Dorsal-ventral (DV) patterning of the Drosophila embryo is initiated by Dorsal, a sequence-specific transcription factor distributed in a broad nuclear gradient in the precellular embryo. Previous studies have identified as many as 70 protein-coding genes and one microRNA (miRNA) gene that are directly or indirectly regulated by this gradient. A gene regulation network, or circuit diagram, including the functional interconnections among 40 Dorsal (Dl) target genes and 20 associated tissue-specific enhancers, has been determined for the initial stages of gastrulation. This study attempts to extend this analysis by identifying additional DV patterning genes using a recently developed whole-genome tiling array. This analysis led to the identification of another 30 protein-coding genes, including the Drosophila homolog of Idax, an inhibitor of Wnt signaling. In addition, remote 5' exons were identified for at least 10 of the ~100 protein-coding genes that were missed in earlier annotations. As many as nine intergenic uncharacterized transcription units (TUs) were identified, including two that contain known microRNAs, miR-1 and -9a. The potential functions of these recently identified genes are discussed and it is suggested that intronic enhancers are a common feature of the DV gene network (Biemar, 2006).
The Dl nuclear gradient differentially regulates a variety of target genes in a concentration-dependent manner. The gradient generates as many as five different thresholds of gene activity, which define distinct cell types within the presumptive mesoderm, neuroectoderm, and dorsal ectoderm. Total RNA was extracted from embryos produced by three different maternal mutants: pipe/pipe, Tollrm9/Tollrm10, and Toll10B. pipe/pipe mutants completely lack Dl nuclear protein and, as a result, overexpress genes that are normally repressed by Dl and restricted to the dorsal ectoderm. For example, the decapentaplegic (dpp) TU is strongly "lit up" by total RNA extracted from pipe/pipe mutant embryos. The intron-exon structure of the transcribed region is clearly delineated by the hybridization signal, most likely because the processed mRNA sequences are more stable than the intronic sequences present in the primary transcript. There is little or no signal detected with RNAs extracted from Tollrm9/Tollrm10 (neuroectoderm) and Toll10B (mesoderm) mutants. Instead, these other mutants overexpress different subsets of the Dl target genes. For example, Tollrm9/Tollrm10 mutants contain low levels of Dl protein in all nuclei in ventral, lateral, and dorsal regions. These low levels are sufficient to activate target genes such as intermediate neuroblasts defective (ind), ventral neuroblasts defective (vnd), rhomboid (rho), and short gastrulation (sog) but insufficient to activate snail (sna). In contrast, Toll10B mutants overexpress genes (e.g., sna) normally activated by peak levels of the Dl gradient in ventral regions constituting the presumptive mesoderm (Biemar, 2006).
To identify potential Dl targets, ranking scores were assigned for the six possible comparisons of the various mutant backgrounds, pipe vs. Tollrm9/Tollrm10, pipe vs. Toll10B, Tollrm9/Tollrm10 vs. Toll10B, Tollrm9/Tollrm10 vs. pipe, Toll10B vs. Tollrm9/Tollrm10, and Toll10B vs. pipe, using the TiMAT software package. As a first approximation, only hits with a median fold difference of 1.5 and above were considered. For further analysis, the top 100 TUs were selected for each of the comparisons, with the exception of Tollrm9/Tollrm10 vs. pipe for which the TiMAT analysis returned only 43 hits that meet the cutoff. To refine the search for TUs specifically expressed in the mesoderm, where levels of nuclear Dl are highest, only those present in the Toll10B vs. Tollrm9/Tollrm10 and Toll10B vs. pipe, but not pipe vs. Tollrm9/Tollrm10 comparisons were selected. For TUs induced by intermediate and low levels of nuclear Dl in the neuroectoderm, those present in both the Tollrm9/Tollrm10 vs. Toll10B and Tollrm9/Tollrm10 vs. pipe, but not pipe vs. Toll10B comparisons were selected. For TUs restricted to the dorsal ectoderm, only those present in the pipe vs. Tollrm9/Tollrm10 and pipe vs. Toll10B, but not Tollrm9/Tollrm10 vs. Toll10B, were selected. Finally, the TUs corresponding to annotated genes already identified in the previous screen were eliminated to focus on annotated genes not previously considered as potential Dorsal targets, as well as transcribed fragments (transfrags) not previously characterized. Using these criteria, 45 previously annotated protein-coding genes were identified, along with 23 uncharacterized transfrags. Of the 45 protein-coding genes, 29 exhibited localized patterns of gene expression across the DV axis, whereas the remaining 16 were not tested (Biemar, 2006).
A 5' exon was identified for crossveinless-2 (cv-2), a component of the Dpp bone morphogenetic protein (BMP) signaling pathway. cv-2 binds BMPs and functions as both an activator and inhibitor of BMP signaling. It is specifically required in the developing wing disk to generate peak Dpp signaling in the presumptive crossveins. cv-2 is also expressed in the dorsal ectoderm of early embryos, but its role during embryonic development has not been investigated. The whole-genome tiling array identified a 5' exon located ~10 kb 5' of the transcription start site of the cv-2 TU. Using RT-PCR and in situ hybridization assays, it was confirmed that the exon is part of the cv-2 transcript. It is possible that the exon resides near an embryonic promoter that is inactive in the developing wing discs. Future studies will determine whether this 5' exon influences the timing or levels of Cv-2 protein synthesis (Biemar, 2006).
The putative cv-2 transcript is expressed in a dynamic pattern, including expression in and near the developing CVs, that is identical to a combination of the lacZ expression patterns seen in both of the cv-2 enhancer trap lines. Moreover, this expression is lost or reduced in cv-2 mutants. In late third instar wing discs, both enhancer trap lines show weak general expression and slightly heightened expression along the anteroposterior (AP) boundary in the region of dpp expression. The cv-23511 enhancer trap line also shows strong expression in three distinct regions just distal to the prospective notum, as identified by the position of notal bristle precursors. In pupal wings, cv-23511 is expressed generally at low levels, but was also found at the tips of the LVs and, beginning at 25 hours after pupariation, in a broad region surrounding the CVs. The second enhancer trap line, cv-2225-3, retains a slight emphasis along the AP boundary in pupal wings, but lacks CV expression (Conley, 2000).
The putative cv-2 transcript is expressed in a pattern resembling a combination of the enhancer traps. There is weak general expression at all stages. Expression in late third instar wing discs is also strong in the three regions near the prospective notum, and weakly emphasizes the AP boundary. At 19-21 hours after pupariation, expression is stronger in broad regions in and around the developing CVs; this expression resolves into narrower regions by 28 hours after pupariation. Heightened expression is also observed at the tips of the LVs in pupal wings. This pattern is thus very similar to that of anti-p-Mad staining at these stages (Conley, 2000).
The cv-2 enhancer traps express lacZ in other imaginal tissues. The pattern in leg and antennal discs is similar to that of dpp; expression in both is higher along the dorsal or lateral sides, respectively, of the AP boundary. Fainter expression is also observed in eye discs posterior to the morphogenetic furrow (Conley, 2000).
Morphologically, the LVs first become visible from 4-8 hours after pupariation. The dorsal and ventral surfaces of the everting wing disc come together to form the wing blade, and LV proveins form as broad gaps or lacunae between the dorsal and ventral epithelia. The pattern of LV proveins differs from the mature vein pattern: the veins are broader and somewhat incomplete, and proximally the L3 and L4 proveins fuse into a central, single provein. Molecular markers of veins, such as rhomboid-lacZ (rho-lacZ), argos-lacZ, anti-Delta (anti-Dl), and an antibody against the activated form of MAPK, or of interveins, such as anti-Drosophila Serum Response Factor (DSRF, also known as Blistered), define the LV proveins as early as mid-late third instar. However, even with these markers, distinct L3 and L4 proveins are difficult to detect in the proximal region, where L3 and L4 fuse. The provein lacunae are lost from 8-16 hours after pupariation as the wing inflates, separating the dorsal and ventral surfaces. Only as the dorsal and ventral surfaces of the wing reappose, at approximately 16-26 hours after pupariation, are the definitive veins visible. MAPK activity remains high in the definitive veins, but is lost from the veins and become high in the intervein regions after 30 hours after pupariation (Conley, 2000).
The development of the ACV and PCV differ; a temporary, ACV-like provein is formed during the formation of the LV proveins, but no PCV provein is visible. The L3 and L4 proveins join at the future site of the ACV, as shown by heightened rho-lacZ expression in the cells of the ACV campaniform sensillum. Anti-Dl and anti-DSRF detected a distinct, CV-like structure extending between L3 and L4 in this region, but no PCV-like gene expression could be detect at 5-6 hours after pupariation. However, the ACV-like provein is no longer visible with anti-Dl at 16-20 hours after pupariation, even though the LVs can be identified at these stages (Conley, 2000).
The definitive ACV and PCV first become visible at 19-22 hours after pupariation as broad regions with slightly reduced anti-DSRF staining; this reduction is only rarely observed at 19-20 hours after pupariation The DSRF suppression begins during the morphological formation of the definitive CVs. By 23-26 hours after pupariation, narrow, well-defined CVs are visible with anti-Dl, anti-DSRF and anti-MAPK*. Although the reduction of anti-DSRF in the CVs precedes detectable levels of anti-MAPK* staining, DSRF is suppressed in a cell-autonomous manner by Egfr signaling so its reduction may correspond to increased Egfr activity. rho-lacZ and argos-lacZ do not appear in the CVs until 28-32 hours after pupariation. wingless-lacZ is expressed in the developing CVs, beginning at 25-26 hours after pupariation. wingless plays only a weak role in the formation of the CVs (Conley, 2000).
To follow the signaling mediated by Dpp and Gbb, an antiserum specific to the phosphorylated, activated form of Mothers Against Dpp (Mad), the receptor-activated Smad in Drosophila (anti-p-Mad) was used. No patterned anti-p-Mad staining is detected during early pupal stages (5-6 hours after pupariation), when the temporary ACV-like provein is formed. However, at 19 and 21 hours after pupariation, nuclear staining is observed in broad regions at the future sites of the CVs and in the tips of the LVs; some weaker staining is also observed in the portion of the LVs near the CV attachment sites (including the portion of L4 between the ACV and PCV) and along L2. Interestingly, the CV anti-p-Mad staining typically precedes the reduction of anti-DSRF staining in the CVs, suggesting that Mad activation precedes the suppression of DSRF expression mediated by Egfr. At 24 and 26 hours after pupariation anti-p-Mad-stains nuclei along all the LVs, but staining is still stronger near the CVs. At approximately 36 hours after pupariation, staining is observed throughout the wing, but again is stronger in the CVs. Strong staining is also observed in both the nuclei and axons of the PNS of the wing at 18-36 hours after pupariation (Conley, 2000).
To confirm that Mad activation is induced by BMP-like signaling, Sog, which can inhibit BMP-like signaling was overexpressed in pupal wings. en-Gal4 was used to drive UAS-sog in the posterior of the wing. Adult wings lack the ACV and the PCV, but these levels of Sog are not sufficient to block formation of the ends of the LVs; as expected, heightened anti-p-Mad staining was not detected near the PCV in 19-20 hours after pupariation pupal wings, while staining in the ACV was reduced and in the LVs was largely normal (Conley, 2000).
Localized ligand expression cannot apparently account for the early stages of Mad activation in the CVs. dpp is first expressed along the LVs at 18-20 hours after pupariation, but is not detected in the CVs until some time after 24 hours after pupariation, as shown by in situ hybridization and dpp lacZ enhancer traps. As in imaginal discs, anti-Gbb staining appears largely uniform at 21 and 24 hours after pupariation, and is not higher in the CVs. The localized activation of Mad is apparently not due to localized receptor expression. Expression of the BMP receptor tkv is ubiquitous during pupal stages, but is lower in the veins and higher in the cells immediately flanking the veins; the lowered expression is thought to be mediated by Dpp signaling. tkv-lacZ expression is slightly reduced in the CVs at 22.5 hours after pupariation (Conley, 2000).
In many developing systems, the fate of a cell is determined by its position in a time-independent spatial distribution of a morphogen. However, during dorsal-ventral patterning in the Drosophila embryo, an initial low-level signal refines to a narrow, high-intensity band. This refinement suggests that cells respond to the local transient morphogen distribution that results from interactions between bone morphogenetic proteins (BMPs), their receptors, the BMP-binding proteins Sog and Tsg, the metalloprotease Tld, and a putative, positively regulated component that locally enhances surface binding of BMPs within the region of high signaling. A computational model was developed for dorsal surface patterning; when positive feedback of a cell surface BMP-binding protein is incorporated, bistability in the kinetic interactions transduces the transient BMP distribution into a switch-like spatial distribution of the BMP-bound receptor. Inclusion of positive feedback leads to the observed contraction of signaling, because cells near the dorsal midline outcompete adjacent lateral cells for limited amounts of BMP. In the model, cells interpret the morphogen distribution by differentiating according to the history of their exposure rather than to a threshold concentration in a static spatial gradient of the morphogen (Umulis, 2006).
An open problem in the interpretation of morphogen gradients is understanding how cells respond to a transiently evolving extracellular morphogen distribution. One possibility is that a steady-state gradient forms, and a time control cue is released to signal that cells should respond to that gradient according to their spatial position. Another idea is that the cellular state changes in response to the time-dependent morphogen distribution. The model described in this study, that incorporates both positive feedback and degradation/internalization of BMP, suggests a mechanism in which cells respond to a transient extracellular gradient by producing a factor that stabilizes signaling for cells that have been exposed to the ligand sufficiently, while at the same time reducing the extracellular concentration of morphogen to reinforce the low-level signaling fate of adjacent cells. In this case, cells respond not only to the level but also to the time they are exposed to the extracellular signal. Initially the cellular response tracks the BMP gradient, but in later stages the positive feedback loop establishes a switch-like distribution of cells that have either a high- or low-signaling state. To illustrate this phenomenon, consider an evolving extracellular gradient that rises and falls before stabilizing at a steady-state distribution. An interpretation is presented of the changing gradient (the level of signaling receptors), in which two distinct regions are dealt with: a high-signaling region and a low-signaling region. Suppose that exposure is limited by reducing BMP production after 35 min. The response is initially the same as before, but due to the rapid decrease in morphogen, the response returns to a low-signaling state. The final distribution of morphogen is nearly the same in the two cases, and the standard morphogen model would suggest that the output response would be the same, yet the history of the morphogen gradient is different, and this leads to steady-state responses that are very different (Umulis, 2006).
The key to establishing such a system is the induction of a positive feedback component. Although the identity of the component that provides this function in the embryo is not yet known, this study shows that a cell surface-bound BMP-binding protein (SBP) such as Crossveinless 2 (Cv-2) can provide that activity. Cv-2 binds BMP molecules, is induced by positive feedback from BMP signaling, and at least one form binds to the surface. Remarkably, posterior cross-vein development uses a related set of extracellular modulators to achieve a similar spatial localization of BMP ligands. In that case, the ligands are Dpp and Gbb, and the modulators are Sog, the Tsg-related factor Cv, and the Tolloid-related factor Tlr. A major molecular difference between the posterior cross-vein pathway and signaling in the embryo is that the former also requires the extracellular protein Cv-2. Cv-2 contains cysteine-rich domains related to those found in Sog as well as a partial Von Willebrand domain. Recent data demonstrates that this protein not only binds Dpp and Gbb, but also binds to cell surfaces via the Von Willebrand domain and more importantly, its expression is induced by Dpp signaling. Although Cv-2 is required for promoting high-level BMP signaling in the cross-vein and during zebrafish gastrulation, its loss does not seem to affect early embryonic development. Nevertheless, it serves to exemplify the type of molecule that could be involved in a positive feedback loop. There are several other CR-containing proteins in Drosophila, and numerous examples are found in vertebrate systems. In addition, the exact binding motif is likely to be unimportant. Other extracellular BMP-binding proteins such as the proteoglycan Dally, small leucine-rich proteoglycan family members such as Tsukushi, and the glycosylphosphatidylinositol-linked protein Dragon could potentially act as positive feedback modulators of BMP signaling. Other possibilities include a molecule that modifies the affinity of the receptor through an intra- or extra-cellular mechanism. However, such a mechanism has to both enhance pMad signaling and lead to accumulation of Dpp on the surface. One additional observation of note is that Tsg has recently been suggested to facilitate binding of BMPs to the cell surface. Although no enhanced association of BMPs with receptors in the presence of Tsg has been found it is possible that Tsg could mediate this effect through enhancement of binding of BMPs to one of these alternative cell surface BMP-binding proteins (Umulis, 2006).
To achieve the 'constancy of the wild-type,' the developing organism must be buffered against stochastic fluctuations and environmental perturbations. This phenotypic buffering has been theorized to arise from a variety of genetic mechanisms and is widely thought to be adaptive and essential for viability. In the Drosophila blastoderm embryo, staining with antibodies against the active, phosphorylated form of the bone morphogenetic protein (BMP) signal transducer Mad, pMad, or visualization of the spatial pattern of BMP-receptor interactions reveals a spatially bistable pattern of BMP signaling centered on the dorsal midline. This signaling event is essential for the specification of dorsal cell fates, including the extraembryonic amnioserosa. BMP signaling is initiated by facilitated extracellular diffusion that localizes BMP ligands dorsally. BMP signaling then activates an intracellular positive feedback circuit that promotes future BMP-receptor interactions. This study identified a genetic network comprising three genes that canalizes this BMP signaling event. The BMP target eiger (egr) acts in the positive feedback circuit to promote signaling, while the BMP binding protein encoded by crossveinless-2 (cv-2) antagonizes signaling. Expression of both genes requires the early activity of the homeobox gene zerknullt (zen). Two Drosophila species lacking early zen expression have high variability in BMP signaling. These data both detail a new mechanism that generates developmental canalization and identify an example of a species with noncanalized axial patterning (Gavin-Smith, 2013).
This study has identified a genetic network that acts as a phenotypic stabilizer of a spatially bistable patterning process. The minimal bistable systems allowed by theory require a nonlinear activation rate and a linear degradation rate. It is believed that the identified network defined in this study represents the minimal genetic components required for bistability of BMP signaling in D. melanogaster. In turn, bistability canalizes dorsal patterning. During amnioserosa specification, egr provides positive feedback, conferring nonlinearity, while cv-2> acts as a linear negative regulator of the signaling pathway. The loss of both components reveals the inherent noise of facilitated extracellular diffusion of BMP ligands, as without egr and cv-2, embryos manifest a huge range of signaling domain breadth and intensity. The data also reveal that amnioserosa specification in D. melanogaster is robust on multiple levels, with different mechanisms ensuring robustness in various Drosophila species (Gavin-Smith, 2013).
First, egr or bsk RNAi embryos have normal amounts of amnioserosa and minimal embryonic lethality despite the 2-fold reduction in signaling intensity. This demonstrates that amnioserosa specification is robust to decreases of BMP signaling and the wild-type level of BMP signaling in D. melanogaster is much higher than necessary. Second, the D. melanogaster embryo can tolerate at least a 250% increase or a 20% decrease in amnioserosa cell number without compromising viability. Lastly, the variability in amnioserosa cell number in D. yakuba embryos is equivalent to that in D. melanogaster embryos, indicating that amnioserosa specification in D. yakuba is robust against variable BMP signaling intensity. Therefore, in D. yakuba embryos, either less BMP signaling is required to direct amnioserosa specification or a second mechanism downstream of BMP signaling intensity maintains robust amnioserosa specification (Gavin-Smith, 2013).
Finally, as a counterpoint to the predicted ubiquity and selective maintenance of developmental canalization, D. santomea has been identified as a noncanalized wildtype species. D. santomea both has highly variable cell fate specification and is not robust to genetic variants found in its wild population. The identification of this noncanalized species may permit further investigation of the evolutionary factors allowing for this diversity in developmental trajectories (Gavin-Smith, 2013).
The cv-21 allele, a visible, spontaneous allele that segregates from several natural populations, generates the strongest wing phenotype; wings typically lack most or all of the PCV and, in some cases, the posterior of the ACV. cv-2225-3 flies typically lack part or all of the PCV. cv-23511 phenotypes overlap wild type. Phenotypes of all three alleles are strengthened when placed over a deficiency; cv-21/Df(2)Pu-D17 always lack the PCV, variably lack some or all of the ACV and, on occasions, lack the ends of the LVs. A small percentage of lethal P-element excisions were also generated, but none have appreciably stronger wing phenotypes than their parent P-element lines. No other defects have been detected in adults or embryos in any of these lines (Conley, 2000).
dpp and gbb mutations both disrupt CV formation. Weak cv-2 alleles are strengthened by dpp and gbb loss-of-function mutations. cv-2225-3/cv-23511 flies never lack the entire PCV, but 50% of gbb 4 cv-2225-3/cv-2 3511 flies lack the entire PCV. Similarly, cv-23511/Df(2R)Pu-D17 only rarely disrupt the ACV, but dppd6 cv-23511/Df(2R)Pu-D17 commonly does. However, cv-2 cannot dominantly enhance earlier dpp-dependent patterning in the wings: dppd5 Df(2R)Pu-D17 /dpphr4 wings look no worse than dppd5/dpphr4 wings. To provide a more direct link between cv-2 and Dpp and Gbb signaling, Mad activation was examined in mutant pupal wings. In cv-21 adults, the PCV is more reliably disrupted than the ACV; the anti-p-Mad staining normally found near the PCV in 19, 22, 26 and 36 hours after pupariation wings is lost or disrupted in cv-21 homozygotes, as is the reduction of anti-DSRF in the PCV. In adults of the stronger allelic combination cv-21/Df(2R)Pu-D17, the ACV is also often lost along with the ends of some of the LVs. Interestingly, no disruption of the ACV or LV anti-p-Mad staining cv-21/Df(2R)Pu-D17 pupal wings is detected at 21 or 25 hours after pupariation; only at 36 hours after pupariation is staining lost from the ACV. This indicates that cv-2 is required not only to initiate Mad activity in the PCV, but also to maintain that activity in the ACV (Conley, 2000).
Expression of the cv-2 message is altered in cv-21 and cv-2225-3 lines. At late third instar, the heightened expression observed in the three regions near the notum is largely lost in cv-2225-3 and consistently reduced in cv-21. Mutant adults have no obvious defects in the notum or hinge, so this loss is unlikely to be a secondary effect of a structural change. Expression near the CVs was also reduced in both these lines. However, general expression is not totally lost from either cv-2225-3 or cv-21, consistent with their hypomorphic phenotypes (Conley, 2000).
EP elements contain multiple UAS sequences coupled to a heat-shock promoter; once inserted into the genome, these can be used in conjunction with Gal4 enhancer traps to drive the expression of neighboring genes. Several EP lines contain EP insertions upstream of cv-2, close to the insertion site of the cv-23511 P element. Crossing the EP(2)1103 line to either patched-Gal4 or engrailed-Gal4 lines results in strong misexpression of the cv-2 transcript at levels equal to or greater than endogenous levels. Adults from these crosses are fully viable and no wing phenotypes were apparent. This suggests that wild-type levels of Cv-2, while required for CV formation, are not normally sufficient to induce ectopic venation. However, the stronger ap-Gal4 driver does generate a range of wing phenotypes; the most severely affected wings have ectopic venation near L2, vein deltas at the tips of the LVs, thickening of the ACV and, rarely, the partial loss of the PCV (Conley, 2000).
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date revised: 2 February 2014
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