The Interactive Fly

Zygotically transcribed genes

Dorsal-Ventral Patterning Genes and BMP Signaling

Transcriptional regulation of genes involved in dorsal-ventral patterning

Protein interactions in DV patterning

Comprehensive identification of Drosophila dorsal-ventral patterning genes using a whole-genome tiling array

Evolution of the dorsal-ventral patterning network in the mosquito: Altered expression of sog and tld correlates with a broader domain of Dpp signaling, when compared with Drosophila


Dorsal-Ventral Patterning Genes and Dpp Signaling

  • Transcription factors

  • Ventral lateral system or spitz group

  • Secreted factors in the BMP system

  • Receptors for Dpp, Screw and Gbb

  • DPP and Baboon pathways

  • Others

    Transcriptional regulation of genes involved in dorsal-ventral patterning

    Dorsoventral (DV) patterning of the Drosophila embryo is initiated by a broad Dorsal (Dl) nuclear gradient, which is regulated by a conserved signaling pathway that includes the Toll receptor and Pelle kinase. What are the consequences of expressing a constitutively activated form of the Toll receptor, Toll(10b), in anterior regions of the early embryo? Using the bicoid 3' UTR, localized Toll(10b) products result in the formation of an ectopic, anteroposterior (AP) Dl nuclear gradient along the length of the embryo. The analysis of both authentic Dorsal target genes and defined synthetic promoters suggests that the ectopic gradient is sufficient to generate the full repertory of DV patterning responses along the AP axis of the embryo. For example, mesoderm determinants are activated in the anterior third of the embryo, whereas neurogenic genes are expressed in central regions. These results raise the possibility that Toll signaling components diffuse in the plasma membrane or syncytial cytoplasm of the early embryo (Huang, 1997).

    The Huang (1997) paper also clearly summarizes what is known about the regulation of genes involved in dorsal/ventral patterning. There are five distinct thresholds of gene activity in response to the Dorsal nuclear gradient in early embryos. The type I target gene folded gastrulation is activated only in response to peak levels of the Dl gradient, so that expression is restricted to a subdomain of the presumptive mesoderm. The PE enhancer from the twist promoter region exhibits a similar pattern of expression. This enhancer contains a cluster of low-affinity Dl binding sites that restrict expression to the ventral-most regions of early embryos. The type II target gene snail contains a series of low-affinity Dl-binding sites, as well as binding sites for the bHLH activator, Twist. The Dl and Twist proteins appear to make synergistic contact with the basal transcription complex, so that snail is activated throughout the presumptive mesoderm in response to both peak and high levels of the Dl gradient. The ventral midline arises from the mesoderm, which is derived from the ventral-most regions of the neuroectoderm. Mesectoderm differentiation is controlled by the bHLH-PAS gene, sim. Some of the E(spl) complex also exhibit early expression in the presumptive mesectoderm. A synthetic enhancer containing high-affinity Dl-binding sites and Twist binding sites exhibits expression in this region. The type IV target gene rhomboid is expressed in lateral stripes that encompass the ventral half of the presumptive neuroectoderm. These stripes are regulated by a 300-bp enhancer (NEE) that contains high-affinity Dl-binding sites, Twist-binding sites, and "generic" E-box sequences that appear to bind ubiquitously distributed bHLH activators (Daughterless and Scute), which are present in the unfertilized egg. The fifth and final threshold response is defined by the lowest levels of the Dl gradient. The zerknullt target gene is repressed by high and low levels of the gradient, so that expression is restricted to the presumptive dorsal ectoderm. The zen promoter region contains high-affinity Dl-binding sites and closely linked "corepressor" sites. Efficient occupancy of the Dl sites appears to depend on strong, cooperative DNA-binding interactions between Dl and the corepressors. The same low levels of Dl that repress zen also repress sog. The sim, E(spl), rho and sog expression patterns are restricted to the neurogenic ectoderm and excluded from the ventral mesoderm by Snail, which encodes a zinc finger repressor (Huang, 1997).

    This study also provides evidence that neurogenic repressors may be important for the establishment of the sharp mesoderm/neuroectoderm boundary in the early embryo. About half of the embryos carrying the Toll anteriorly expressed transgene exhibit a ventral gap in the endogenous ventral expression pattern of snail behind the ectopic anterior staining pattern. Although the identity of the repressor creating this gap is unknown, it is conceivable that members of the E(spl) complex encode putative snail repressors because previous studies have shown that the m7 and m8 genes are expressed in the lateral neuroectoderm of early embryos. Proteins coded for by these genes are known to repressors. These proteins might be regulated by the gene hierarchy responsible for D/V polarity (Huang, 1997).


    Protein interactions in DV patterning

    Developmental patterning relies on morphogen gradients, which generally involve feedback loops to buffer against perturbations caused by fluctuations in gene dosage and expression. Although many gene components involved in such feedback loops have been identified, how they work together to generate a robust pattern remains unclear. The network of extracellular proteins that patterns the dorsal region of the Drosophila embryo by establishing a graded activation of the bone morphogenic protein (BMP) pathway has been studied. The BMP activation gradient itself is robust to changes in gene dosage. Computational search for networks that support robustness shows that transport of the BMP class ligands (Scw and Dpp) into the dorsal midline by the BMP inhibitor Sog is the key event in this patterning process. The mechanism underlying robustness relies on the ability to store an excess of signaling molecules in a restricted spatial domain where Sog is largely absent. It requires extensive diffusion of the BMP-Sog complexes, coupled with restricted diffusion of the free ligands. Dpp is shown experimentally to be widely diffusible in the presence of Sog but tightly localized in its absence, thus validating a central prediction of a theoretical study (Eldar, 2002).

    Graded activation of the BMP pathway subdivides the dorsal region of Drosophila embryos into several distinct domains of gene expression. This graded activation is determined by a well-characterized network of extracellular proteins, which may diffuse in the perivitelline fluid that surrounds the embryo. The patterning network is composed of two BMP class ligands (Scw and Dpp), a BMP inhibitor (Sog), a protease that cleaves Sog (Tld) and an accessory protein (Tsg), all of which are highly conserved in evolution and are used also for patterning the dorso-ventral axis of vertebrate embryos. Previous studies have suggested that patterning of the dorsal region is robust to changes in the concentrations of most of the crucial network components. For example, embryos that contain only one functional allele of scw, sog, tld or tsg are viable and do not show any apparent phenotype. Misexpression of scw or of tsg also renders the corresponding null mutants viable (Eldar, 2002).

    To check whether robustness is achieved at the initial activation gradient, signaling was monitored directly by using antibodies that recognize specifically an activated, phosphorylated intermediate of the BMP pathway (pMad). Prominent graded activation in the dorsal-most eight cell rows was observed for about 1h, starting roughly 2h after fertilization at 25°C. This activation gradient was quantified in heterozygous mutants that were compromised for one of three of the crucial components of the patterning network, Scw, Sog or Tld. Whereas homozygous null mutants that completely lack the normal gene product have a deleterious effect on signaling, the heterozygotes, which should produce half the amount of the gene product, were indistinguishable from wild type. Similarly, overexpression of the Tld protein uniformly in the embryo did not alter the activation profile. The activation profile at 18°C is the same as that at 25°C. This robustness to temperature variations is marked, considering the wide array of temperature dependencies that are observed in this temperature span. By contrast, the profile of pMad is sensitive to the concentration of Dpp. The dosage sensitivity of Dpp is exceptional among morphogens and is singled out as being haploid-insufficient (Eldar, 2002).

    No apparent transcriptional feedback, which might account for the robustness of dorsal patterning, has been identified so far. Robustness should thus be reflected in the design of interactions in the patterning network. To identify the mechanism underlying robustness, a general mathematical model of the dorsal patterning network was formulated. For simplicity, initial analysis was restricted to a single BMP class ligand (Scw or Dpp), a BMP inhibitor (Sog) and the protease (Tld). The general model accounted for the formation of the BMP-Sog complex, allowed for the diffusion of Sog, BMP and BMP-Sog, and allowed for the cleavage of Sog by Tld, both when Sog is free and when Sog is associated with BMP. Each reaction was characterized by a different rate constant (Eldar, 2002).

    Extensive simulations were carried out to identify robust networks. At each simulation, a set of parameters (rate constants and protein concentrations) was chosen at random and the steady-state activation profile was calculated by solving three equations numerically. A set of three perturbed networks representing heterozygous situations was then generated by reducing the gene dosages of sog, tld or the BMP class ligand by a factor of two. The steady-state activation profiles defined by those networks were solved numerically and compared with the initial, nonperturbed network. A threshold was defined as a given BMP value (corresponding to the value at a third of the dorsal ectoderm in the nonperturbed network). The extent of network robustness was quantified by measuring the shift in the threshold for all three perturbed networks. Over 66,000 simulations were carried out, with each of the nine parameters allowed to vary over four orders of magnitude (Eldar, 2002).

    As expected, in most cases (97.5%) the threshold position in the perturbed networks was shifted by a large extent (>50%). In most of those nonrobust cases, the BMP concentration was roughly uniform throughout the dorsal region. By contrast, Sog was distributed in a concentration gradient with its minimum in the dorsal midline, defining a reciprocal gradient of BMP activation. Thus, the key event in this nonrobust patterning mechanism is the establishment of a concentration gradient of Sog, which was governed by diffusion of Sog from its domain of expression outside the dorsal region, coupled with its cleavage by Tld inside the dorsal region. Although such a gradient has been observed, it is also compatible with other models (Eldar, 2002).

    A small class of networks (198 networks, 0.3%) was identified in which a twofold reduction in the amounts of all three genes resulted in a change of less than 10% in the threshold position. Notably, in all of these robust cases, BMP was redistributed in a sharp concentration gradient that peaked in the dorsal midline. In addition, this concentration gradient decreases as a power-low distribution with an exponent n = 2, which indicates the uniqueness of the robust solution. In these cases, Sog was also distributed in a graded manner in the dorsal region. Analysis of the reaction rate constants of the robust networks showed a wide range of possibilities for most parameters. But two restrictions were apparent and defined the robust network design: (1) in the robust networks the cleavage of Sog by Tld was facilitated by the formation of the complex Sog-BMP; (2) the complex BMP-Sog was broadly diffusible, whereas free BMP was restricted (Eldar, 2002).

    To identify how robustness is achieved, an idealized network was considered by assuming that free Sog is not cleaved and that free BMP does not diffuse. The steady-state activation profile defined by this network can be solved analytically; the solution reveals two aspects that are crucial for ensuring robustness. First, the BMP-Sog complex has a central role, by coupling the two processes that establish the activation gradient: BMP diffusion and Sog degradation. This coupling leads to a quantitative buffering of perturbations in gene dosage. Second, restricted diffusion of free BMP enables the system to store excess BMP in a confined spatial domain where Sog is largely absent. Changes in the concentration of BMP alter the BMP profile close to the dorsal midline but do not change its distribution in most of the dorsal region (Eldar, 2002).

    The complete system, comprising Sog, Tld, Tsg, both Scw and Dpp, and their associated receptors was examined next. Two additional molecular assumptions are required to ensure the robustness of patterning. First, Sog can bind and capture the BMP class ligands even when the latter are associated with their receptors. Second, Dpp can bind Sog only when the latter is bound to Tsg. Indeed, it has been shown that, whereas Sog is sufficient for inhibiting Scw, both Tsg and Sog are required for inhibiting Dpp. This last assumption implies that Tsg functions to decouple the formation of the Scw gradient from the parallel generation of the Dpp gradient, ensuring that Scw and Dpp are transported to the dorsal midline independently by two distinct molecular entities (Eldar, 2002).

    The complete model was solved numerically for different choices of rate constants. In particular, the effect of twofold changes in gene dosage was assessed. The steady-state activation profiles can be superimposed, indicating the robustness of the system. In addition, with the exception of Dpp, the expression of all other crucial network components can be altered by at least an order of magnitude before an effect on the position of a given threshold is observed. In the model, the lack of robustness to Dpp stems from its insufficient dosage. Note that the time taken to reach steady state is sensitive to these concentrations of protein. For the wide range of parameters that were used, however, the adjustment time does not exceed the patterning time. Flexible adjustment time thus facilitates the buffering of quantitative perturbations (Eldar, 2002).

    This analysis has identified two principle molecular features that are essential for robust network design: first, free Sog is not cleaved efficiently -- an assumption that is supported by the in vitro finding that Sog cleavage by Tld requires BMP; second, the diffusion of free BMP is restricted. This is the central prediction of the theoretical study, namely, that Scw diffusion requires Sog, whereas Dpp diffusion requires both Sog and Tsg. Although several reports suggest that in wild-type embryos both Dpp and Scw are widely diffusible, their ability to diffuse in a sog or tsg mutant background has not been examined as yet (Eldar, 2002).

    To monitor the diffusion of Scw or Dpp, the even-skipped (eve) stripe-2 enhancer (st2) was used to misexpress Dpp or Scw in a narrow stripe perpendicular to the normal BMP gradient. In transgenic embryos, dpp or scw RNA was detected in a stripe just posterior to the cephalic furrow. Initially the stripe was about 12 cells wide at early cleavage cycle 14, but refined rapidly to about 6 cells by late cycle 14. The st2-dpp and st2-scw embryos were viable, despite the high expression of these proteins as compared with their endogenous counterparts (Eldar, 2002).

    The activation of the BMP pathway was monitored either by staining for pMad or by following dorsal expression of the target gene race, which requires high activation. Scw is a less potent ligand than is Dpp. This experimental setup could not be used to study Scw diffusion properties because expressing st2-scw did not alter the pattern of pMad or race expression in wild-type or sog-/- embryos. By contrast, expression of st2-dpp led to an expansion of both markers in a region that extends far from the st2 expression domain, indicating a wide diffusion of Dpp in a wild-type background. Conversely, on expression of st2-dpp in sog-/- or in tsg-/- embryos, both markers were confined to a narrow stripe in the st2 domain. The width of this stripe was comparable to that of st2-dpp expression, ranging from 6 to 12 cells, indicating that Dpp does not diffuse from its domain of expression in the absence of Sog or Tsg. Taken together, these results show that both Sog and Tsg are required for Dpp diffusion, as predicted by the theoretical analysis (Eldar, 2002).

    The computation ability of biochemical networks is striking when one considers that they function in a biological environment where the amounts of the network components fluctuate, the kinetics is stochastic, and sensitive interactions between different computation modules are required. Studies have examined the effect of these properties on cellular computation mechanisms, and robustness has been proposed to be a 'design principle' of biochemical networks. The applicability of this principle to morphogen gradient patterning has been shown during early development. Quantitative analysis can be used to assess rigorously the robustness of different patterning models and to exclude incompatible ones. The remaining, most plausible model points to crucial biological assumptions and serves to postulate the central feedback mechanisms. Applying the same modelling principles to other systems might identify additional 'design principles' that underlie robust patterning by morphogen gradients in development (Eldar, 2002).

    Comprehensive identification of Drosophila dorsal-ventral patterning genes using a whole-genome tiling array

    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). Chip/chip in vivo binding data is at Berkeley Drosophila Transcription Network Project

    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).

    The previous microarray screen relied on high cutoff values for the identification of authentic DV genes. For example, only genes exhibiting 6-fold up-regulation in pipe/pipe mutant embryos were tested by in situ hybridization for localized expression in the dorsal ectoderm. Many other genes displayed >2-fold up-regulation but were not explicitly tested for localized expression. The whole-genome tiling array permitted the use of much lower cutoff values. For example, CG13800, which was identified by conventional microarray screens, falls just below the original cutoff value but displays 5-fold up-regulation in pipe/pipe mutants in the analysis. In situ hybridization assays reveal localized expression in the dorsal ectoderm. This pattern is greatly expanded in embryos derived from pipe/pipe mutant females, as expected for a gene that is either directly or indirectly repressed by the Dl gradient. Genes exhibiting even lower cutoff values were also found to display localized expression. Among these genes is a Wnt homologue, Wnt2, which is augmented only 2.25-fold in mutant embryos lacking the Dl nuclear gradient (Biemar, 2006).

    The 4-fold cutoff value used in the previous screen for candidate protein-coding genes expressed in the neuroectoderm also excluded genes expressed in this tissue. The Trim9 gene exhibits just a 2-fold increase in mutant embryos derived from Tollrm9/Tollrm10 females. Nonetheless, in situ hybridization assays reveal localized expression in the neuroectoderm of WT embryos. As expected, expression is expanded in Tollrm9/Tollrm10 mutant embryos. Another gene, CG9973, displays just 1.8-fold up-regulation but is selectively expressed in the neuroectoderm. CG9973 encodes a putative protein related to Idax, an inhibitor of the Wnt signaling pathway. Idax inhibits signaling by interacting with the PDZ domain of Dishevelled (Dsh), a critical mediator of the pathway. A Wnt2 homologue is selectively expressed in the dorsal ectoderm. Recent studies identified a second Wnt gene, WntD, which is expressed in the mesoderm. Thus, the CG9973/Idax inhibitor might be important for excluding Wnt signaling from the neuroectoderm. Such a function is suggested by the analysis of Idax activity in vertebrate embryos (Biemar, 2006).

    Additional genes were also identified that are specifically expressed in the mesoderm. Among these is CG9005, which encodes an unknown protein that is highly conserved in different animals, including frogs, chicks, mice, rats, and humans. It displays <2-fold up-regulation in Toll10B embryos but is selectively expressed in the ventral mesoderm of WT embryos. Expression is expanded in embryos derived from Toll10B mutant females (Biemar, 2006).

    Other protein-coding genes were missed in the previous screen because they were not represented on the Drosophila Genome Array used at the time. These include, for instance, CG8147 in the dorsal ectoderm and CG32372 in the mesoderm (Biemar, 2006).

    An interesting example of the use of tiling arrays to identify tissue-specific isoforms is seen for the bunched (bun) TU. bun encodes a putative sequence-specific transcription factor related to mammalian TSC-22, which is activated by TGFβ signaling. It was shown to inhibit Notch signaling in the follicular epithelium of the Drosophila egg chamber. Three transcripts are expressed from alternative promoters in bun, but it appears that only the short isoform (bun-RC) is specifically expressed in the dorsal ectoderm. A number of bun exons are ubiquitously transcribed at low levels in the mesoderm, neuroectoderm, and dorsal ectoderm. However, the 3'-most exons are selectively up-regulated in pipe/pipe mutants. It is conceivable that Dpp signaling augments the expression of this isoform, which in turn, participates in the patterning of the dorsal ectoderm (Biemar, 2006).

    In addition to protein-coding genes, the tiling array also identified uncharacterized TUs not previously annotated. Some of them are associated with ESTs, providing independent evidence for transcriptional activity in these regions. For 14 of these transfrags (61%), visual inspection of neighboring loci using the Integrated Genome Browser suggested coordinate expression of a neighboring protein-coding region (i.e., overexpressed in the same mutant background). The N-Cadherin gene (CadN) has a complex intron-exon structure consisting of ~20 different exons. The strongest hybridization signals are detected within the limits of exons, but an unexpected signal was detected ~10 kb upstream of the 5'-most exon. It is specifically expressed in the mesoderm, suggesting that it represents a previously unidentified 5' exon of the CadN gene. Support for this contention stems from two lines of evidence: (1) in situ hybridization using a probe against the 5' exon detects transcription in the presumptive mesoderm, the initial site of CadN expression; (2) using primers anchored in the 5' transfrag as well as the first exon of CadN, confirmation was obtained by RT-PCR that the recently identified TU is part of the CadN transcript. This recently identified 5' exon appears to contribute to the 5' leader of the CadN mRNA. It is possible that this extended leader sequence influences translational efficiency as seen in yeast. Because there seems to be a considerable lag between the time when CadN is first transcribed and the first appearance of the protein, it is suggested that this extended leader sequence might inhibit translation. An interesting possibility is that it does so through short upstream ORFs, as has been shown for several oncogenes in vertebrates (Biemar, 2006).

    A 5' exon was also 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).

    In addition to the identification of 10 5' exons associated with previously annotated genes such as CadN and cv-2, three other transfrags appear to correspond to 3' exons, and nine of the RNAs seem to arise from autonomous TUs. Three of these represent annotated computational RNA (CR) genes: CR32777, CR31972, and CR32957. CR32777 corresponds to roX1, which is ubiquitously expressed at the blastoderm stage, hence it represents a false positive. The other two potential noncoding RNAs were recently identified independently in two other studies, and although the expression of CR32957 could not be detected by in situ hybridization, CR31972 transcripts are detected in the mesoderm. There is no evidence that these transcripts are processed into miRNAs, but noncoding genes corresponding to known miRNA loci were also identified in the screen. Transfrag 22 corresponds to the miR-9a primary transcript (pri-mir9a) and is detected in both the dorsal- and neuroectoderm. Expression of pri-mir9a is ubiquitous in embryos derived from pipe/pipe or Tollrm9/Tollrm10 females. Transfrag 8 corresponds to pri-mir1, which is present in the mesoderm (Biemar, 2006).

    A third noncoding transcript (Transfrag 12) maps next to a known miRNA, miR-184. It is selectively expressed in the mesoderm and overexpressed in Toll10B mutants. The mesodermal expression of miR-184 has been reported. It is possible that Transfrag 12 corresponds to pri-mir-184, and that secondary structures in the miRNA region preclude detection on the array. This is seen for several other miRNA precursors expressed at various stages during embryogenesis. Alternatively, Transfrag 12 might represent the fragment resulting from Drosha cleavage of the pri-mir-184 to produce the miR-184 precursor hairpin (pre-miR-184). A similar situation has been observed for the iab4 locus. Like miR-1, miR-184 is selectively expressed in the ventral mesoderm. It will be interesting to determine whether the two miRNAs jointly regulate some of the same target mRNAs (Biemar, 2006).

    The identity of the last three transfrags is less clear. Visual inspection using the Integrated Genome Browser suggests expression of Transfrag 10 in the mesoderm, Transfrag 21 in the neuroectoderm, and Transfrag 11 in both the dorsal ectoderm and neuroectoderm. However, in situ hybridization assays confirm the predicted expression pattern only for Transfrag 11. Computational analyses designed to estimate the likelihood of translation suggest a protein-coding potential for Transfrag 10 [Likelihood Ratio Test (LRT) P < 0.001] and possibly Transfrag 11 (LRT P < 0.01), whereas Transfrag 21 could not be analyzed because of lack of conservation in other Drosophila species (Biemar, 2006).

    This work has attempted to identify nonprotein coding genes involved in patterning the DV axis of the Drosophila embryo using an unbiased approach to survey the entire genome. This study, along with earlier analyses, identified as many as 100 protein-coding genes and five to seven noncoding genes that are differentially expressed across the DV axis of the early Drosophila embryo. Roughly half of the noncoding RNAs correspond to miRNAs, although <1% of the annotated genes in the Drosophila genome encode miRNAs. Future studies will determine how these RNAs impinge on the DV regulatory network (Biemar, 2006).

    Recent studies have identified large numbers of noncoding transcripts in the mouse and human genomes. If the present study is predictive, less than one-fourth of the transcripts correspond to novel noncoding RNAs of unknown function, akin to CR31972 and Transfrag 11 expressed in the mesoderm and ectoderm, respectively. Most of the noncoding transcripts are likely to derive from intronic sequences because of the occurrence of cryptic remote 5' exons as seen for the CadN and cv-2 genes. At least 10% of the DV protein-coding genes were found to contain such exons. As a result, these genes contain large tracts of intronic sequences that might encompass regulatory DNAs such as tissue-specific enhancers. The FGF8-related gene, thisbe (ths), represents such a case. A neurogenic-specific enhancer that was initially thought to reside 5' of the TU actually maps within a large intron because of the occurrence of a remote 5' exon. It is suggested that such exons are responsible for the evolutionary "bundling" of genes and their associated regulatory DNAs. Gene duplication events are more likely to retain this linkage when regulatory DNAs map within the TU. In contrast, enhancers mapping in flanking regions can be uncoupled from their normal target gene by chromosomal rearrangements (Biemar, 2006).

    Evolution of the dorsal-ventral patterning network in the mosquito: Altered expression of sog and tld correlates with a broader domain of Dpp signaling, when compared with Drosophila.

    The dorsal-ventral patterning of the Drosophila embryo is controlled by a well-defined gene regulation network. This study addressed how changes in this network produce evolutionary diversity in insect gastrulation. Focus was placed on the dorsal ectoderm in two highly divergent dipterans, the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae. In D. melanogaster, the dorsal midline of the dorsal ectoderm forms a single extra-embryonic membrane, the amnioserosa. In A. gambiae, an expanded domain forms two distinct extra-embryonic tissues, the amnion and serosa. The analysis of approximately 20 different dorsal-ventral patterning genes suggests that the initial specification of the mesoderm and ventral neurogenic ectoderm is highly conserved in flies and mosquitoes. By contrast, there are numerous differences in the expression profiles of genes active in the dorsal ectoderm. Most notably, the subdivision of the extra-embryonic domain into separate amnion and serosa lineages in A. gambiae correlates with novel patterns of gene expression for several segmentation repressors. Moreover, the expanded amnion and serosa anlage correlates with a broader domain of Dpp signaling as compared with the D. melanogaster embryo. Evidence is presented that this expanded signaling is due to altered expression of the sog gene (Goltsev, 2007).

    A variety of dorsal patterning genes were examined in A. gambiae embryos in an effort to determine the basis for the formation of distinct ectodermal derivatives. For example hindsight (hnt; also known as peb - Flybase) is expressed along the dorsal midline of D. melanogaster embryos, while tailup (tup) is expressed in a broader pattern that encompasses both the presumptive amnioserosa and dorsolateral ectoderm. The hnt expression pattern seen in A. gambiae is similar to that detected in D. melanogaster, although there is a marked expansion in the dorsal-ventral limits of the presumptive extra-embryonic territory. By contrast, the tup pattern in A. gambiae is dramatically different from that seen in D. melanogaster -- it is excluded from the prospective serosa and restricted to the future amnion (Goltsev, 2007).

    The T-box genes Dorsocross1 (Doc1) and Doc2 are involved in amnioserosa development and expressed along the dorsal midline and in a transverse stripe near the cephalic furrow of gastrulating D. melanogaster embryos. The Doc1 and Doc2 orthologues in A. gambiae exhibit restricted expression in the presumptive amnion, similar to the tup pattern. The expression patterns of the two genes are identical but only Doc1 is shown. They are initially expressed in a broad dorsal domain but come to be repressed in the serosa. There is also a head stripe of expression comparable to the D. melanogaster pattern. Additional dorsal-ventral patterning genes are also expressed in a restricted pattern within the developing amnion. Overall, the early expression patterns of tup, Doc1 and Doc2 (and additional patterning genes) foreshadow the subdivision of the dorsal ectoderm into separate serosa and amnion lineages in Anopheles (Goltsev, 2007).

    In D. melanogaster, the patterning of the dorsal ectoderm depends on Dpp and Zen, along with a variety of genes encoding Dpp signaling components, such as the Thickveins (Tkv) receptor. Most of the corresponding genes are expressed in divergent patterns in A. gambiae embryos. For example, dpp and tkv are initially expressed throughout the dorsal ectoderm, but become excluded from the presumptive serosa and restricted to the amnion. By contrast, both genes have broad, nearly uniform expression patterns in the dorsal ectoderm of D. melanogaster embryos (Goltsev, 2007).

    There is an equally dramatic change in the zen expression pattern. In A. gambiae, expression is restricted to the presumptive serosa territory, even at the earliest stages of development. By contrast, zen is initially expressed throughout the dorsal ectoderm of cellularizing embryos in D. melanogaster, and becomes restricted to the dorsal midline by the onset of gastrulation. Thus, the dpp/tkv and zen expression patterns are essentially complementary in A. gambiae embryos, but extensively overlap in Drosophila (Goltsev, 2007).

    The loss of dpp, tkv, Doc1, Doc2 and tup expression in the presumptive serosa of A. gambiae embryos raises the possibility that zen activates the expression of one or more repressors in the serosa. It is unlikely that Zen itself is such a repressor since the expression of the A. gambiae zen gene in transgenic Drosophila embryos does not alter the normal development of the amnioserosa (Goltsev, 2007).

    Different segmentation genes were examined in an effort to identify putative serosa-specific repressors. For example, the gap gene hunchback (hb) is initially expressed in the anterior regions of A. gambiae embryos, in a similar pattern to that seen in D. melanogaster, but by the onset of gastrulation a novel pattern arises within the presumptive serosa. hb expression has also been seen in the developing serosa of other insects, including a primitive fly (Clogmia) and the flour beetle, Tribolium (Goltsev, 2007).

    Two additional segmentation genes behave like hb, empty spiracles (ems) and tramtrack (ttk). ems is involved in head patterning in D. melanogaster. Its expression is limited to a single stripe in anterior regions of cellularizing D. melanogaster embryos. Staining is seen in a comparable anterior region of A. gambiae embryos, but a second site of expression (not seen in Drosophila) is also detected in the presumptive serosa (Goltsev, 2007).

    Ttk is a maternal repressor that helps establish the expression limits of several pair-rule stripes. It is ubiquitously expressed throughout the early D. melanogaster embryo, but has a tightly localized expression pattern within the presumptive serosa of A. gambiae embryos. Thus, novel patterns of ems and ttk expression are consistent with the possibility that serosa-specific repressors help subdivide the dorsal ectoderm into separate serosa and amnion lineages in A. gambiae embryos (Goltsev, 2007).

    The analysis of dorsal-ventral patterning genes identified two critical differences between the pre-gastrular fly and mosquito embryos. First, there are separate serosa and amnion lineages in A. gambiae, but just a single amnioserosa in D. melanogaster. Second, there is an expansion in the limits of the dorsal ectoderm in A. gambiae as compared with the D. melanogaster embryo. Localized repressors might help explain the former observation of separate lineages, but do not provide a basis for the expansion of the dorsal ectoderm (Goltsev, 2007).

    In D. melanogaster, the limits of Dpp signaling are established by the repressor Brinker and the inhibitor Sog. Genetic studies suggest that Sog is the more critical determinant in early embryos. It is related to Chordin, which inhibits BMP signaling in vertebrates, and is expressed in broad lateral stripes encompassing the entire neurogenic ectoderm. The secreted Sog protein directly binds Dpp, and blocks its ability to interact with the Tkv receptor. However, Sog-Dpp complexes are proteolytically processed by the Tolloid (Tld) metalloprotease, which is expressed throughout the dorsal ectoderm of early Drosophila embryos. Tld helps ensure that high levels of the Dpp signal are released at the dorsal midline located far from the restricted source of the inhibitor Sog (Goltsev, 2007).

    The expression patterns of the sog and tld genes in A. gambiae are very different from those seen in D. melanogaster. sog expression is primarily detected in the ventral mesoderm, although low levels of sog transcripts might extend into the ventral-most regions of the neurogenic ectoderm. This pattern is more restricted across the dorsal-ventral axis than the D. melanogaster sog pattern. tld expression is restricted to lateral regions of A. gambiae embryos and is excluded from the dorsal ectoderm, which is the principal site of expression in Drosophila. These significant changes in the sog and tld expression patterns might account, at least in part, for the expanded limits of Dpp signaling in the dorsal ectoderm of A. gambiae embryos (Goltsev, 2007).

    Direct evidence for broader Dpp signaling was obtained using an antibody that detects phosphorylated Mad (pMad), the activated form of Mad obtained upon induction of the Tkv receptor. In D. melanogaster pMad expression is restricted to the dorsal midline. This is the domain where Sog-Dpp complexes are processed and peak levels of Dpp interact with the receptor Tkv. The spatial limits of the sog expression pattern are decisive for this restricted domain of pMad activity. Just a twofold reduction in the levels of Sog (sog/+ heterozygotes) causes a significant expansion in pMad expression (Goltsev, 2007).

    There is a marked expansion of the pMad expression domain in A. gambiae embryos as compared with Drosophila. The domain encompasses the entire presumptive serosa and extends into portions of the presumptive amnion. The dpp and tkv expression patterns are downregulated in the presumptive serosa, nonetheless, the pMad staining pattern clearly indicates that this is the site of peak Dpp signaling activity. The early expression of both dpp and tkv encompasses the entire dorsal ectoderm. It would appear that peak Dpp signaling is somehow maintained in the developing serosa even after the downregulation of dpp and tkv expression in this tissue. A similar scenario is seen in the Drosophila embryo, in that there is downregulation of both dpp and tkv expression along the dorsal midline of gastrulating embryos (Goltsev, 2007).

    To determine the basis for expanded Dpp signaling a sog enhancer was identified and characterized in A. gambiae. The D. melanogaster enhancer is located in the first intron of the sog transcription unit. It is ~300 bp in length and contains four evenly spaced, optimal Dorsal binding sites. These sites permit activation of sog expression by low levels of the Dorsal gradient; however, closely linked Snail repressor sites inactivate the enhancer in the ventral mesoderm. A putative A. gambiae enhancer was identified by scanning the sog locus for potential clusters of Dorsal binding sites. The recently developed cluster-draw program was used for this purpose since it successfully identified a sim enhancer in the honeybee, Apis mellifera, which is even more divergent than Anopheles. The best putative Dorsal binding cluster was identified within the first intron of the A. gambiae sog locus. Several genomic DNA fragments were tested for enhancer activity, but only this cluster was found to activate gene expression in transgenic Drosophila embryos (Goltsev, 2007).

    Two different genomic DNA fragments, 3.7 kb and 1.1 kb, that encompass the intronic binding cluster were tested in transgenic embryos. Both fragments were attached to a lacZ reporter gene containing the core eve promoter from D. melanogaster, and both direct lacZ expression in the presumptive mesoderm. They exhibit the same restricted dorsal-ventral limits of expression as that seen for the endogenous sog gene in A. gambiae, although the smaller fragment produces ventral stripes whereas the larger fragment directs a more uniform pattern. The change in the dorsal-ventral limits -- broad expression in D. melanogaster and restricted expression in A. gambiae -- might be due to the quality of individual Dorsal binding sites in the two enhancers (Goltsev, 2007).

    Therefore, s comprehensive analysis of dorsal-ventral patterning genes in the A. gambiae embryo reveals elements of conservation and divergence in the gastrulation network of D. melanogaster. There is broad conservation in the expression of regulatory genes responsible for the patterning of the mesoderm and neurogenic ectoderm, including sequential expression of sim, vnd and ind in the developing nerve cord. By contrast, there are extensive changes in the expression of regulatory genes that pattern the dorsal ectoderm. These changes foreshadow the subdivision of the dorsal ectoderm into separate serosa and amnion lineages in A. gambiae (Goltsev, 2007).

    The major difference in the early patterning of the mesoderm in flies and mosquitoes concerns the manner in which mesoderm cells enter the blastocoel of gastrulating embryos. In D. melanogaster, there is a coherent invagination of the mesoderm through the ventral furrow, much like the movement of bottle cells through the blastocoel of Xenopus embryos. By contrast, there is no invagination of the mesoderm in A. gambiae. Instead, the mesoderm undergoes progressive ingression during germband elongation. This type of ingression is seen in D. melanogaster mutants lacking fog signaling. The A. gambiae genome lacks a clear homologue of fog, and it is therefore conceivable that fog represents an innovation of the higher Diptera that was only recently incorporated into the D. melanogaster dorsal-ventral patterning network (Goltsev, 2007).

    D. melanogaster is somewhat unusual in having an amnioserosa, rather than separate serosa and amnion tissues as seen in most insects. In certain mosquitoes the serosa secretes an additional proteinaceous membrane that provides extra protection against desiccation. The changes in gene expression in the D. melanogaster and A. gambiae dorsal ectoderm provide a basis for understanding the evolutionary transition of two dorsal tissues in A. gambiae into a novel single tissue in higher dipterans (Goltsev, 2007).

    The D. melanogaster amnioserosa expresses a variety of regulatory genes, including Doc1/2 and tup. The expression of most of these genes is restricted in the presumptive amnion of the A. gambiae embryo. zen is the only dorsal patterning gene, among those tested, that exhibits restricted expression in the serosa. Several segmentation genes have a similar pattern, and one of these, ttk, encodes a known repressor. Ectopic expression of Ttk causes a variety of patterning defects in Drosophila embryos, including disruptions in head involution and germband elongation that might arise from alterations in the amnioserosa. It is proposed that zen activates ttk in the serosa of A. gambiae embryos. The encoded repressor might subdivide the dorsal ectoderm into separate serosa and amnion tissues by inhibiting the expression of Doc1/2 and tup in the serosa. The loss of this putative zen-ttk regulatory linkage might be sufficient to allow Dpp signaling to activate tup and Doc1/2 throughout the dorsal ectoderm, thereby transforming separate serosa and amnion tissues into a single amnioserosa. According to this scenario, the loss of zen binding sites in ttk regulatory sequences might be responsible for the evolutionary transition of the amnioserosa (Goltsev, 2007).

    The formation of separate amnion and serosa tissues is not the only distinguishing feature of A. gambiae embryos when compared with D. melanogaster. There is also a significant expansion in the overall limits of the dorsal ectoderm. This can be explained, in part, by distinct patterns of sog expression. The broad expression limits of the Sog inhibitor are responsible for restricting Dpp/pMad signaling to the dorsal midline of the D. melanogaster embryo. This pattern depends on a highly sensitive response of the sog intronic enhancer to the lowest levels of the Dorsal gradient. The Dorsal binding sites in the sog enhancer are optimal sites, possessing perfect matches to the idealized position weighted matrix of Dorsal recognition sequences. By contrast, the A. gambiae intronic sog enhancer contains low-quality Dorsal binding sites, similar to those seen in the regulatory sequences of genes activated by peak levels of the Dorsal gradient, such as twist. The binding sites in the D. melanogaster sog enhancer have an average score of ~10. By contrast, the best sites in the A. gambiae sog enhancer have scores in the 6.5-7 range, typical of enhancers that mediate expression in the mesoderm in response to high levels of the Dorsal gradient. Although every potential regulatory sequence in the A. gambiae sog locus was not explicitly tested, none of the putative Dorsal binding clusters in the vicinity of the gene possess the quality required for activation by low levels of the Dorsal gradient in the neurogenic ectoderm. Thus, the narrow limits of sog expression in A. gambiae embryos can be explained by the occurrence of low-quality Dorsal binding sites, along with the loss of Snail repressor sites (Goltsev, 2007).

    The altered sog expression pattern is probably not the sole basis for the expansion of the dorsal ectoderm. A. gambiae embryos also exhibit a significant change in the tld expression pattern. tld is expressed throughout the dorsal ectoderm in D. melanogaster, but restricted to the neurogenic ectoderm of A. gambiae. Tld cleaves inactive Tsg-Sog-Dpp complexes to produce peak Dpp signaling along the dorsal midline of Drosophila embryos. It is proposed that the altered tld pattern in combination with altered sog leads to two dorsolateral sources of the active Dpp ligand in mosquito embryos. The sum of these sources might produce a step-like distribution of pMad across dorsal regions of mosquito embryos. This broad plateau of pMad activity might be responsible for the observed expansion of the dorsal ectoderm territory, and the specification of the serosa (Goltsev, 2007).

    In Drosophila, tld is regulated by a 5' silencer element that prevents the gene from being expressed in ventral and lateral regions in response to high and low levels of the Dorsal gradient. This silencing activity is due to close linkage of Dorsal binding sites and recognition sequences for 'co-repressor' proteins. Preliminary studies suggest that Dorsal activates the A. gambiae tld gene, possibly by the loss of co-repressor binding sites in the 5' enhancer (Goltsev, 2007).

    It is proposed that there are at least two distinct threshold readouts of Dpp signaling in the dorsal ectoderm of A. gambiae embryos. Type 1 target genes, such as hb, ems, ttk and zen, are activated by high levels and thereby restricted to the presumptive serosa. Type 2 target genes, such as tup and Doc1/2, can be activated - in principle - by both high and low levels of Dpp signaling in the presumptive serosa and amnion. However, these target enhancers contain binding sites for one or more type 1 repressors expressed in the serosa. The favorite candidate repressor is Ttk. Perhaps the type 2 tup enhancer contains optimal pMad activator sites as well as binding sites for the localized repressor Ttk, which keeps tup expression off in the serosa and restricted to the amnion. As discussed earlier, the simple loss of ttk regulation by the Dpp signaling network might be sufficient to account for the evolutionary conversion of separate serosa and amnion tissues into a single amnioserosa. Localization of this single tissue within a restricted domain along the dorsal midline would arise from concomitant dorsal shifts in the sog and tld expression patterns (Goltsev, 2007).

    References

    Biemar, F., et al. (2006). Comprehensive identification of Drosophila dorsal-ventral patterning genes using a whole-genome tiling array. Proc. Natl. Acad. Sci. 103(34): 12763-8. Medline abstract: 16908844

    Eldar, A., et al. (2002). Robustness of the BMP morphogen gradient in Drosophila embryonic patterning. Nature 419: 304-308. 12239569

    Goltsev, Y., Fuse, N., Frasch, M., Zinzen, R. P., Lanzaro, G. and Levine, M. (2007). Evolution of the dorsal-ventral patterning network in the mosquito, Anopheles gambiae. Development 134(13): 2415-24. Medline abstract: 17522157

    Huang, A. M., Rusch, J., and Levine, M. (1997). An anteroposterior Dorsal gradient in the Drosophila embryo. Genes Dev. 11(15): 1963-1973. Medline abstract: 9271119


    For maternal genes that establish DV polarity see the dorsal group

    Zygotically transcribed genes

    Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

    The Interactive Fly resides on the
    Society for Developmental Biology's Web server.