BIOLOGICAL OVERVIEW

dHb9 (FlyBase designation: Extra-extra [Exex]), the Drosophila homolog of vertebrate Hb9, encodes a factor central to motorneuron (MN) development. Exex regulates neuronal fate by restricting expression of Lim3 and Even-skipped (Eve), two homeodomain (HD) proteins required for development of distinct neuronal classes. Exex and Lim3 are activated independently of one another in a virtually identical population of ventrally and laterally projecting MNs. Surprisingly, Exex represses Lim3 cell nonautonomously in a subset of dorsally projecting MNs, revealing a novel role for intercellular signaling in the establishment of neuronal fate in Drosophila. Evidence is provided that Exex and Eve regulate one another's expression through Groucho-dependent crossrepression. This mutually antagonistic relationship bears similarity to the crossrepressive relationships between pairs of HD proteins that pattern the vertebrate neural tube (Broihier, 2002).

To identify genes required for proper neuronal fate specification in the Drosophila embryonic CNS, EMS saturation mutagenesis of the third chromosome was conducted and a screen was performed for changes in the CNS expression pattern of Eve. Changes in Eve expression were assayed because Eve is expressed in a stereotyped pattern of eight dorsally projecting MNs and 12 interneurons in each abdominal hemisegment, and because eve is a known regulator of neuronal fate (Broihier, 2002).

Four alleles of exex were identifed. exex mutant embryos display a highly specific phenotype in which two ectopic Eve-expressing neurons develop per hemisegment. These ectopic Eve-positive neurons appear during late stage 11 in the vicinity of the Eve-positive neurons aCC/pCC. By stage 14, one ectopic Eve-expressing neuron is found adjacent to aCC/pCC, while the other migrates posteriorly and laterally to adopt a stereotyped mediolateral position (Broihier, 2002).

To examine more closely the cell fate changes that occur in exex mutant embryos, the lineal origin of the ectopic Eve-positive neurons was determined. Since in exex mutants, the ectopic Eve-expressing neurons arise immediately adjacent to the sibling aCC/pCC neurons, it was hypothesized that, like aCC/pCC, the ectopic Eve-positive neurons derive from the NB1-1 lineage. To test this, assays were carried out to determine whether an Eve-ß-gal reporter gene normally expressed solely by the aCC/pCC and RP2 neurons is also expressed by the ectopic Eve-positive neurons in exex mutant embryos. In support of the model, both ectopic Eve-positive neurons express ß-gal in exex mutant embryos, indicating that the ectopic Eve-positive neurons likely arise within the NB1-1 lineage. These data indicate that exex regulates neuronal fate by repressing eve expression in the NB1-1 lineage (Broihier, 2002).

These data support a role for exex in the cell-autonomous and nonautonomous regulation of several factors required for the development of distinct neuronal fates. Expression of murine Hb9, a vertebrate homolog of exex is restricted to MNs whose axons exit from the ventral side of the neural tube (v-MNs) (Thaler, 1999). v-MNs and V2 interneurons arise from common progenitors characterized by coexpression of Lim3 and Gsh4 (Sharma, 1998). This shared lineage necessitates the presence of factors that differentiate v-MNs and V2 interneurons. Hb9 activity contributes to the v-MN/V2 interneuron distinction, since V2 interneuron-specific gene expression is derepressed in Hb9 mutant mice (Arber, 1999; Thaler, 1999). Interestingly, MNs whose axons emerge from the dorsal side of the neural tube (d-MNs) and arise from an MN-specific progenitor pool do not require Hb9 function (Thaler, 1999). The restriction of Hb9 expression to those MNs arising from Lim3/Gsh4-positive progenitors suggests that Hb9 function is required only in MNs that need to actively suppress an alternate genetic program (Broihier, 2002 and references therein).

In Drosophila, many NBs produce both MNs and interneurons, suggesting a widespread requirement for factors that function to arbitrate between alternate genetic programs. The data suggest that exex acts cell autonomously to repress Eve in neurons in the NB1-1 lineage, whereas exex acts cell nonautonomously to repress lim3 in dorsally projecting U MNs. Inappropriate expression of eve and lim3 in exex mutants is consistent with exex contributing to proper neuronal fate by suppressing the expression of key determinants of neuronal identity. These results also hint at the possibility that Exex regulates cell fate in a manner analogous to its vertebrate homologs (Broihier, 2002).

Several cell fate changes have been characterized in exex mutant embryos, and these phenotypes have been paired with exex function in distinct neurons. However, Exex is expressed in approximately 30 neurons, and regulatory targets have been identified in only a handful of these cells, strongly suggesting that additional targets exist. Given the enormous complexity of the genetic regulatory network that dictates neuronal fate, the power of Drosophila genetics should provide an indispensable tool for identifying Exex-interacting genes -- as well as other key determinants of neuronal identity (Broihier, 2002).

In the vertebrate neural tube, Hb9, Lim3/4, and Isl1 are elements of a combinatorial code directing neuronal identity and axonal pathfinding (Arber, 1999; Sharma, 1998; Thaler, 1999). Hb9 and Lim3/4 have been shown to be expressed in all MNs exiting the neural tube ventrally, though Lim3/4 are only transiently expressed in these MNs. In the Drosophila CNS, functional analysis and reporter construct expression data have supported roles for Lim3 and Islet in regulating the projections of ventrally projecting neurons. Islet expression has been proposed to be required for the identities of ISNb and ISNd MNs, while Lim3 expression in only ISNb MNs is thought (Thor, 1997; Thor, 1999) to resolve ISN neurons into ISNb and ISNd classes (Broihier, 2002).

This analysis of the Lim3 protein expression pattern argues against its proposed role in distinguishing ISNb trajectories from those of ISNd. Lim3 is expressed much more broadly than suggested by a lim3 reporter gene (Thor, 1999). Lim3 is coexpressed with Exex in five of the six major motor axon branches. In addition, Lim3 but not Exex is expressed in the TN motor axon branch (Thor, 1999). Lim3 is then expressed in neurons that populate all motor axon branches. Thus, differential expression of Lim3 is insufficient to explain how neurons choose between ISNb and ISNd (Broihier, 2002).

One question that then arises is why the motor axon phenotypes of exex and lim3 mutants are specific to the ISNb nerve branch when these factors are expressed widely in MNs. It is possible that the ISNb is generally more sensitive to genetic perturbations than other motor axon branches. Consistent with this, guidance molecules with broad CNS expression patterns display motor axon phenotypes largely confined to ISNb. Alternatively, the axonal phenotypes may be ISNb specific because Exex and Lim3 are expressed in a higher percentage of ISNb-projecting neurons than neurons projecting in other nerve branches. For example, eight MNs that project dorsally in the ISN are Eve positive and Exex/Lim3 negative (Broihier, 2002).

While these data argue against the simple combinatorial code proposed to regulate axon pathway choice, it is still certainly true that a neuron's fate is established largely by the combination of transcription factors it expresses. However, the fact that Exex, Lim3, and Isl are coexpressed in a large number of neurons with different identities indicates that individual neuronal identities are not defined by the mere presence or absence of these factors. Clearly additional as yet unidentified factors are required to create the tremendous cellular diversity found in the CNS (Broihier, 2002).

Additional layers of complexity also likely exist within the combinatorial code. For example, the levels and timing of expression of individual transcription factors may play important roles in directing different cellular fates. Consistent with this possibility, while Exex and Lim3 have largely overlapping expression patterns, their relative levels and duration of expression vary between neurons. The data establish that these two factors act largely in parallel to establish neuronal identity. It will therefore be critical to determine whether Exex and Lim3 act independently on distinct targets or together as members of one transcriptional complex. In this context, it is possible that changes in the relative levels of Exex/Lim3 would alter the composition and functional properties of these complexes. Clearly, future research that identifies additional genes with roles in neuronal fate determination and integrates their functions into the regulatory network that controls neuronal diversity will provide a more lucid picture of the genetic and molecular basis of neuronal diversity (Broihier, 2002).

The data demonstrate that a crossinhibitory interaction between Exex and Eve contributes to their mutually exclusive expression patterns -- Eve is expressed in dorsally projecting MNs, and Exex is expressed in more ventrally projecting MNs. Furthermore, functional studies demonstrate that Eve and Exex regulate axonal trajectories of dorsally and ventrally projecting axons, respectively. Together, these results suggest that the crossrepressive relationship between Exex and Eve helps to ensure that neurons in these two populations acquire distinct identities (Broihier, 2002).

The mutual antagonism of Eve and Exex is similar to the relationship between pairs of HD factors whose crossrepressive interactions are central to neural tube patterning. In the vertebrate neural tube, domains of HD protein expression in distinct progenitor domains are established in response to a Shh gradient. Crossrepressive interactions between these HD factors then appear to refine and maintain the progenitor domains. These proteins likely function as transcriptional repressors and may require the corepressor Groucho (Gro) (Broihier, 2002).

The results suggest that Eve and Exex also mediate their crossrepressive interaction in a Gro-dependent manner. The ability of Eve to repress Exex depends on its Gro-interaction domain, implicating Gro in the Eve side of this crossinhibitory interaction. In support of the idea that Exex acts through Gro to repress Eve, a potential Gro-interaction domain has been identified in Exex. Clearly, the significance of this conserved domain with respect to Exex function must be tested in vivo. Nonetheless, these results highlight the significant mechanistic conservation of neuronal fate specification between Drosophila and vertebrates (Broihier, 2002).

The mutually exclusive expression patterns of Eve and Exex arise in part through a crossinhibitory interaction between the two proteins. exex mutant embryos display several additional Eve-positive neurons, and eve mutants exhibit several additional Exex-positive neurons, arguing that the Eve and Exex expression patterns are established largely independently and then refined by the mutually repressive interaction. In the future, it will be important to identify upstream regulators of eve and exex to understand the manner in which these distinct patterns of gene expression arise. Research in this area is likely to be of general relevance since in Drosophila and vertebrates, Hb9 and Lim3 are coexpressed in nearly identical populations of MNs (Arber, 1999; Thaler, 1999; Broihier, 2002). These data argue that Hb9/Lim3-positive MNs constitute an evolutionarily conserved MN population. Given this, significant overlap is expected between the upstream regulators of Exex/Lim3 in Drosophila and vertebrates (Broihier, 2002).

GENE STRUCTURE

cDNA clone length - 2355

Bases in 5' UTR - 175

Exons - 3

Bases in 3' UTR - 692

PROTEIN STRUCTURE

Amino Acids - 525

Structural Domains

Comparative sequence analysis indicates exex codes for the Drosophila homolog of the vertebrate HD proteins MNR2/Hb9. Within the HD, Exex is 90% identical and 95% similar to MNR2/Hb9. The next most closely related Drosophila HD protein, Deformed, shares only 68% identity with Hb9/MNR2, indicating that exex is the sole Hb9/MNR2 homolog in Drosophila. Outside the HD, the largest region of homology between Exex and Hb9 bears some sequence similarity to the TN domain of Nkx and Dbx class HD proteins. The TN domain has been shown to mediate the repressive ability of these proteins and to interact with the Groucho corepressor. These data and the requirement of exex to repress eve suggest dHb9 functions as a transcriptional repressor during CNS development (Broihier, 2002).

date revised: 2 September 2002

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