Thoracic Limb Primordium Arises in the Neurectoderm and Is Divided Into Distinct Domains That Will Give Rise to Imaginal and Neural Derivatives
An early marker for the thoracic limb primordium is the homeodomain transcription factor Dll. Dll is first detectable at late stage 10. Between stages 12 and 13, the population of Dll-expressing cells is subdivided into domains that express either neural or imaginal markers. The neural derivative gives rise to the Keilin's organ, whereas the imaginal components give rise to the leg and wing discs. Using the svp and ASC neural markers, we found that the ventral half of the primordium arises within the neurogenic ectoderm. Because the dorsal-most cells of the primordium are those that become the wing disc, the portion of the primordium that lies outside of the neurogenic ectoderm likely gives rise to the wing disc. Cells that lie within the neurectoderm probably give rise to the leg disc and Keilin's organ. Based on analysis of Dll expression in several phyla, it has been proposed that the earliest role of Dll was in the development of the nervous system and that its role in patterning the proximodistal axes of appendages evolved later (Panganiban et al., 1997). The relationship between the developing nervous system and the appendages in more primitive organisms than Drosophila is unknown, but it may be that the relationship between these structures is of broader evolutionary significance.
Not all Dll-expressing cells of the primordium are born at the same time. Instead, cells that activate the early embryonic Dll enhancer de novo are produced continuously between stages 10 and 12 in the ventral portion of the primordium (Goto and Hayashi, 1997a). We have shown here that the Dll-producing domain lies within the neurogenic ectoderm. Dll-expressing cells subsequently migrate dorsally. As they move away from this hypothetical “source,” they cease utilization of the Dll early embryonic enhancer and either switch to the late embryonic enhancer or cease transcription of Dll entirely. The latter population gives rise to the wing disc, whereas the former gives rise to the leg disc and Keilin's organ. The location of the primordium at the boundary between the neurectoderm and non-neural ectoderm and the early expression of Dll in the migrating cells resembles Dlx expression in the specification and migration of neural crest cells in vertebrates. Dlx family members are among the earliest markers for the neural crest (reviewed in Merlo et al., 2000). A second intriguing parallel between the Drosophila thoracic limb primordium and the vertebrate neural crest is that the Esg-related transcription factors Snail and Slug have been implicated in the specification and migration of the neural crest, whereas Sna and Esg have been implicated in the development of the wing cells that migrate away from the leg disc and neuroectoderm (reviewed in Hemavathy et al., 2000). We, therefore, propose that the vertebrate neural crest and the thoracic imaginal discs may be derived from a common set of cells present in the last common ancestor of protostomes and deuterostomes.
Fate Mapping of the Thoracic Limb Primordium
Fate maps of the leg/Keilin's organ limb primordium have been proposed (Goto and Hayashi, 1997b; Kubota et al., 2003). However, these maps do not distinguish between distal leg and Keilin's organ precursors. Our data indicate that the primordium has three distinct populations of cells that are likely to correspond to proximal leg disc, distal leg disc, and Keilin's organ (Fig. 8A). Previous models include specification of proximal leg disc at an intermediate dorsoventral position within the early primordium, and their subsequent ventral migration to encircle the cells that give rise to the distal leg (Goto and Hayashi, 1997b). We have not obtained evidence for this ventral migration in our own experiments. Instead, we observe esg expression as a discrete ring in the primordium by stage 14.
Figure 8. Proposed fate map of the thoracic limb primordium and summary of genetic interactions involved in its subdivision. A: A summary of the fate map of the thoracic limb primordium of the second thoracic segment. The dorsal-most derivative of the primordium is the wing disc (blue). The more ventral portions of the primordium give rise to two structures, the leg disc (green and purple) and the Keilin's organ (red). The Keilin's organ precursors arise centrally and express Dll, Ct, and Cpo but lack the imaginal determinant Esg. The leg disc precursors may be further subdivided into the cells giving rise to the presumptive distal (green) and putative proximal (purple) domains of the leg imaginal disc. Distal leg disc cells are marked by their coexpression of Dll and Esg proteins (green), whereas proximal cells express only Esg (purple). B: The genetic relationships between the various genes involved in specifying the thoracic limb primordium. The wg and dpp pathways serve key roles in specification of the primordium through initiating the expression of determinants such as Dll and esg (Cohen, 1990; Goto and Hayashi, 1997b). Graded levels of these factors probably serve to subdivide these structures. High levels of Dpp activity (shown with the solid arrow) are required for specification of the wing disc and lower levels (shown with the dashed arrow) are required for specification of the leg disc (Raz and Shilo, 1993; Goto and Hayashi, 1997b; Kubota et al., 2000). Dpp dorsally limits primordium formation (Goto and Hayashi, 1997b). After specification, subdomains are patterned through the activities of several genes. The Keilin's organ is patterned by the activities of Dll and the ASC, which also prevent these cells from adopting imaginal identity. Members of the ASC are under Dll control within the Keilin's organ, and it is unclear whether Dll plays a direct role in repressing imaginal development or works only by means of the ASC (question mark over the arrow between Dll and esg). Imaginal components are patterned by esg (proximal and distal leg) and esg, vg, and sna (wing disc; Hayashi et al., 1993; Fuse et al., 1996; and this work). esg also is involved in preventing imaginal cells from adopting neural identity. Dll patterns the distal part of the leg disc but is not required for specification of imaginal identity. At later stages, ct is expressed in the Keilin's organ precursors under the control of Dll and the ASC. ct is required for establishment of external sensory structures (Wieschaus et al., 1984; Blochlinger et al., 1988; Blochlinger et al., 1990; and this work), and cpo is required for neural function (Bellen et al., 1992).
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Lineage tracing experiments of Dll-expressing cells suggest that not all of the cells of the leg imaginal disc have expressed Dll (Campbell and Tomlinson, 1998; Weigmann and Cohen, 1999; Andrews and Boekhoff-Falk, unpublished observations). We also have noted the presence of a small cluster of cells that express esg but not Dll on the ventral side of the primordium. As Dll is not required for formation of proximal leg structures or leg disc-derived body wall (Cohen et al., 1993; Campbell and Tomlinson, 1998), we propose that these cells that express esg and hth but not Dll will give rise to proximal leg disc-derived structures, whereas those that express esg, hth, and Dll give rise to the distal-most portions of the leg disc, with medial domains being specified during larval development. This explanation is consistent with the timing of activation of the medial patterning gene dachshund (dac) during larval development (Mardon et al., 1994; Lecuit and Cohen, 1997).
A major difference between our proposed fate map and those put forth previously is the placement of presumptive distal leg cells within the esg-expressing population (Fig. 8A). We think this strategy is justified for two reasons. First, esg has been implicated in the maintenance of diploidy of imaginal cells (Hayashi et al., 1993). Thus, cells that lack esg expression are unlikely to be imaginal. Second, the central cells that express Dll but lack Esg express ASC. In ASC mutants, these central cells die (this work). Yet mutating the ASC yields the full complement of distal leg segments (Garcia-Bellido and Santamaria, 1978). Thus, the central ASC-expressing cells are unlikely to contribute to the leg imaginal disc. Instead, they are probably Keilin's organ precursors. Consistent with this view, these central cells express Dll and ct as well as ASC genes, and the Keilin's organ is lost in animals mutant for any of these (Wieschaus et al., 1984; Bodmer et al., 1987; Dambly-Chaudiere and Ghysen, 1987; Cohen and Jurgens, 1989; and this work).
A second difference in our proposed fate map is the placement of presumptive proximal leg outside of the Dll expression domain. We believe this positioning is justified based on both Dll phenotypes and lineage analysis and on the requirement for esg in imaginal tissues. Specifically, Dll is not required for the formation of proximal leg disc-derived structures (Cohen et al., 1993) nor has lineage analysis revealed Dll expression in proximal leg disc-derived structures at any stage of development (Campbell and Tomlinson, 1998; Weigmann and Cohen, 1999; Andrews and Boekhoff-Falk, unpublished observation). Thus, the proximal leg disc is likely to arise from cells that express esg but lack Dll. Such cells can be found ventral to the Dll-expressing population.
There are two caveats to our proposed fate map. First, esg lineage data are not available. Thus, we do not know whether all esg-expressing cells of the primordium become imaginal. Nor do we know whether esg expression marks all of the presumptive imaginal cells. Second, although the cell death observed in ASC mutants together with their normal distal leg segmentation suggest that ASC is not required to make the distal leg, it is possible that distal leg normally arises from ASC-expressing cells and that these cells are regenerated in the mutants. Nonetheless, we believe the available data strongly support the fate put forth here.
Fate Determination Within the Thoracic Limb Primordium Requires the Activities of Dll, the ASC, ct, and esg
This study constitutes the first genetic analysis of the roles of Drosophila Dll in a component of the developing embryonic nervous system. Larvae lacking Dll, the ASC or ct are missing Keilin's organ. In embryos null for either Dll or the ASC, the number of cells in the thoracic primordia expressing ct and cpo is significantly reduced and the number of cells expressing esg appears to increase. Therefore, Dll and the ASC function upstream of ct and cpo in specification of the neural portion of the primordium and are required to repress imaginal development in these cells. Nonetheless, Dll and the ASC are not sufficient to convert imaginal precursors to Keilin's organ fate. Although members of the ASC are required for cell survival in late embryonic development, the loss of Keilin's organ cells in ASC nulls appears to be independent of cell death, because Keilin's organ marker expression is reduced well before the onset of cell death.
We note that, although Dll is upstream of the ASC in the Keilin's organ precursors, loss of Dll results in less apoptosis than does loss of the ASC. We hypothesize that this finding could be due to Dll functioning nearer the top of the genetic hierarchy in specification of these cells. In the absence of Dll, cells may more readily adopt a new fate than they do when their fate specification is interrupted later by loss of the ASC. When interrupted later, it is possible that these cells become “confused” as to their fate and become targeted for removal by means of cell death. Consistent with this explanation, cells were observed in the ASC null embryos that expressed both Keilin's organ and imaginal markers. This finding was not observed in Dll null embryos. It may be that targeting of mixed fate cells for death is a common phenomenon in developmental biology. A recent study of the otic placode in zebrafish noted that loss of the later-expressed pax genes resulted in a more severe phenotype than did loss of dlx, even though dlx-3 and dlx-7 are both required for formation of the placode (Solomon and Fritz, 2002) and probably upstream of pax. The cause of the more severe phenotype in the pax mutants was found to be due to cell death, consistent with our model.
Dll is required for both ASC and ct expression. Whether Dll is required simply for ASC expression or also is required later in parallel with the ASC for ct and cpo expression is unclear. The Dll- and ASC-dependent repression of esg appears to be limited to the Keilin's organ precursors because ectopic expression of ASC family members within the thoracic limb primordium does not repress esg, expand the zone of ct and cpo expression, or increase the number of sensory hairs in the differentiated Keilin's organ. Other factors, therefore, are likely to be required. Dll collaboration with ASC could constitute an ancient regulatory mechanism, because the vertebrate Dlx1 and Dlx2 genes cooperate with ASC homologs to regulate formation of γ-aminobutyric acid-ergic neurons in the developing brain (Letinic et al., 2002).
In the absence of ct, the thoracic limb primordium expresses other Keilin's organ markers, including Dll and Cpo. This finding indicates that ct plays a relatively late role in differentiation of the Keilin's organ. ct may specify Keilin's organ precursors as ES class. However, the mechanism appears to be more complicated than a simple choice between ES and chordotonal identity, because ato is not derepressed in ct nulls, nor do the Keilin's organs adopt chordotonal morphologies.
What is the function of the Keilin's organ and the reason for its association with the developing leg imaginal disc? The Keilin's organ remains associated with the leg disc throughout larval development, and it is thought that axons from the Keilin's organ to the PNS serve pathfinding functions for later-developing leg neuron axons (Jan et al., 1985; Tix et al., 1989). Enervation of the regenerating limb of the newt precludes distal patterning, and Dlx3 expression is dependent on limb innervation (Mullen et al., 1996). Thus, Dll and Dlx could serve related or analogous roles in axon-induced limb patterning in both flies and vertebrates. As the Keilin's organ cells invaginate before leg disc cells, it is possible that the neural cells assist the migration of the presumptive leg disc to its final position within the larva. The Keilin's organ, thus, may play roles both in development of the leg disc and in differentiation and patterning of the adult leg.
esg is required for the maintenance of diploidy of at least some imaginal tissues (Fuse et al., 1994; Hayashi, 1996). It has been hypothesized that esg may be the critical imaginal determinant (Hayashi et al., 1993). Within the thoracic limb primordium, the expression patterns of esg and the neural markers ct and cpo are mutually exclusive. Thus, either esg is not required to make distal leg, or ct and cpo mark only the Keilin's organ precursors. In the absence of esg, we observed some expansion of ct or cpo domains, indicating that esg may play a role in repressing Keilin's organ fates. However, the Keilin's organ of esg null animals is not increased in size, and TUNEL analysis indicates that there is no extra cell death in esg mutant primordia.
Consistent with the loss of function analysis, ectopic expression of esg is capable of repressing ct and cpo in the presumptive Keilin's organ. The effect of ectopic esg is probably due to repression of ASC genes because the expression of l'sc is severely reduced in the thoracic limb primordium at stage 11 in embryos expressing ectopic esg. esg and other snail family members have the ability to bind to the E-box recognized by bHLH transcription factors such as the ASC proteins (Hayashi et al., 1993; Fuse et al., 1999). Also, the Scutoid mutation in Drosophila, in which there is a loss of sensory bristles from the scutellum, was shown recently to be a gain of function mutation of snail (Fuse et al., 1999). Thus, esg repression of ct and cpo may be mediated by competition with ASC products for E-box binding sites.
There appears to be a developmental switch at play in the developing primordium (Fig. 8B). Cells within the primordium have the potential to adopt either neural or imaginal identity. Within the imaginal precursors, we propose that esg is required for choosing imaginal identity, whereas within the Keilin's organ precursors, repression of esg is essential for neural differentiation to proceed. How, then, are these different potentials established? It has been shown that the imaginal components of the second thoracic primordium, the wing and leg discs, are specified in part due to opposing gradients of Dpp and EGF activity (Kubota et al., 2000). It is possible that high levels of EGF signaling could promote Keilin's organ development. Consistent with this possibility, a high level of rhomboid transcript is accumulated in a cluster of cells in the center of the primordium at stage 11 (Kubota et al., 2000). This accumulation appears to be in approximately the same domain where l'sc is expressed at this stage. Rhomboid is required for the activation of spitz, an activating ligand for the DER receptor (Schweitzer et al., 1995; Wasserman and Freeman, 1998). Thus, it is possible that expression of the ASC in the Keilin's organ precursors is dependent on up-regulation of the EGF pathway in these cells. These precursors, in turn, may maintain neural identity and repress imaginal development. Outside of this domain, cells may adopt either dorsal or ventral imaginal disc identity in response to the relative levels of Dpp and EGF signaling. However, we do not observe marker expression for the three derivatives of the primordium in a pattern directly consistent with this model. Nor does this model of opposing signaling gradients along the dorsoventral axis explain how the Keilin's organ becomes specified as a cluster of cells in the center of the thoracic limb primordium. Clearly, questions still remain with respect to the mechanisms underlying subdivision of the primordium. Examination of the expression patterns of imaginal versus Keilin's organ markers in animals mutant for components of the Dpp, Wg, and EGF signaling pathways could provide additional insights into these intriguing cell fate decisions.