See the embryonic expression pattern of exes at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
To investigate the role of Exex during CNS development, Exex-specific antibodies were raised. Embryonic expression of Exex initiates in the posterior midgut primordium at stage 7. By stage 9, Exex protein is present in the primordia of the anterior and posterior midgut and persists in anterior and posterior regions of the endoderm throughout embryogenesis. In the CNS, Exex protein expression is first detected during stage 11 in one-to-two mitotic GMCs and approximately 15 neurons per hemisegment. Exex expression in the CNS peaks at stage 14, when it is strongly expressed in approximately 30 neurons per hemisegment, including the well-characterized RP1, RP3-5 MNs, and dMP2 and MP1 interneurons. Thus, in the CNS, Exex expression is expressed almost exclusively in a distinct population of postmitotic MNs and interneurons, consistent with Exex regulating neuronal identity (Broihier, 2002).
A key distinguishing trait of neurons is their axonal trajectory. Thus, the axonal trajectories of Exex-positive neurons were traced to investigate whether Exex identifies specific subpopulations of CNS neurons. To create an Exex-dependent axonal marker, targeted transposition was employed to convert a exexLacZ enhancer trap to a exexGal4 enhancer trap. The exexGal4 driver was used to express GAP-GFP and it was confirmed that GFP expression faithfully recapitulates the Exex expression pattern with the exception that the peripheral LBD neuron expresses ExexGal4 but not Exex. Within the CNS, it was found that Exex-positive interneurons project axons in three distinct longitudinal fascicles (Broihier, 2002).
The trajectory of Exex-positive MNs was traced into the periphery and it was found that Exex-positive neurons populate five of the six motor axon branches. In Drosophila, motor axons exit the CNS in the ISN, SN, and transverse nerve (TN). The main branch of the ISN innervates the dorsal and lateral body wall musculature. Axons in two branches of the ISN (ISNb and ISNd) defasciculate from the ISN to innervate distinct groups of ventral body wall muscles. Similarly, the primary branch of the SN, SNa, innervates a lateral muscle group, and axons in its minor branch, SNc, extend along SNa until their choice point where they defasciculate and innervate ventral muscles. Exex-positive motor axons were found to extend in the ISN, ISNb, ISNd, Sna, and SNc nerves. While Exex-positive motor axons are observed in the ISN, they do not project to the most dorsal muscle regions. In addition, no Exex-positive axons were detected in the TN. These data demonstrate that Exex-positive axons populate five of the six major nerve branches and that Exex is expressed in the majority of ventrally and laterally projecting MNs. Interestingly, dorsally projecting MNs express Eve, but apparently not Exex, suggesting that Exex and Eve identify distinct populations of MNs (Broihier, 2002).
Expression of HB9 mRNA and protein is indistinguishable during embryogenesis and is restricted to the gut and CNS. Endoderm expression is conserved from sea urchins to vertebrates, and neuronal expression is conserved from amphioxus to vertebrates. Drosophila HB9 CNS expression begins during late stage 10 in ~12 cells per hemisegment and increases to ~30 cells per hemisegment at stage 15 and later. None of these cells express the glial marker Repo, indicating that all HB9 cells are neurons or GMCs. The timing of expression of HB9 and the size of cells expressing HB9 indicate that cells are likely to be postmitotic. To confirm this, a double-labeling of HB9 and phosphohistone H3, a marker for cells undergoing mitosis, was performed. One or two cells were found in stereotyped mediolateral positions that express phosphohistone H3 and express HB9 weakly; it is concluded that HB9 can be expressed just before GMC division but is usually restricted to postmitotic neurons. HB9 is also detected in the third instar larval brain (Odden, 2002).
On the basis of vertebrate studies, it was expected that HB9 would be expressed specifically in motoneurons. Thus, embryos were double-labeled for HB9 and various motoneuron markers, including Eve (labels all dorsally projecting motoneurons), Islet-Tau:myc (labels all ventrally projecting motoneurons), and Eagle (labels a subset of ventrally projecting motoneurons). HB9 was found to be coexpressed widely with Islet in most ventrally projecting neurons, including the well characterized RP1, RP3, RP4, and RP5 motoneurons that project via segmental nerve B (SNb) and segmental nerve D (SNd) to ventral muscles 6, 7, 12, and 13. To help distinguish exactly which Islet+ motoneurons are HB9+, a lim3:lacZ transgene was used to identify all Islet-positive motoneurons that project via SNb. HB9 is coexpressed with Lim3 and Islet in the four RP motoneurons, two lateral SNb-projecting motoneurons, and a single ventral neuron that has not been described previously. HB9 is not expressed in all ventrally projecting motoneurons, however, because it is not detected in the Eagle+ GW motoneuron that projects to muscle 15 or the Eagle+ motoneuron derived from neuroblast 2-4 that projects via segmental nerve a (SNa) to muscle 8. In addition, HB9 is not expressed in any of the Eve+ motoneurons that innervate dorsal muscle targets. Thus, HB9 is not expressed in all motoneurons but rather is restricted to a small subset of motoneurons that includes those projecting via SNb and SNd to ventral muscle targets (Odden, 2002).
To determine whether HB9 expression is limited to motoneurons like its counterparts in vertebrates, expression was examined in several identified interneurons. The best-characterized interneurons in the CNS are the serotonergic interneurons derived from neuroblast 7-3, for which there are numerous markers. Using several of these markers in combination with HB9, it was found that HB9 is expressed in the two serotonergic interneurons (EW1/EW2) and a third lineally related interneuron (EW3); each of these interneurons projects contralaterally across the posterior commissure. In addition, FasII, a transmembrane protein that labels all motoneurons and a few interneurons, was assayed, reasoning that any HB9+, FasII- cells would be interneurons. Only a ventral cluster of six HB9+ cells fails to express FasII, indicating that these HB9+ cells are interneurons. In summary, HB9 is expressed in at least nine interneurons (three Islet+ Lim3- Eagle+ EW neurons and a ventral cluster of six Islet- Lim3- Eagle- FasII- neurons) (Odden, 2002).
The ability of exex to regulate neuronal fate by repressing eve places exex within the genetic regulatory network that governs neuronal fate. To begin to illuminate the role exex plays in this network, exex was characterized at the molecular level. Standard meiotic mapping positioned exex between ru and h on the genetic map, and deficiency analysis localized exex to cytological position 66B1-2. The subsequent completion of sequencing of the Drosophila genome facilitated a candidate gene approach to identify exex. Predicted genes in the region were screened for a CNS expression pattern by RNA in situ hybridization, and one gene, CG8254, expressed in the embryonic CNS, was identified. To determine if this gene corresponds to exex, the CG8254 coding region was sequenced from larvae homozygous for each exex allele. It was found that each exex allele contains a distinct nonsense mutation in the CG8254 coding region. These data and the finding that exexKK30 homozygous mutant embryos fail to produce detectable Exex protein demonstrate that the exex locus corresponds to CG8254 (Broihier, 2002).
The identical phenotypes of ExexKK30 homozygous and ExexKK30/Df(pblNR) transheterozygous embryos identify ExexKK30 as a null allele. Interestingly, Exex protein is present at wild-type levels in embryos homozygous for ExexJJ154, an allele predicted to encode the entire protein except the C-terminal 32 amino acids. Since ExexJJ154 embryos exhibit similar, albeit more severe, CNS phenotypes than ExexKK30embryos, the ExexJJ154 allele likely has dominant-negative activity (Broihier, 2002).
The widespread expression of Exex in MNs suggested that exex regulates MN differentiation. To address this, MAb 1D4 against Fasciclin II was used to visualize MN projections in embryos mutant for the null allele, ExexKK30. The overall organization of motor axon projections is normal, and pathfinding aberrations were not detected in either SN branch or in the ISN or ISNd. However, the ISNb branch exhibits two predominant phenotypes both resulting in a lack of innervation of the ventral muscle field. In 41% of hemisegments, the ISNb defasciculates from the ISN and enters the ventral musculature, where the axons stall and growth cones accumulate. In 19% of hemisegments, the ISNb fails to defasciculate from the ISN and extends dorsally with the ISN. Since Exex is expressed in the ISNb-projecting RP MNs, the aberrant pathfinding of ISNb in exex mutants suggests that exex promotes the differentiation of these neurons (Broihier, 2002).
Loss-of-function analysis indicates that exex is necessary for the proper axonal trajectories of a subset of ventrally projecting MNs. To test whether Exex misexpression is sufficient to reroute motor axons, Exex was misexpressed via the UAS/Gal4 system. Embryos in which Exex is misexpressed in all postmitotic neurons via the elavGal4 driver display highly penetrant axonal phenotypes. In these embryos, all motor axons fuse with the ISN prior to exiting the CNS. Thus, only a single nerve branch, a thickened ISN, forms in these embryos. The thickness of the ISN decreases dramatically in the lateral muscle region, suggesting that most axons acquire a laterally projecting ISN identity. Consistent with this, the ISN terminates prematurely in the dorsal body wall and often branches aberrantly in this region. The defects in dorsal MN projections likely arise as a result of the ability of Exex misexpression to abolish Eve in dorsally projecting MNs. It is concluded that Exex misexpression forces MNs to acquire an ISN-projecting identity and preferentially induces these MNs to project to the lateral body wall region. In combination with the loss-of-function analysis, these data demonstrate that proper levels of Exex activity are required to direct the normal pattern of motor axon outgrowth (Broihier, 2002).
The ISNb MN phenotypes of Exex exhibit similarity to those of Lim3 and Islet. Lim3 and Islet are two LIM-HD proteins that are required for the development of ISNb-projecting axons (Thor, 1997; Thor, 1999). As noted, ISNb-MNs express Exex and require Exex function for their differentiation, suggesting that Exex might interact with Lim3 and Islet to regulate neuronal fate. To investigate this, the relative expression patterns and genetic interactions between these genes were examined. To this end, Lim3- and Islet-specific antibodies were generated because prior expression analyses of Lim3 and Islet used gene-specific reporter constructs (Thor, 1997; Thor, 1999) and such reporter constructs often identify only a subset of a gene's expression profile (Broihier, 2002).
It was found that Lim3 is expressed in about 40 neurons per hemisegment -- this is many more neurons than previously identified by reporter gene expression. Of particular interest, Lim3 is expressed in all Exex-positive neurons as well as in several lateral Exex-negative neurons, including the Eve-positive EL interneurons. Therefore, like Exex, Lim3 is expressed in MNs projecting in the primary and secondary branches of both the SN and ISN. Since previous work has demonstrated that Lim3 is expressed in the TN nerve (Thor, 1999), it is concluded that Lim3 is expressed in all motor axon branches. These results suggest that all ventrally and laterally projecting MNs may express Lim3 (Broihier, 2002).
Despite the near identity of the Exex and Lim3 expression patterns, Exex and Lim3 do not activate each other's expression in these cells. Exex expression initiates normally in lim3 mutants and Lim3 expression in Exex-expressing cells also initiates normally in exex mutants. These data demonstrate that Exex and Lim3 are activated independently of one another in coexpressing cells and suggest that they act in parallel to specify neuronal identity. In addition, the striking similarity of the Exex and Lim3 expression patterns suggests coregulation of Lim3 and Exex by a largely overlapping set of transcriptional regulators (Broihier, 2002).
More limited overlap is found in the expression patterns of Exex and Islet. Islet is expressed in roughly 30 neurons per hemisegment, the majority of which are located laterally in the CNS. Exex and Islet are coexpressed in three discrete neuronal populations: the medial ISNb-projecting RP MNs, a pair of mediolateral interneurons corresponding to the serotonergic interneurons of the CNS, and a compact cluster of six lateral neurons. As observed for Exex and Lim3, Exex and Islet do not regulate each other's expression -- Islet expression is normal in exex mutant embryos and Exex expression is normal in isl mutant embryos. These results indicate that exex and isl do not fall into a simple linear hierarchy and suggest they act in parallel to specify neuronal fate (Broihier, 2002).
To investigate whether exex and Islet act in parallel, isl;exex double mutants were constructed and axonal organization was analyzed in these embryos. isl or exex single mutant embryos exhibit no overt defects in the overall architecture of the CNS. In contrast, isl;exex double mutant embryos exhibit clear defects in the organization of the axonal scaffold. For example, the anterior and posterior commissures are thinner than in wild-type and frequently only one commissure forms per segment. In addition, the longitudinal connectives are thinner than in wild-type and often veer toward or away from the midline (Broihier, 2002).
The defects in axonal organization in isl;exex double mutants have suggested these embryos might exhibit pronounced defects in motor axon projections. Whereas the axonal phenotypes of both single mutants are confined to the ISNb nerve branch, double mutant embryos display widespread defects. In isl;exex double mutants, the organization of motor axons into five nerve branches usually occurs, though axonal outgrowth is substantially delayed relative to wild-type. In addition, the penetrance of ISNb phenotypes in isl;exex double mutant embryos is dramatically higher than in exex single mutants. In 96% of hemisegments, the ISNb either bypasses the ventral muscle domain and extends along the ISN, or stalls shortly after it defasciculates from the ISN. Furthermore, defects are observed in the main ISN branch. In 32% of hemisegments, ISN axons defasciculate inappropriately, giving the ISN a 'frayed' appearance. At lower frequency (5%), the ISNs from adjacent hemisegments fuse. The ISN phenotypes are consistent with the presence of Exex-positive axons in the ISN and demonstrate that like ISNb, the ISN is sensitive to exex levels. Since it is unclear whether Isl is expressed in ISN-projecting neurons, the ISN phenotype in isl;exex embryos may result from loss of isl and exex activity either in common or distinct neuronal populations. In conclusion, the widespread axonal phenotypes in isl;exex double mutant embryos indicate that isl and exex act in parallel to regulate neuronal differentiation. Furthermore, the fact that the isl;exex double mutant reveals a role for exex in regulating ISN-projecting axons suggests that exex may genetically interact with other factors to control the outgrowth of additional motor axon branches (Broihier, 2002).
Expression analyses indicate that Exex and Lim3 are expressed widely in ventrally and laterally projecting MNs. In contrast, Eve has been shown to be expressed in dorsally projecting MNs, suggesting that Exex/Lim3 and Eve might label nonoverlapping MN populations. This is, in fact, what is observed since Exex and Eve label mutually exclusive neuronal subsets. Lim3 and Eve also identify nonoverlapping sets of MNs, since they are only coexpressed in the EL interneurons. Together with other expression analyses, these data show that Exex/Lim3 are expressed in the majority of Eve-negative MNs and demonstrate that Exex/Lim3 and Eve identify distinct MN classes (Broihier, 2002).
Drosophila HB9 is detected in a subset of motoneurons with ventral muscle targets and in a small group of interneurons, including the well characterized serotonergic interneurons. RNA interference knockdown of HB9 levels leads to defects in motoneuron ventral muscle target recognition, ectopic expression of a marker for dorsally projecting motoneurons (Even-skipped), and defects in serotonergic interneuronal projections. Conversely, ectopic HB9 expression causes an expansion of ventral motoneuron projections and repression of Even-skipped. Thus, Drosophila HB9 is required in a subset of motoneurons and interneurons for establishing proper axon projections but does not have a general role in distinguishing motoneuron and interneuron cell types (Odden, 2002).
The CNS contains three primary cell types: motoneurons, interneurons, and glia. Drosophila genes expressed specifically in all motoneurons have not been described, although a growing number of genes are known to be expressed in subsets of motoneurons. The Even-skipped (Eve) homeodomain transcription factor is expressed in dorsally projecting motoneurons and a subset of interneurons; loss of function and misexpression experiments show that it is necessary and sufficient for dorsal axon projections in motoneurons. The Huckebein (Hkb) zinc finger transcription factor is expressed in a subset of dorsally and ventrally projecting motoneurons and a subset of interneurons; it is required for motoneuronal pathfinding and target recognition. Islet and Lim3 are LIM (lin-11, isl-1, mec-3) homeodomain transcription factors that are expressed in overlapping subsets of ventrally projecting motoneurons and a subset of interneurons, in which they regulate motoneuronal pathfinding and target recognition. Together, these studies have led to the model that the motoneuron population consists of small groups of motoneurons that are each specified by a distinct 'combinatorial code' of transcription factors. It is unknown whether any additional transcription factors promote a general motoneuron identity (Odden, 2002).
On the basis of recent vertebrate studies, the HB9/MNR2 gene family is a prime candidate for a general determinant of somatic motoneuron cell type. In vertebrates, chick MNR2 and mouse HB9 are expressed in presumptive somatic motoneuron progenitors. Mouse HB9 maintains expression in somatic motoneurons, whereas chick HB9 is expressed only in postmitotic somatic motoneurons. Together, vertebrate HB9/MNR2 transcription factors are expressed in all somatic motoneurons and excluded from interneurons. Chick MNR2 or HB9 misexpression in interneurons causes a decrease in interneuronal markers; in addition, chick HB9 misexpression causes an increase in motoneuron markers. Mice lacking HB9 have somatic motoneurons with a hybrid motoneuron/interneuron fate; they extend motoneurons into the muscle field but transiently express interneuronal markers. Thus, vertebrate HB9/MNR2 genes are expressed specifically in somatic motoneurons and are essential for distinguishing motoneuron/interneuron cell types (Odden, 2002 and references therein).
Drosophila HB9 is the sole fly ortholog of the HB9/MNR2 gene family. Drosophila HB9 differs from its vertebrate orthologs in several ways: it is not expressed in all somatic motoneurons, it is expressed in a subset of interneurons, and it is required for the proper development of both interneurons and motoneurons (Odden, 2002).
BLAST searches of the Drosophila genome with the entire coding region of chick HB9 and chick MNR2 protein sequences identified a single related Drosophila gene (CG8254). PCR was used to amplify a 751 nt genomic DNA fragment, which was used to screen a pNB40 cDNA library and obtain one full-length clone; conceptual translation of the cDNA yielded a protein identical to that predicted by the Drosophila genome sequence project (Odden, 2002).
To determine the function of HB9 in ventrally projecting motoneurons, RNAi was used to knock down the levels of HB9. Greater than 90% of injected embryos showed extremely low HB9 protein levels; thus, these HB9RNAi embryos can be considered strong hypomorphs for HB9 expression. Control RNAi injections of buffer or other CNS genes showed no effect on HB9 expression (Odden, 2002).
HB9RNAi embryos were assayed for expression of the ventrally projecting motoneuron determinant Islet and the dorsally projecting motoneuron determinant Eve. No change was observed in the expression of the number of cells expressing the islet-tau:myc transgene, despite strong coexpression of Islet and HB9 in a subset of ventrally projecting motoneurons. In contrast, HB9RNAi embryos show a derepression of the Eve dorsal motoneuron determinant: there are consistently two ectopic Eve+ neurons, one located adjacent to the Eve+ dorsally projecting aCC motoneuron and the other located near the Eve+ dorsally projecting U1-5 motoneurons. It was reasoned that if loss of HB9 leads to ectopic Eve expression, then perhaps misexpression of HB9 would inhibit Eve expression. The Gal4/UAS system was used to misexpress HB9, using either ptc,en-Gal4 (which drives HB9 expression in all neuroectoderm and newly formed neuroblasts) or sca-Gal4 (which drives HB9 expression in all newly formed neuroblasts and the initial GMCs and neurons in their lineages). Both drivers result in precocious and ubiquitous expression of HB9 throughout the CNS, and both result in a nearly complete elimination of Eve expression in all dorsally projecting motoneurons (aCC, RP2, U1-5) and the pCC intersegmental interneuron. Interestingly, there is an increase in the number of Eve+ EL local interneurons, showing that the inhibition of Eve expression is not a general effect but depends on the cell type in which HB9 is expressed. There is no change in Islet expression after misexpression of HB9. It is concluded that HB9 restricts Eve expression to a specific subset of motoneurons, in which Eve promotes innervation of dorsal muscle targets. However, no role is found for HB9 in regulating islet gene expression in ventrally projecting motoneurons (Odden, 2002).
HB9 is expressed in ventrally projecting motoneurons: HB9RNAi embryos were assayed for defects in ventral motoneuron axon outgrowth, pathfinding, and muscle target recognition. Focus was placed on the four HB9+ RP1/3/4/5 motoneurons, which project out of the CNS via the SNb to form three distinct synaptic endings between the ventral muscles 7, 6, 13, and 12. In wild-type embryos, three well defined synaptic endings were observed at the 7/6, 6/13, and 13/12 muscle clefts in 90% of the hemisegments assayed. In HB9RNAi embryos, general CNS morphology is normal; axons project out of the SNb nerve at approximately the normal time and terminate within the appropriate ventral muscle field but fail to establish synaptic endings at the 7/6, 6/13, and 13/12 muscle clefts in 65% of the hemisegments assayed. In the affected segments, axons have expanded growth cones and terminate in an abnormally broad pattern. In addition to defects in ventral muscle target recognition, a slight increase was observed in the thickness of the intersegmental nerve (ISN) innervating dorsal muscles, which may reflect ectopic dorsal motor projections from the two extra Eve+ neurons, because it is known that ectopic Eve induces dorsal axon projections. Thus, HB9 is required for ventral muscle target recognition by the well characterized RP motoneurons (Odden, 2002).
To determine whether misexpression of HB9 leads to an increase in ventral muscle target innervation at the expense of dorsal muscle projections (the opposite of the HB9RNAi phenotype), scabrous-Gal4 was used to misexpress HB9 throughout the CNS (but not muscles) and motoneuron projections were scored with FasII. Indeed, a striking increase in the thickness of the SNb projections to ventral muscles was observed, with many hemisegments showing the normal synaptic endings in three muscle clefts. A clear decrease was observed in the number of motoneuron projections to dorsal muscles. The loss of dorsal projections is a result of multiple defects, including stalling of the ISN in the ventral muscle field or ISN neurons forming apparent synapses on the more ventral transverse nerve, the ISN and the SNb were never observed fasciculating together. It is concluded that HB9 promotes the targeting of motoneuron projections to ventral muscles and inhibits motoneuronal projections to dorsal muscle targets (Odden, 2002).
Drosophila HB9, unlike vertebrate HB9/MNR2 genes, is expressed in a subset of interneurons. To determine whether HB9 plays a role in interneuronal axon targeting similar to its role in motoneurons, the eagle-kinesin:lacZ transgene was used as an axonal marker for the HB9+ EW1/EW2 serotonergic interneurons and the lineally related EW3 interneuron. In wild-type embryos, the EW1-EW3 axons are fasciculate tightly, project anteriorly, turn medially to cross the midline, and synapse within the contralateral neuropil. In HB9RNAi embryos, the EW1-EW3 neurons project anteriorly as usual but are occasionally defasciculated and fail to cross the midline. No change was observed in the contralateral projections of other interneurons (e.g., the HB9- EL interneurons), suggesting that EW1-EW3 axon pathfinding defects are not caused indirectly by midline defects. It is concluded that HB9 is required in EW1-EW3 interneurons to promote contralateral axon projections. Misexpression of HB9 causes many general defects in the CNS, some of which, such as broken longitudinal connectives, can be attributed to interneuronal defects (Odden, 2002).
Thus HB9 is expressed in a subset of ventrally projecting motoneurons but not in their target muscles, and HB9 is required for proper muscle target recognition by these motoneurons. It is possible that the HB9 phenotype is caused by loss of Islet or Lim3 expression, because both Islet and Lim3 are required to establish normal ventral axon projections, and HB9 expression overlaps with Islet and Lim3. However, the HB9 loss and gain of function has no effect on Islet expression, and the HB9 and lim3 phenotypes are clearly different (lim3 mutants show a rerouting of SNb motoneurons into the SNd; this is not observed in HB9RNAi embryos). Instead, a model is favored in which HB9, Islet, and Lim3 have independent functions in establishing RP motoneuronal axon projections. lim3 mutants have pathfinding defects in which SNb motoneurons are diverted into the SNd nerve and terminate outside their normal muscle field; islet mutants also show pathfinding and fasciculation defects, in addition to target recognition defects. HB9 RNAi causes target recognition defects but not fasciculation defects or pathfinding defects (Odden, 2002).
Misexpression of HB9 is sufficient to promote thickening of ventral motor projections and reduced innervation of dorsal muscles. This phenotype could arise in several ways. (1) HB9 could transform interneurons into ventrally projecting motoneurons. This is thought to be unlikely, because no transformation is seen of apterous- or islet-expressing interneurons into motoneurons after HB9 misexpression; moreover, HB9 is normally expressed in some interneurons without turning them into motoneurons. (2) HB9 could transform dorsal motoneurons into ventral motoneurons. This seems unlikely, because dorsal motoneurons are clearly extending in the ISN, and although it is truncated, it is not fused with the SNb nerve. (3) The SNb motoneurons could be slightly defasciculated, which would make them appear thicker than normal. Transmission electron microscopy to count axons in the SNb would be necessary to test this model. Finally, the model is favored that HB9 induces SNa motoneurons to fasciculate into the SNb nerve root and innervate the SNb muscle target field, because misexpression of HB9 leads to the loss of the SNa nerve root in parallel with the thickening of the SNb nerve root. The SNa normally extends past the SNb target muscles en route to its own target muscles, so misexpression of HB9 might lead to precocious termination at the SNb target muscles (Odden, 2002).
HB9 is expressed in the serotonergic EW1 and EW2 interneurons and a third lineally related EW3 interneuron and is required to establish their normal contralateral projections. It is suspected that HB9 is acting autonomously in these interneurons rather than leading to defects at the midline that block contralateral projections, because HB9 is not expressed in midline cells of the CNS, and other interneurons (ELs) and motoneurons (RPs) show normal contralateral projections. The generation of hb9 mutant clones in the serotonergic interneurons will be necessary to distinguish between a cell autonomous or cell nonautonomous function of HB9 in regulating interneuronal axon projections. Interestingly, many transcription factors known to regulate motoneuron development, including Eve, Islet, Lim3, and Hkb, are also expressed in a subset of interneurons. islet and hkb are both expressed in the serotonergic interneurons, and each is required for proper axon pathfinding and neurotransmitter synthesis; lim3 is required for axon pathfinding of a different subset of interneurons. An open question is how interneurons can maintain expression of motoneuronal determinants such as HB9, Islet, Lim3, and Eve without fasciculating with motoneurons or exiting the CNS (Odden, 2002).
The expression and function of HB9 in interneurons is unexpected, because all known vertebrate HB9/MNR2 genes are expressed only in motoneurons within the CNS. Perhaps there are small groups of HB9/MNR2+ interneurons in vertebrates that have evaded detection; alternatively, Drosophila may have co-opted an ancestral motoneuronal determinant, HB9, for a parallel function in interneurons. Detailed analysis of HB9 expression patterns in additional organisms will be necessary to resolve this question (Odden, 2002).
Drosophila HB9 is expressed primarily in postmitotic neurons, whereas vertebrate HB9 family members are expressed in progenitor cells and in differentiated cells. This difference in the timing of HB9 expression may reflect differences in the timing of motoneuron cell fate commitment in each organism. In mouse and chick embryos, a domain of ventral spinal cord neural precursors goes through a phase in which they generate only motoneurons; during this period, the precursors express HB9. In Drosophila, there are no known precursors that generate solely motoneurons; virtually every motoneuron derives from a terminal cell division that produces one motoneuron and a non-motoneuron sibling cell. Thus, it is not surprising that Drosophila motoneuron determinants either are restricted to postmitotic cells (Islet and HB9) or are expressed just before the terminal division but downregulated rapidly in the non-motoneuronal sibling (Eve) (Odden, 2002).
The expression of Drosophila HB9 may help illuminate how Drosophila motoneurons are related evolutionarily to vertebrate motoneurons. It has been proposed that Drosophila motoneurons fall into two nonoverlapping groups: Islet+ motoneurons that are homologous to vertebrate somatic and visceral motoneurons and Eve+ motoneurons that have no vertebrate motoneuron counterpart. The Drosophila Islet+ motoneurons contain somatic motoneurons projecting to ventral muscle targets and a visceral TMNp motoneuron that projects to the heart; the Eve+ motoneurons project to dorsal muscles. Drosophila HB9 expression provides additional support for this model. Drosophila HB9 is expressed in many Islet+ somatic motoneurons but not in the Islet+ TMNp visceral motoneuron, similar to the observed restriction of vertebrate HB9/MNR2 expression to somatic motor neurons but not visceral motor neurons. These data are consistent with a model in which Drosophila Islet+ HB9+ somatic motoneurons are homologous to vertebrate Islet+ HB9+ somatic motoneurons, the Drosophila Islet+ TMNp visceral motoneuron is homologous to vertebrate Islet+ visceral motoneurons, and the Drosophila Eve+ motoneurons have no vertebrate motoneuron counterpart. It is noted, however, that murine Even-skipped (Evx1/2) CNS expression is restricted to the locally projecting commissural V0 interneurons. Perhaps this class of vertebrate interneurons has unrecognized similarities to Drosophila Eve+ motoneurons and/or interneurons (Odden, 2002).
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date revised: 15 October 2007
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