The mutually exclusive expression patterns of Eve and Exex and the ability of Exex to repress Eve led to an investigation of whether Eve exhibits a reciprocal ability to repress Exex. Whether eve represses exex was tested by following Exex in eve1D mutant embryos. This temperature-sensitive allele allowed the circumvention of the early requirement for eve during embryonic segmentation. On average, two ectopic Exex-positive neurons were observed in each hemisegment of eve mutant embryos. The position of these neurons identifies one as RP2 and the other as likely aCC or pCC. Therefore, eve exhibits a reciprocal ability to repress exex in a subset of dorsally projecting MNs (Broihier, 2002).
During segmentation, Eve has been shown to act as a transcriptional repressor and contains two domains with repressive capability -- one dependent on the corepressor Groucho (Gro) and one Gro independent. To determine whether Eve requires Gro to repress Exex in the CNS, Exex expression was assayed in eve null embryos that contain an eve transgene deleted for the Gro-interaction domain. In these embryos, Exex is derepressed in RP2 and one of the corner cells. Since this phenotype is essentially identical to that of eve1D mutants, it is concluded that Eve represses Exex in a Gro-dependent manner. These results demonstrate that Eve/Evx proteins act through Gro to regulate cell fate in the CNS (Broihier, 2002).
To investigate if Eve is also sufficient to repress Exex, Eve was misexpressed in all postmitotic neurons. In these embryos, Exex expression is abolished, demonstrating that Eve is a potent repressor of Exex expression in the CNS. Thus, Eve is both necessary and sufficient to repress Exex. Taken together, these genetic studies demonstrate crossrepressive interactions between exex and eve function to delimit the expression of Exex to ventral and lateral MNs—and Eve to dorsal MNs. Since both Exex and Eve are key cell fate determinants, this mutually repressive relationship likely helps to consolidate distinct MN fates (Broihier, 2002).
Nervous system-specific eve mutants were created by removing regulatory elements from a 16 kb transgene capable of complete rescue of normal eve function. When transgenes lacking the regulatory element for either RP2+a/pCC, EL or U/CQ neurons were placed in an eve-null background, eve expression was completely eliminated in the corresponding neurons, without affecting other aspects of eve expression. Many of these transgenic flies are able to survive to fertile adulthood. In the RP2+a/pCC mutant flies: (1) both RP2 and aCC show abnormal axonal projection patterns, failing to innervate their normal target muscles; (2) the cell bodies of these neurons are positioned abnormally; and (3) in contrast to the wild type, pCC axons often cross the midline. The Eve HD alone is able to provide a weak, partial rescue of the mutant phenotype, while both the Groucho-dependent and -independent repressor domains contribute equally to full rescue of each aspect of the mutant phenotype. Complete rescue is also obtained with a chimeric protein containing the Eve HD and the Engrailed repressor domain. Consistent with the apparent sufficiency of repressor function, a fusion protein between the Gal4 DNA-binding domain and Eve repressor domains is capable of actively repressing UAS target genes in these neurons. A key target of the repressor function of Eve is Drosophila Hb9 (Extra-extra), the derepression of which correlates with the mutant phenotype in individual eve-mutant neurons. Finally, homologs of Eve from diverse species are able to rescue the eve mutant phenotype, indicating conservation of both targeting and repression functions in the nervous system (Fujioka, 2003).
The requirement for Eve in axonal guidance is somewhat more stringent in aCC than in RP2 neurons. Although a significant fraction of mutant RP2s initially extend axons in the same direction as wild-type RP2s, essentially none of mutant aCCs do so. In addition, unlike for RP2s, the aCC phenotype is not significantly rescued by either the HD alone or the HD with the N terminus (which provides no detectable repression activity, but might stabilize the protein). In aCC, as in RP2, the phenotype is partially rescued by including either repressor domain, and the Engrailed repressor domain is able to provide full activity. Furthermore, Eve repressor domains are able to actively repress a UAS target gene in aCC neurons. These data indicate that the primary function of eve in aCC is to actively repress target genes. The more stringent requirements in aCC versus RP2 suggest that there may be different target genes in these two motoneurons, although Drosophila Hb9 is a common target (Fujioka, 2003).
Drosophila Hb9 and Eve are expressed in a non-overlapping pattern in the wild-type CNS, and ectopic Eve expression represses Hb9, indicating that Hb9 is a target gene of Eve. Hb9 is derepressed in the RP2 mutant in both RP2 and aCC, but not in pCC neurons (the RP2 mutant lacks Eve in all three cell types), showing that there are significant differences in target gene regulation in different neurons, even in those derived from the same GMC (in the case of aCC and pCC) (Fujioka, 2003).
When the Eve HD alone is used to rescue the RP2 mutant, Hb9 is repressed in many of the RP2 neurons, and this seemingly stochastic repression correlates with a more normal axonal morphology. However, effective repression, particularly in aCC, requires active repression domains, with either of the repressor domains of Eve alone providing partial activity (in the context of the Eve HD). Although there is a strong correlation in situations of partial rescue between the axonal phenotypes of individual neurons and derepression of Hb9, this correlation is not 100%. This suggests that there may be other key target genes that mediate Eve neuronal function in addition to Hb9. The level of expression of the antigen (Futsch) of the monoclonal antibody 22C10 is reduced in RP2 and aCC in the absence of Eve. However, the gene encoding this antigen is likely to be an indirect target of Eve, because its expression is activated rather than repressed by Eve (Fujioka, 2003).
Either of the repressor domains of Eve is sufficient to give a similar degree of partial rescue of each of the phenotypes studied in the nervous system, including the repression of Hb9, showing that these repressor domains provide a similar function. In fact, two copies of a transgene expressing either EveDeltaC or EveDeltaR are able to rescue to a similar degree as that of one copy of the wild-type transgene. Thus, the recruitment of either of two apparently distinct co-repressors, Groucho or Atrophin, produces the same net result. The two are used in these neurons in an additive fashion to generate the appropriate level of Eve repressor activity, with no apparent target gene specificity (Fujioka, 2003).
exex mutant embryos display several ectopic Eve-positive neurons. Using the protein-positive ExexJJ154 allele, it was found that these ectopic Eve cells arise from cells that normally express Exex, suggesting that Exex represses Eve cell autonomously. The nonoverlapping expression patterns of Exex and Eve further indicate that Exex acts operationally as an Eve repressor in the CNS. To investigate whether Exex is sufficient to repress Eve, Exex was misexpressed in all postmitotic neurons, and it was found that Exex represses Eve in all Eve-positive neurons except the EL neurons. By late stage 14, only one or two weakly Eve-positive neurons remain in the positions normally occupied by the U, RP2, a/pCC, and fpCC neurons, while the cluster of Eve-positive EL interneurons appears normal. Thus, Exex expression is sufficient to repress Eve expression in all dorsally projecting MNs. The inability of Exex to repress Eve expression in the ELs suggests that the relative ability of Exex to repress Eve is controlled by factors expressed specifically in different neuronal types (Broihier, 2002).
During analysis of Lim3 expression in exex mutant embryos, the presence of a group of ectopic Lim3-positive neurons was observed. Since all Exex-positive neurons normally coexpress Lim3, the presence of ectopic Lim3-positive neurons suggests a cell-nonautonomous effect of Exex on the regulation of Lim3. Surprisingly, double label experiments identify the ectopic Lim3-positive neurons as the six Eve-positive U MNs. This phenotype was visualized using a lim3-tau-myc transgene due to the perdurance of transgene expression in U MNs relative to the more transient expression of endogenous Lim3 in these cells. The transient nature of Lim3 expression in the U MNs is attributed to the ability of Eve to repress Lim3(Broihier, 2002).
The ectopic expression of Lim3 in the U MNs in exex mutants is exciting because neither the U MNs nor their progenitors ever express Exex. These data further support the model that Exex acts cell nonautonomously to repress Lim3 expression in the U MNs. Consistent with this, several groups of Exex-positive neurons surround the U MNs during their development. One or more of these groups of Exex-positive neurons likely serves as the source of the signal received by the U MNs. Taken together, these results uncover a novel role for intercellular signaling in the establishment of neuronal fate in Drosophila (Broihier, 2002).
The homeodomain protein Nkx6 is a key member of the genetic network of transcription factors that specifies neuronal fates in Drosophila. Nkx6 collaborates with the homeodomain protein Hb9/ExEx to specify ventrally projecting motoneuron fate and to repress dorsally projecting motoneuron fate. While Nkx6 acts in parallel with hb9 to regulate motoneuron fate, Nkx6 plays a distinct role to promote axonogenesis; axon growth of Nkx6-positive motoneurons is severely compromised in Nkx6 mutant embryos. Furthermore, Nkx6 is necessary for the expression of the neural adhesion molecule Fasciclin III in Nkx6-positive motoneurons. Thus, this work demonstrates that Nkx6 acts in a specific neuronal population to link neuronal subtype identity to neuronal morphology and connectivity (Broihier, 2004).
In many model systems, MNs that extend axons along common trajectories express similar sets of transcriptional regulators, which in turn regulate key aspects of the differentiation of these MN subtypes. Drosophila MNs are classified by the location of the body wall muscles they innervate. MNs that innervate dorsal body wall muscles in Drosophila express the homeodomain (HD) transcription factor Even-skipped (Eve). Furthermore, genetic analyses indicate that Eve is a key determinant of the fate of dorsally projecting MNs. Eve engages in a cross-repressive interaction with the HD protein Hb9, a determinant of ventrally projecting MNs (Broihier, 2004 and references therein).
Ventrally projecting MNs also express the HD transcription factors Lim3 and Islet. Functional analyses have demonstrated that these three HD factors are required for proper axon guidance of ventrally projecting MNs. The genetic hierarchy governing the fate of ventrally projecting neurons has, however, remained elusive as Lim3, Islet, and Hb9 are expressed independently of each other (Broihier, 2004 and references therein).
To explore the genetic networks behind neuronal diversification in Drosophila, the role of the Drosophila Nkx6 homolog in regulating distinct MN fates was investigated. Genetic interactions were characterized between Nkx6 and factors essential for neuronal fate acquisition. Evidence that Nkx6 collaborates with hb9 (exex – FlyBase) to regulate the fate of distinct neuronal populations. This analysis of hb9 Nkx6 double mutant embryos indicates that ventrally projecting MNs fail to develop properly in these embryos, while expression of eve, a key determinant of dorsally projecting MN identity, expands. In addition, Nkx6 promotes axonogenesis of Nkx6-positive neurons. Consistent with a direct regulatory role in this process, Nkx6 activates the expression of the neural adhesion molecule Fasciclin III in ventrally projecting motoneurons. These data suggest that Nkx6 is a primary transcriptional regulator of molecules essential for axon growth and guidance in a specific neuronal population (Broihier, 2004).
The findings that Nkx6 has roles in both the specification and differentiation of ventrally projecting MNs places Nkx6 in the regulatory circuit that specifies distinct postmitotic neuron fates in the Drosophila CNS. In the mouse, Nkx6 protein function in MN progenitors regulates Hb9 expression in postmitotic MNs. Drosophila Nkx6 is expressed in neural precursors and postmitotic neurons while Hb9 expression is nearly exclusive to postmitotic neurons. However, in contrast to the linear relationship of Nkx6.1/2 and Hb9 in vertebrates, Nkx6 and hb9 were found to act in parallel to specify neuronal fate in Drosophila. Nkx6 and hb9 act in concert both to repress expression of the dorsal MN determinant Eve and to promote expression of Lim3 and Islet in ventrally projecting RP MNs. It will be of interest to extend this genetic analysis to other groups of ventrally projecting MNs. It will be also be important to examine the directness of these genetic interactions. Both Nkx6 and hb9 contain conserved TN domains that in vertebrate HD proteins have been shown to interact with the Groucho co-repressor, suggesting that Nkx6 and hb9 function as transcriptional repressors. This raises the possibility that Nkx6 and hb9 bind to sequences in the eve enhancer and directly repress its transcription. In addition, Nkx6 and hb9 activate lim3/islet gene expression within ventrally projecting MNs, raising the possibility that they do so by repressing an unidentified repressor of ventrally projecting MN identity (Broihier, 2004).
eve represents an appealing candidate for the unidentified repressor in this model. Ectopic Eve expression in RP MNs in hb9 Nkx6 double mutants may repress Lim3 and Islet. Consistent with this, though it was not possible to unambiguously identify the ectopic Eve neurons in hb9 Nkx6 mutants, many of them are situated close to the midline, suggesting they may represent mis-specified RP MNs. Furthermore, pan-neuronal eve expression represses Lim3 and Islet expression in the RP MNs demonstrating that Eve can repress Lim3 and Islet (Landgraf, 1999). A direct test of this model will require resolving the identity of the ectopic Eve neurons in hb9 Nkx6 mutant embryos (Broihier, 2004).
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