil (Kaphingst, 1994).

Distal-less expression is found in wg-expressing cells adjacent to the dpp domains. dll expression is significantly greater in the dorsal domain. The involvement of dll in neurogenesis in Drosophila has yet to be documented (Kaphingst, 1994).

The gut-innervating stomatogastric nervous system of Drosophila, unlike the central and the peripheral nervous system, derives from a compact, single layered epithelial anlage. This anlage is initially defined during embryogenesis by the expression of proneural genes of the achaete-scute complex in response to the maternal terminal pattern forming system. Within the stomatogastric nervous system anlage, the wingless-dependent intercellular communication system adjusts the cellular range of Notch-dependent lateral inhibition to single-out three achaete-expressing cells. Those cells define distinct invagination centers which orchestrate the behavior of neighboring cells to form epithelial infoldings, each headed by an achaete-expressing tip cell. The wingless pathway may act not as an instructive signal, but as a permissive factor which coordinates the spatial activity of morphoregulatory signals within the stomatogastric nervous system anlage (Gonzalez-Gaitan, 1995).

Demonstrating an opposite effect of Wingless in the eye than in the wing, ectopic expression of wingless results in a reduced expression of achaete, and blockage of sensory organ precursor formation. Interommatidial bristles are mechanosensory organs composed of four cells that are derived from a single sensory organ precursor. The process involves achaete and scute. achaete expression is greatly reduced when wingless is expressed in the eye disc. The effect of Wingless is non-cell autonomous, and wingless signaling in the eye involves the same components as in the wing: porcupine, dishevelled, shaggy and armadillo. As in the wing, Wingless can still regulate achaete expression in homozygous clones for a Notch null allele. Thus, a direct role for Notch (as a receptor, for example) in wingless signaling is unlikely. It is unknown whether the effects of Wingless on achaete are direct, or whether wingless signaling modifies negative inputs from other bHLH proteins such as Extramachrocheate and Hairy (Cadigan, 1996).

Wingless and Decapentaplegic cell signaling pathways act synergistically in their contribution to macrochaete (sense organ) patterning on the notum of Drosophila. The analysis of the origin of sense organ precursor prepatterning has focussed on the specification and positioning of the anterior and posterior dorsocentral macrochaetes (aDC, pDC) two large mechanosensory organs located in precise positions relative to surrounding rows of microchaetes. The aDC and pDC SOPs form sequentially on the proximal edge of a single DC proneural cluster where Achaete and Scute expression depends on a cis-activating enhancer sequence, the DC enhancer. Ac expression in the DC proneural cluster requires the activity of wingless. The DC SOPs form adjacent to the stripe of cells expressing wg in the presumptive notum during the third larval instar. To probe the nature of gene interaction required for macrochaetae formation, the Wingless-signaling pathway was ectopically activated by removing Shaggy activity (the homolog of vertebrate glycogen synthase kinase 3) in mosaics. Proneural activity is asymmetric within the Shaggy-deficient clone of cells and shows a fixed polarity with respect to body axis, independent of the precise location of the clone. This asymmetric response indicates the existence in the epithelium of a second signal, possibly Decapentaplegic. Ectopic expression of Decapentaplegic induces extra macrochaetes only in cells that also receive the Wingless signal. Outside the Wg-activated domain, in the medial scutum and prescutum, clones that ectopically express Dpp make only microchaetes. In the Wg-activated domain, within and lateral to the DC meridian, clones of cells ectopically expressing dpp are associated with many extra macrochaetes, which are formed both within and around the Dpp-expressing clones. It is concluded that in areas of the notum where the WG transduction pathway is inactive, Dpp alone is insufficient for macrochaete formation. Activation of Hedgehog signaling generates a long-range signal (Dpp) that can promote macrochaete formation in the Wingless activity domain. This signal depends on decapentaplegic function. Autonomous activation of the Wingless signal response in cells causes them to attenuate or sequester this signal. Extramacrochaetae (a proneural antagonist) is required to limit the anterior/posterior extent of this cluster. If the level of emc is reduced, extra macrochaetes form primarily anterior but also posterior to the normal DCs along the proximal edge of the wg stripe. Further reduction of emc results in additional extra macrochaetes along the dorsal edge of the stripe. These results suggest a novel patterning mechanism that determines sense organ positioning in Drosophila (Phillips, 1999).

Wingless (Wg) and other Wnt proteins play a crucial role in a number of developmental decisions in a variety of organisms. In the ventral nerve cord of the Drosophila embryo, Wg, signaling from row 5 is non-autonomously required for the formation and specification of a neuronal precursor cell, NB4-2. NB4-2 gives rise to a well-studied neuronal lineage, the RP2/sib lineage. While the various components of the Wg-signaling pathway are also required for generating NB4-2, the target gene(s) of this pathway in the signal-receiving cell is not known. In this paper, it is shown that sloppy paired 1 and sloppy paired 2 function as the downstream targets of the Wg signaling to generate the NB4-2 cell. Thus, while the loss-of-function mutations in wg and slp have the same NB4-2 formation and specification defects, these defects in wg mutants can be rescued by expressing slp genes from a heterologous promoter. The fact that slp genes function downstream of the Wg signaling is also indicated by the result that expression of slp genes is lost from the neuroectoderm in wg mutants and that ectopic expression of wg induces ectopic expression of slp. Finally, Gooseberry (Gsb) prevents Wg from specifying NB4-2 identity to the wg-expressing NB5-3. In this paper, it is shown that gsb interacts with slp and prevents Slp from specifying NB4-2 identity in NB5.3. Overexpression of slp overcomes this antagonistic interaction and respecifies NB5-3 as NB4-2. This respecification, however, can be suppressed by a simultaneous overexpression of gsb at high levels. This mechanism appears to be responsible for specifying NB5-3 identity to a row 5 neuroblast and preventing Wg from specifying NB4-2 identity to that neuroblast (Bhat, 2000).

NB4-2 is delaminated from an equivalence group of 4-6 neuroectodermal cells during the second wave of neuroblast delamination in mid stage 9 (approximately 4.5 hours old) of embryogenesis. It is located in the 4th column along the anterior-posterior axis and 2nd row along the medio-lateral axis within each hemisegment. The NB4-2 undergoes its first asymmetric division approximately 1.5 hours after formation to self renew and to generate its first GMC, GMC-1 (this GMC-1 is also called GMC4-2a: the first GMC generated from NB4-2). The GMC-1 divides about 1.5 hours later to generate two cells, the larger RP2 and the smaller sib. The RP2 cell migrates to its specific position within the anterior commissure and projects its axon antero-ipsilaterally to the intersegmental nerve bundle (ISN) and innervates muscle #2 on the dorsal musculature. The sib cell migrates to a position posterior and more dorsal to RP2. NB4-2, GMC-1, RP2 and RP2-sib cells can be reliably identified by their gene expression pattern, physical sizes and position within the half-segment (Bhat, 2000).

In the epidermis, mutation in slp genes result in a fusion of abdominal segments A1-A2, A3-A4, A5-A6 and A7-A8 (characteristic of pair-rule mutants) and replacement of naked cuticle by denticle belts, a wg-type of segment polarity phenotype. During the patterning of the epidermis, slp genes function upstream of wg to maintain wg expression (Cadigan, 1994a and b). Since wg is also required for the formation and specification of NB4-2 identity, it is possible that the effect of loss of slp genes on NB4-2 is mediated via its effect on wg expression. Therefore, to determine the precise temporal requirement of slp for maintaining wg expression during neurogenesis, the expression of wg in slp mutant embryos was first examined. In slp mutants the wg expression begins to fade from the neuroectoderm initially in even-numbered parasegments approximately 3.75 hours of development (stage 7, early germ band extension). This fading is particularly prominent in abdominal segments. By approximately 4.5 hours of development (early stage 9), wg expression in these parasegments is completely lost. By contrast, in odd numbered parasegments, wg expression is nearly as high as in wild type during early stage 9 (approximately 4.5 hours of development), and is only lost by approximately 6-6.5 hours of development (stage 10). These results are consistent with the previous findings (Cadigan, 1994a and b) and show that slp genes function upstream of wg and positively regulate wg expression (Bhat, 2000).

Studies using a temperature-sensitive allele of wg have revealed that Wg activity is required for the specification of NB4-2 identity at approximately 4 hours of development (between early to mid-stage 8, at 22°C). However, in slp mutants the expression of wg is still high in the odd-numbered parasegments around the time of NB4-2 specification and the expression of wg is lost in these parasegments only by approximately 6.5 hours of development (stage 10), nearly 2.5 hours after the specification of NB4-2 identity. Thus the loss of Wg expression from odd-numbered parasegments is well past the temporal requirement of wg for NB4-2 specification. Therefore, at least in the odd-numbered parasegments, the specification of NB4-2 identity in slp mutants must occur earlier than the decay of wg expression. Therefore, it is concluded that the loss of NB4-2 identity in slp mutants is unlikely due to the loss of wg expression, at the least in the odd-numbered parasegments, and possibly in the even-numbered parasegments as well (Bhat, 2000).

While the evidence to support the conclusion that slp genes regulate expression of wg in the epidermis is quite strong (Cadigan, 1994a and b), the evidence that the wg-signaling controls the expression of slp in the CNS is also equally strong. (1) The slp genes are expressed not only in the wg-expressing row 5 cells but also in the Wg-negative, but Wg-receiving row 4 cells. (2) The expression of slp is affected in wg mutant embryos. That is, staining of wg mutant embryos show that the expression of slp is lost from the Wg-receiving row 4 neuroectodermal cells. This result is also supported by the western analysis of embryo extract from wg mutants in which the level of Slp protein is found to be greatly reduced. (3) Consistent with the above result, the ectopic expression of wg induces ectopic expression of slp in the neuroectoderm. (4) In slp mutants, just as in wg mutants, the formation and identity specification of a well-studied neuronal precursor cell, NB4-2, is affected; this defect in wg mutants can be rescued by the expression of slp genes from a heterologous promoter. Moreover, a similar relationship also appears to exist between slp and wg during mesoderm specification. For instance, in both wg and slp mutants, the specification of heart cells (derived from mesoderm) is affected and this defect in wg mutants can be rescued by expressing slp genes from a heterologous promoter. These results therefore indicate that wg is a positive regulator of slp expression not only during neurogenesis but also in other processes such as mesoderm specification (Bhat, 2000).

An intriguing aspect of regulation of slp genes by wg is the finding that this regulation is restricted primarily to the neuroectoderm but not extended to the neuroblasts that are derived from these neuroectodermal cells, with one exception: the NB4-2. Thus, while row 4 neuroectodermal cells in wg mutant are missing slp1 expression, row 4 neuroblasts other than NB4-2 have slp1 expression. The induction/maintenance of slp1 expression in these neuroblasts must, therefore, necessarily be under the control of some other pathway. Alternatively, the Wg-signaling pathway is redundant in these neuroblasts. These results are consistent with the finding that the induction of an ectopic gsb-stripe by gain-of-function wg occurs only in the neuroectodermal cells but not in the underneath neuroblasts. In summary, these results reveal a hitherto unsuspected relationship between wg and slp in the CNS that is the opposite of their relationship in the epidermis (Bhat, 2000).

Using a temperature-sensitive allele of wg, it has been shown that the requirement of Wg in the CNS for NB4-2 formation and specification is between late stage 7 and early stage 8 and precedes Wg requirement for epidermal patterning. Moreover, it is the neuroectodermal expression of wg that regulates NB4-2 formation and identity specification. Thus, while the timing of decay of wg expression in slp mutants in even-numbered parasegments coincides with the requirements of wg for NB4-2 formation and specification, it is not so in the odd-numbered parasegments. Thus, the odd-numbered parasegments in slp mutants have wg expression during the time Wg is required for NB4-2 formation and specification. Since the loss of NB4-2 lineage in slp mutants is not parasegment-specific and the expression of slp in NB4-2 and its precursor neuroectodermal cells is lost in wg mutants, it must be that slp genes are downstream of wg in these CNS cells (Bhat, 2000).

The Wg signal regulates the specification of NB4-2 identity via Armadillo and Pangolin. The Arm-Pan signaling complex must activate certain downstream target gene(s), presumably transcription factors, and these transcription factors then initiate a program that mediates the formation and specification of NB4-2. slp genes function as downstream targets of the wg signaling, regulating both the NB4-2 formation as well as the identity specification during neurogenesis. This conclusion is based on the following facts: (1) the loss of function effect for slp genes has the same effect as the loss of function for wg on NB4-2 lineage; (2) the loss of wg activity in row 5 cells in the CNS leads to a loss of slp expression from the Wg-receiving NB4-2 and its precursor cell; (3) the loss of NB4-2 in wg mutants can be rescued by the expression of slp genes from a heterologous promoter during the time when wg is known to be required for the process. It is acknowledged that the slp genes might be either the direct targets of the Wg-signaling pathway (i.e. Arm-Pan complex directly activating slp genes), or instead there may be additional genes in between pan and the slp genes. While this issue has not been resolved here, the rescue of the NB4-2 lineage defect in wg mutants by expressing slp genes from a heterologous promoter reveals that the Wg-signaling pathway must ultimately activate slp genes, and the slp genes then regulate the formation and specification of NB4-2 (Bhat, 2000).

Determination of cell fate along the anteroposterior axis of the Drosophila ventral midline

The Drosophila ventral midline has proven to be a useful model for understanding the function of central organizers during neurogenesis. The midline is similar to the vertebrate floor plate, in that it plays an essential role in cell fate determination in the lateral CNS and also, later, in axon pathfinding. Despite the importance of the midline, the specification of midline cell fates is still not well understood. This study shows that most midline cells are determined not at the precursor cell stage, but as daughter cells. After the precursors divide, a combination of repression by Wingless and activation by Hedgehog induces expression of the proneural gene lethal of scute in the most anterior midline daughter cells of the neighbouring posterior segment. Hedgehog and Lethal of scute activate Engrailed in these anterior cells. Engrailed-positive midline cells develop into ventral unpaired median (VUM) neurons and the median neuroblast (MNB). Engrailed-negative midline cells develop into unpaired median interneurons (UMI), MP1 interneurons and midline glia (Bossing, 2006).

The determination of midline cells appears to take place during germband elongation, since by germband retraction most midline subsets can be identified by the expression of unique molecules. The anteroposterior position of midline siblings was determined during germband elongation. Midline precursors were labelled with the lipophilic dye DiD or DiI in embryos expressing GFP in the Engrailed domain (en-GAL4/UAS-tauGFP). After division of the precursors, the daughter cells were followed throughout development, recording their segmental position at stage 10 and stage 11. MP1 interneurons, UMI and MNB neurons each arise from one precursor, and their daughter cells occupy fixed anteroposterior positions during germband elongation. The four daughter cells of the two glial precursors can be located either in the middle of the segment or just anterior to the Engrailed domain. VUM neurons arise from three midline precursors, and the six daughter cells of these precursors are located inside the Engrailed domain and immediately posterior to the domain, in the anterior of the next segment (Bossing, 2006).

In summary, the midline glia and MP1 interneurons are the most anterior midline subsets, followed by a second pair of midline glia and a pair of UMIs, and, finally, the VUM and MNB neurons. DiI labelling cannot resolve whether the MP1 interneurons or the midline glia are the most anterior cells. Since determination of the MP1 interneurons depends on Notch/Delta signalling, it is possible that the anteroposterior position of the most anterior midline cells, the midline glia and MP1 interneurons, is random. Interestingly, four VUM neurons and the MNB neurons seem to arise from the anterior compartment of the next posterior segment. These cells initiate Engrailed expression half-way through germband elongation, and, during germband retraction, they join the adjacent anterior segment to become the most posterior midline subsets (Bossing, 2006).

The separation of midline cells into two compartments is an early and crucial step in midline cell determination. During germband elongation, a second phase of Engrailed expression is initiated at the midline in the anterior cells of the next posterior segment. During germband retraction, these cells join the anterior segment where they develop into posterior midline cells. Expression of late Engrailed depends on Hedgehog signalling and the proneural gene lethal of scute. Lethal of scute precedes Engrailed expression and is also activated by Hedgehog. Hedgehog and Wingless signalling counteract each other to define the position of the Lethal of scute cluster, and to divide the 16 midline daughter cells into eight non-Engrailed- and eight Engrailed-expressing cells (Bossing, 2006).

It has generally been believed that the determination of the different subsets of midline cells occurs before the precursors undergo their simultaneous division at stage 8. This view is challenged by the observation that expression of the proneural gene lethal of scute, and the subsequent expression of Engrailed, is initiated in midline daughter cells at stage 10, about one hour after the precursors divide. In the neuroectoderm, proneural genes confer neural competence to a cluster of ectodermal cells. Lateral inhibition by Notch/Delta signalling then limits the expression of proneural genes to a single cell, which delaminates from the ectoderm and becomes a neural precursor (neuroblast). Because the only neuroblast at the ventral midline (median neuroblast, MNB) originates from the proneural Lethal of scute cluster, it seems likely that the MNB is selected by lateral inhibition from a cluster of midline daughter cells. However, the process of lateral inhibition in the midline differs from that in the adjacent neuroectoderm. In the neuroectoderm, a single cell delaminates and the remaining cells of the cluster cease proneural expression and give rise to the epidermis. The proneural cluster in the midline consists of three pairs of siblings generated by the division of three separate precursors. Labelling of single precursors shows that, during the selection of the MNB, only one of the two labelled siblings enlarges, but both delaminate from the embryo. In contrast to the neuroectoderm, the remaining cells of the midline cluster continue to express Lethal of scute after delamination of the MNB. This extended proneural expression might be necessary to maintain neural competence in the non-delaminating cells that develop into VUM neurons (Bossing, 2006).

The results cannot exclude the possibility that some of the midline subsets are determined as precursors, but at least two of the five midline subsets, the VUM neurons and the MNB, are determined after precursor cell division. There are striking similarities between the development of the ventral midline of Drosophila and grasshopper embryos. In grasshopper, Engrailed expression can be detected in the MNB, its progeny and the midline precursors MP4 to MP6, which each give rise to two neurons with projection patterns comparable to the Drosophila VUM neurons. Hence, the same types of midline cells express Engrailed in grasshopper and Drosophila, but in grasshopper Engrailed expression is initiated in all midline precursors prior to division (Bossing, 2006).

In the ectoderm from stage 10 onwards, Wingless, Engrailed and Hedgehog maintain the expression of one another by a feedback loop: Wingless maintains Engrailed expression, Engrailed is needed for the expression of Hedgehog and Hedgehog maintains Wingless expression. In the developing CNS, Wingless and Hedgehog expression seem to be independent of each other. At the ventral midline there are two separate stages of Engrailed expression: the early phase is maintained by Wingless; the late phase does not require Wingless and is instead activated at stage 10 by Hedgehog signalling and Lethal of scute. In the ectoderm, Wingless and Hedgehog act in concert to maintain Engrailed expression, but at the midline Wingless and Hedgehog act in opposition: Wingless represses and Hedgehog activates Lethal of scute expression (Bossing, 2006).

Wingless may repress Lethal of scute expression indirectly, via its maintenance of early Engrailed. As in the ectoderm, midline Engrailed represses expression of the Hedgehog receptor Patched and the Hedgehog signal transducer Cubitus interruptus. It is possible that early Engrailed-expressing midline cells are not able to receive the Hedgehog signal. However, ectopic expression of Hedgehog is able to induce Lethal of scute in all midline cells, suggesting that Wingless may repress Lethal of scute by a yet unknown mechanism. Recently it has been reported that a vertebrate wingless orthologue, Wnt2b, can maintain the naïve state of retinal progenitors by attenuating the expression of proneural and neurogenic genes (Bossing, 2006).

The differentiation of midline cells was studyed in wingless and hedgehog mutants. Consistent with earlier reports, many midline cells become apoptotic in both mutants. The surviving midline cells are not integrated into the CNS and show no morphological differentiation. The reduction in the number of Engrailed-positive midline cells in hedgehog mutant embryos may be mainly due to the loss of midline cell identity. In hedgehog mutants, midline cells lose the expression of Sim, the master regulator of midline development. As described for sim mutants, the loss of midline identity results in increased cell death and misspecification of the surviving midline cells as ectoderm (Bossing, 2006).

Ectopic expression of Hedgehog in the neuroectoderm and the developing CNS induces the expression of Lethal of scute and, approximately 40 minutes later, the expression of late Engrailed in all midline cells. It seems likely that Lethal of scute is an early target of Hedgehog signalling, and its activation may only require release from repression by the short form of Cubitus interruptus. By contrast, the delay in induction of late Engrailed in the same midline cells indicates that Engrailed activation may not only require release from repression, but also activation by the long form of Cubitus interruptus (Bossing, 2006).

Uniformly high levels of ectopic Hedgehog prevent the differentiation of most midline subsets and cause increased cell death. A single source of ectopic Hedgehog, achieved by cell transplantation, does not result in midline cell death, but reveals that the differentiation of the MP1 interneurons is more sensitive to Hedgehog levels than is the differentiation of midline glia. No other midline subsets are affected. It seems likely that Hedgehog not only activates Lethal of scute and late Engrailed, but also acts as a morphogen to control the differentiation of the MP1 neurons and midline glia (Bossing, 2006).

The phenotypes caused by ectopic Hedgehog are due to the induction of Engrailed in all midline cells. Expression of ectopic Hedgehog and ectopic Engrailed blocks the differentiation of midline glia and MP1 interneurons, and also prevents the formation of the anterior commissure. Labelling single midline precursors enabled examination of cell fates in embryos expressing ectopic Engrailed in the midline. The frequency of clones obtained indicates that ectopic Engrailed expression does not transform non-Engrailed-expressing midline subsets (MP1 interneurons, midline glia and UMI) into Engrailed-expressing subsets (VUM and MNB). Instead, embryos expressing midline Engrailed show increased cell death. In particular, the MP1 interneurons seem to be affected and were never obtained during this analysis. The low frequency of midline glia also points to apoptosis caused by expression of Engrailed. Surviving midline glia are not able to differentiate properly and cannot enwrap the remaining, posterior, commissure. All other midline subsets, including the UMIs, are able to differentiate, but they show a variety of axonal pathfinding defects that may result from the loss of anterior midline subsets and the absence of the anterior commissure (Bossing, 2006).

It is likely that genes other than hedgehog and wingless are crucial for midline cell determination. In these experiments, non-Engrailed-expressing midline subsets are never transformed into Engrailed-expressing subsets, or vice versa. gooseberry-distal may be one of these genes. From the blastoderm stage, Gooseberry-distal is expressed by two midline precursors and their four daughter cells. During early embryogenesis Gooseberry-distal expression at the midline does not depend on Wingless and Hedgehog. The anterior Gooseberry-distal cells also express Wingless and most likely give rise to the UMIs. The posterior Gooseberry-distal pair also express early Engrailed and Hedgehog, and develop into the most anterior VUM neurons. At stage 10, Hedgehog activates the expression of Lethal of scute and Engrailed in midline cells posterior to the Gooseberry-distal domain. Lateral inhibition by Notch/Delta signalling selects one cell from the Lethal of scute cluster to become the MNB. The remaining cells become VUM neurons. At stage 10, the absence of Engrailed in the six midline cells anterior to the Gooseberry-distal domain defines a cell cluster that will give rise to midline glia and MP1 interneurons. Based on the expression of Odd, Delta mutants have an increased number of MP1 interneurons, up to six per segment. In Notch mutants, midline glial-specific markers are absent and the number of cells expressing a neuronal marker increases. Therefore, Notch/Delta signalling appears to determine midline glial versus MP1 interneuron cell fates in the anterior cluster. In the current model, midline cell determination takes place mainly after the division of the precursors. Although the initial determination of midline cells appears to be directed by a small number of genes, a far larger number is needed to control the differentiation of the various midline subsets. This work, and the recent identification of more than 200 genes expressed in midline cells, is the beginning of a comprehensive understanding of the differentiation of the ventral midline (Bossing, 2006).

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