short gastrulation


DEVELOPMENTAL BIOLOGY

Embryonic

sog is expressed in a broad lateral stripe of cells that abuts the dorsal territory of dpp-expressing cells (Francois, 1994). The disappearance of SOG ventrally may be a direct result of repression by Snail. The ventral boundary of SOG includes the mesectoderm as cells expressing single minded also express sog. sog transcripts localize predominantly apically. sog expression is progressively lost dorsally, and by germ-band extention, sog transcripts are confined to the ventral midline. At full germ band elongation, sog is expressed in patches dorsal to the tracheal pits. After germ band retraction, sog is expressed externally, in ventral epidermal cells in a pattern resembling the outline of future denticle belts, and internally, in the endoderm of the gut and in the esophagus (Francois, 1994).

Embryonic development of the Drosophila corpus cardiacum

The development of the Drosophila neuroendocrine gland, the corpus cardiacum (CC) was investigated, along with the role of regulatory genes and signaling pathways in CC morphogenesis. CC progenitors segregate from the blastoderm as part of the anterior lip of the ventral furrow. Among the early genetic determinants expressed and required in this domain are the genes giant (gt) and sine oculis (so). During the extended germ band stage, CC progenitor cells form a paired cluster of 6–8 cells sandwiched in between the inner surface of the protocerebrum and the foregut. While flanking the protocerebrum, CC progenitors are in direct contact with the neural precursors that give rise to the pars intercerebralis, the part of the brain whose neurons later innervate the CC. At this stage, the CC progenitors turn on the homeobox gene glass (gl), which is essential for the differentiation of the CC. During germ band retraction, CC progenitors increase in number and migrate posteriorly, passing underneath the brain commissure and attaching themselves to the primordia of the corpora allata (CA). During dorsal closure, the CC and CA move around the anterior aorta to become the ring gland (see Image). Signaling pathways that shape the determination and morphogenesis of the CC are decapentaplegic (dpp) and its antagonist short gastrulation (sog), as well as hedgehog (hh) and heartless (htl; a Drosophila FGFR homolog). Sog is expressed in the midventral domain from where CC progenitors originate, and these cells are completely absent in sog mutants. Dpp and hh are expressed in the anterior visceral head mesoderm and the foregut, respectively; both of these tissues flank the CC. Loss of hh and dpp results in defects in CC proliferation and migration. Htl appears in the somatic mesoderm of the head and trunk. Although mutations of htl do not cause direct effects on the early development of the CC, the later formation of the ring gland is highly abnormal due to the absence of the aorta in these mutants. Defects in the CC are also caused by mutations that severely reduce the protocerebrum, including tailless (tll), suggesting that additional signaling events exist between brain and CC progenitors. The parallels between neuroendocrine development in Drosophila and vertebrates are discussed (De Velasco, 2004).

In the larva, the ring gland forms a large and conspicuous structure located anterior to the brain and connected to the brain by a pair of tracheal branches and the paired nerve of the corpus cardiacum (NCC). Three different glands, the corpus allatum (CA; dorsally), prothoracic gland (laterally), and corpus cardiacum (CC; ventrally) form part of the ring gland. By far, most of its volume is taken up by the prothoracic gland whose cells, the source of ecdysone, grow in size and number as larval development progresses, whereas the cells of the CC remain small and do not appear to proliferate. Both the CC and CA, as well as axons innervating the ring gland, are FasII positive from the late embryonic stage onward. Labeling of the CC is stronger and starts earlier (stage 11) than that of the CA (stage 15), which makes it easy to distinguish between the two structures in the embryo. Another convenient marker of the CC is adipokinetic hormone (AKH), which is expressed exclusively in the CC from late embryonic stages onward (De Velasco, 2004).

The ring gland of the mature embryo is situated posterior to the brain hemispheres. The CC and CA occupy their positions ventral and dorsal to the aorta, respectively. The prothoracic gland cannot yet be recognized as a separate entity, possibly due to the fact that its precursors are small and few in number. Cells of the CC number around eight on each side and are arranged in a U-shape around the floor of the aorta. All cells are spindle shaped and send short processes ventromedially where they meet and form a bundle attached to the ventral wall of the aorta (subaortic processes) (De Velasco, 2004).

Several signaling pathways, notably Shh, BMP, and BMP antagonists, Wnt and FGF, specify the fate map of the head in vertebrates and also control later morphogenetic events shaping head structures. The same signaling pathways are active at multiple stages in Drosophila head development, and the pattern of activity and requirement of these pathways in regard to CC development was therefore investigated (De Velasco, 2004). .

The first signal acting zygotically in the Drosophila head is the BMP homolog Dpp, which forms a dorsoventral gradient across the blastoderm. The homolog of the BMP antagonist Chordin, short gastrulation (Sog), is expressed in the ventral blastoderm, overlapping with the ventral furrow. Loss of sog results in the absence of the CC, while the SNS is still present, which reflects ventral origin of the CC. Sog seems to be the only signal, of those tested, required for CC determination, since mutation of all other pathways does not eliminate the CC but merely effects its size, shape, or location (De Velasco, 2004).

Following its early widespread dorsal expression, Dpp becomes more confined during gastrulation to a narrow mid-dorsal stripe and an anterior cap that corresponds to parts of the anlagen of the esophagus and epipharynx. From this domain segregates the most anterior population of head mesoderm cells that give rise to the visceral muscle of the esophagus and which maintain Dpp expression. The visceral mesoderm of the esophagus flanks both CC and SNS. Loss of Dpp causes absence of the SNS; the CC is still present and expresses AKH but does not migrate posteriorly (De Velasco, 2004).

Activity of the MAPK signaling pathway is widespread in the Drosophila head from gastrulation onward. Beside a wide anterior and posterior domain traversing the lateral and dorsal domain of the head ectoderm, the primordia of the foregut, including the SNS, and head mesoderm show a dynamic MAPK activity. At least two RTKs, EGFR and FGFR/heartless, drive the MAPK pathway in the embryonic head. EGFR is responsible for activation in the ectoderm and foregut. Loss of EGFR causes widespread cell death in the head and the absence of the SNS. The CC is still present, although reduced in size. Activation of MAPK by Heartless (Htl) occurs in a narrow anterior domain of head mesoderm that gives rise to the dorsal pharyngeal muscles. The foregut, SNS, and CC develop rather normally in htl mutants. However, the CC shows variable defects in shape and location, which are most likely due to the absence of the aorta and CA, both of which are derivatives of the dorsal mesoderm, which is defective in htl loss of function and to which the CC is normally attached (De Velasco, 2004).

Larval

The Drosophila EGF receptor (Egfr) is required for the specification of diverse cell fates throughout development. How the activation of Egfr controls the development of vein and intervein cells in the Drosophila wing has been examined. Two distinct events are involved in the determination and differentiation of wing vein cells: (1) the establishment of a positive feedback amplification loop, which drives Egfr signaling in larval stages (at this time, rhomboid, in combination with vein, initiates and amplifies the activity of Egfr in vein cells); (2) the late downregulation of Egfr activity [at this point, the inactivation of MAPK in vein cells is necessary for the maintenance of the expression of decapentaplegic (dpp) and becomes essential for vein differentiation. Subsequently, Egfr becomes activated in intervein territories. During the time that dpp is expressed in vein territories, MAPK activity builds up in intervein territories, probably due to the presence of Vn, a weak Egfr activator. As a consequence, aos expression relocates to intervein territories. Together, these temporal and spatial changes in the activity of Egfr constitute an autoregulatory network that controls the definition of vein and intervein cell types (Martin-Blanco, 1999).

Several types of cell-cell communication have been proposed to be required during the latter stages of pupal wing development. The dpp gene encodes a member of the TGFbeta superfamily and is expressed during early pupal development in vein primordia. A class of loss-of-function dpp alleles and certain combinations of Dpp receptor mutants lead to vein-loss phenotypes. Mosaic analysis of dpps allele show that mitotic clones affect the differentiation of veins. Meanwhile, the effects of overexpression of dpp or an active form of its receptor thick veins (tkv) indicate that Dpp directs vein differentiation through activation of Tkv in pupal stages. The initiation of dpp expression in pupal stages depends on the activity of early acting genes, and in particular Egfr activity. However, although Egfr signaling is downregulated in vein territories during pupariation, dpp expression is maintained through an autoregulatory loop and remains high in vein cells until their final differentiation. Interestingly, in intervein cells, dpp expression is not activated in response to the Egfr activity described above. On the contrary, these cells express short gastrulation (sog), a gene that exerts an opposing effect to dpp. sog plays a role restricting vein formation to the center of the provein regions. dpp and sog interact antagonistically during vein differentiation. Ectopic activation of Egfr signaling in pupal stages abolishes dpp expression from veins. This suppression of dpp correlates with the loss of veins observed in this condition; it is reminiscent of the effect of Sog overexpression in pupal wings. Moreover, vein plexates induced by compromising Egfr activity in pupal wings, associate with a broadening of dpp-expressing areas (Martin-Blanco, 1999).

It is suggested that Egfr signaling downregulation from vein territories allows dpp to autoregulate dpp expression. It remains to be determined whether sog expression depends on Egfr in intervein territories, or is a consequence of the activity of intervein-specific genes such as blistered. The model presented here on how a single receptor (Egfr), triggering a conserved signal transduction pathway, is used reiteratively to implement two different cell fates in the development of the fly wing serves to reconcile many observations that have been made regarding cell fate specification in the wing. This may well provide a paradigm for the regulation of Egfr signal transduction in other developmental events (Martin-Blanco, 1999).

Hexagonal packing of Drosophila wing epithelial cells by the planar cell polarity pathway

The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but they are poorly understood. This problem was investigated in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented by the planar cell polarity pathway. They are constructed by a hexagonal array of wing epithelial cells. Wing epithelial cells are irregularly arranged throughout most of development, but they become hexagonally packed shortly before hair formation. During the process, individual cell boundaries grow and shrink, resulting in local neighbor exchanges, and Cadherin is actively endocytosed and recycled through Rab11 endosomes. Hexagonal packing depends on the activity of the planar cell polarity proteins. It is proposed that these proteins polarize trafficking of Cadherin-containing exocyst vesicles during junction remodeling. This may be a common mechanism for the action of planar cell polarity proteins in diverse systems (Classen, 2005).

How might Cadherin or other junctional material be added to a growing boundary? In other epithelia, Cadherin is dynamically endocytosed and recycled to modulate cell adhesion. To test whether this might happen in the pupal wing, the temperature-sensitive shibire (shi) mutation of dynamin was used. Dynamin is required for scission of endocytic vesicles and vesicles formed from Rab11 recycling endosomes. A total of 30-45 min after shifting to 34°C, gaps form in junctional E-Cadherin in shi mutant wings that are not found in wild-type control wings, even after 3 hr of temperature shift. Similar results are obtained in clones of shi mutant cells. The gaps form exclusively in intervein regions, and they occur primarily at or adjacent to vertices. Similar results were obtained for Armadillo, another adherens junction protein. In contrast, the septate junction protein Coracle and basolaterally localized CD2GFP were undisturbed by loss of Dynamin. After 3 hr at 34°C, shi mutant cells show even larger gaps in Cadherin. By 6 hr, cell-free areas are seen in the intervein region by Cadherin staining. After these animals are restored to 18°C, emerging adults have holes in wing intervein regions. None of these changes are observed when temperature shifts are performed on third instar larvae, even for longer times. Loss of Cadherin is not a consequence of cell death; Cadherin is lost before Caspase is found in the nucleus. These data suggest that Dynamin is required to maintain uniform localization of adherens junctions, but not septate junctions or basolateral proteins, during repacking. Development of holes in intervein regions where Cadherin gaps form suggests that the loss of junctional proteins disturbs epithelial integrity (Classen, 2005).

To precisely define the stage at which Dynamin is required to maintain Cadherin, shi mutants were systematically shifted to 34°C during a sliding 6 hr window starting just after pupariation and ending after hair formation. The frequency and placement of holes in the adult wing were quantified as a read-out because antibody penetration is prevented by the cuticle throughout much of pupal development. Although a variety of phenotypes were observed, only temperature shifts initiated between pupal stage P2A and mid-P2C (before hair formation) cause holes in the wing. These data show that epithelial repacking is temporally coincident with the requirement for Dynamin (Classen, 2005).

To confirm that Cadherin enters the endocytic pathway at the time of hexagonal repacking, GFP-Cadherin-expressing pupal wings (stage P2B) were stained with FM4-64. FM4-64 labels the plasma membrane and endosomes that form after its addition. The majority of pupal wing cells contain multiple internal spots of GFP-Cadherin that colocalize with FM4-64 after 15-30 min. Thus, Cadherin is actively endocytosed during repacking (Classen, 2005).

To ask which type of endosomes contained Cadherin, flies that ubiquitously expressed YFPRab11 or CFPRab5 at low levels were used. Rab11 labels recycling endosomes, and Rab5 marks early endosomes. Cadherin was observed in both types of endosomes, supporting the idea that it is endocytosed and recycled (Classen, 2005).

In MDCK cells, Cadherin is delivered through Rab11 endosomes (Lock, 2005). To ask whether this occurs in the wing, Rab11 function was disturbed by short-term expression of the dominant-negative Rab11SN. A total of 3 hr after initiating Rab11SN expression, Cadherin begins to be lost from the junctional region -- a phenotype similar to that of the shi mutant. These cells are not apoptotic. No gaps form when Rab11SN is expressed for similar times in larval wing discs. Thus, Rab11 is required to deliver Cadherin to junctions, and this requirement is acute during epithelial repacking. Loss of junctional E-Cadherin in dynamin mutant cells may reflect Dynamin's function at Rab11 endosomes (Classen, 2005).

The exocyst is a multiprotein complex that mediates polarized membrane delivery from recycling endosomes and from the golgi in many different cell types. In the thorax, E-Cadherin delivery from recycling endosomes to the zonula adherens depends on exocyst components (Langevin, 2005). To test whether E-Cadherin was recycled via the exocyst during repacking in the wing, a mutation was utilized in Sec5 (sec5E13) that has been suggested to preferentially perturb recycling. Cadherin accumulates in internal vesicles and along the plasma membrane in sec5E13 mutant cells. Accumulation of internal vesicles suggests that delivery of Cadherin is slowed. It is not known whether higher levels of peripheral Cadherin staining reflect accumulated unfused vesicles, or whether Sec5 may also function at some other step in Cadherin trafficking (Classen, 2005).

To ask whether perturbing endocytosis and recycling causes defective cell packing, shi mutant wings were examined shortly after the shift to the restrictive temperature. Compared with wild-type shifted to the same temperature, shi tissue was less hexagonal and had a higher variability in the length of individual cell contacts. This is consistent with the possibility that Dynamin-dependent recycling of junctional components is needed to remodel junctions; however, packing may have been perturbed by some other Dynamin-dependent process (Classen, 2005).

To test whether turnover of Cadherin itself was required for hexagonal packing, expression of an E-Cadherin:α-Catenin fusion protein was induced at the time of repacking. A similar vertebrate construct is not regulated by β-catenin, causes abnormally stable adhesiveness, and inhibits motility in L cells. Expression of this construct disrupts hexagonal packing and increases the variability of cell contact lengths. This is consistent with the idea that junction remodeling depends on the disassembly of E-Cadherin-mediated contacts, although additional effects mediated by irreversible linkage to the actin cytoskeleton cannot be ruled out (Classen, 2005).

A link between the PCP pathway and epithelial repacking is suspected, because repacking occurs at the time that these proteins are thought to polarize. Therefore neighbor number and junction length variability was quantified at the time of hair outgrowth in different PCP mutants. For prickle (pk-sple13/26), neighbor number was quantitated over time (Classen, 2005).

pk-sple13/26 wings begin repacking at the same time as wild-type; however, the process is less successful. Whereas wild-type wings reduce the percentage of pentagonal cells from 34% to 13% by the time that hairs begin to emerge, pk-sple13/26 wings retain 21%. Thus, about 40% of the pentagonal cells that normally assemble boundaries with new neighbors (and become hexagonal) fail to do so in pk-sple mutants. Consistent with this, pk-sple wing epithelia contain abnormally high numbers of four-way vertices between cells. pk1 mutant wings are even more irregularly packed than pk-sple13/26 wings. A total of 62% of the pentagonal cells that would normally become hexagonal fail to assemble boundaries with new neighbors in pk1 wings. Even four-sided cells accumulate significantly in pk1 mutant wings. Individual cell contact lengths are also much more variable; while pk-sple13/26 boundary lengths were 9% more variable than wild-type, those of pk1 were 42% more variable. These data are consistent with the earlier observation that adult pk wings frequently contain pentagonal cells. These data suggest that the assembly of new cell boundaries and regularization of junction length do not occur efficiently in the absence of products of the Pk-Sple locus (Classen, 2005).

Packing defects of the hypomorphic Flamingo (fmi) allele, fmi(stan)3, are mild but significant. The null allele fmiE59 produces much stronger defects. The variability of individual junctional lengths in these cells is more than twice that of wild-type, and only 69% of fmiE59 mutant cells become hexagonal, compared with 78% in wild-type. Pentagonal cells persisted in fmiE59 mutants (27% compared with 13% in wild-type). This suggests that the majority of pentagonal cells fail to assemble boundaries with new neighbors when Fmi is missing (Classen, 2005).

The packing geometry was examined of two different frizzled (fz) alleles, fzR52 and fzP21. fzP21 mutant wings fall into two classes. While the majority of wild-type and PCP mutant wings initiate hair formation by 42 hr after puparium formation (APF) (at 22°C), a subset of fzP21 mutant wings does not. Since these wings were not apoptotic (as indicated by Caspase staining), they were included in the analysis and quantified separately. Even at 50 hr APF, their packing is much more irregular than that of wild-type . Defects in fzP21 mutant wings that do initiate hair formation by 42 hr APF are milder but still significant. fzR52 homozygotes do not produce viable pupae in these experiments, and homozygous mutant clones are small. These clones have even stronger packing defects than those of fzP21, suggesting that little repacking occurs in fzR52 homozygous tissue. Thus, Fz is needed to develop regular hexagonal packing (Classen, 2005).

stbm6 and dgo380 mutant wings have milder, but significant, alterations in the ratio of pentagons, hexagons, and heptagons and of four-way vertices. Both mutants, however, affect junction length variability more strongly than pk-sple13/26. Taken together, these data indicate that PCP mutant cells fail to efficiently assemble boundaries with new neighbors and cannot regularize their packing geometry (Classen, 2005).

To ask whether interfering with PCP polarity could alter the geometry of packing in wild-type cells, cells were examined surrounding PCP mutant clones with either autonomous (fmiE59) or nonautonomous (fzR52) effects on polarity. The frequency of pentagons, hexagons, and heptagons was examined in fzR52 and fmiE59 mutant clones, and in the areas of disturbed and normal Fmi polarity surrounding both. The mutant cells within both fzR52 and fmiE59 clones are abnormally packed. However, whereas the packing defects caused by Fmi clones are predominantly restricted to the clone and directly adjacent cells, Fz clones alter packing over long distances in wild-type tissue in the same regions where Fmi polarity is disturbed. The abnormal packing of wild-type cells surrounding fzR52 clones is unlikely to be a consequence of altered cell packing within the mutant clone, because fmiE59 mutant clones pack just as abnormally, but do not perturb packing in the surrounding tissue. This suggests that dominant reorientation of Fmi polarity by frizzled mutant clones disturbs the repacking of wild-type cells (Classen, 2005).

To investigate how the PCP proteins were localized during repacking, pupal wings were imaged for Fmi before, during, and after hexagonal packing. Since it is thought that PCP proteins do not polarize until shortly before hair formation, it was surprised to find that the subcellular distribution of Fmi is polarized in many areas of the wing before junction remodeling is initiated, even in late third instar wing discs and prepupal wings. Fz-GFP is distributed similarly. This polarity may have been missed because it exhibits less long-range coherence in imaginal discs and prepupal wings than it does later (Classen, 2005).

In prepupal wings, Fmi polarity is roughly proximal-distal in the region surrounding L3. Coherent Fmi polarity is lost at the beginning of the pupal period: this is exactly the time at which junction remodeling initiates. Although polarity is not coherent, Fmi is not uniformly distributed along cell boundaries. This can be clearly seen when Fmi localization is compared to that of E-Cadherin (Classen, 2005).

At pupal time TP1, Fmi polarization begins in vein cells as they contract their apical cross-section. Intervein regions contain only small groups of cells with coherent polarity, and the axes of these groups are not always proximal-distal. By TP2, Fmi polarity is coherent between larger groups of cells, although the axis of polarity is still mixed. Fmi polarity is aligned in large coherent domains along the proximal-distal axis by TP4, when hexagonal packing is completed, and it remains unchanged at TP5 when hairs emerge. In summary, PCP proteins polarize during larval and prepupal stages, alignment of polarity between cells is disturbed when junction remodeling begins, and long-range polarity is reestablished as hexagonal packing is completed. Early polarization of PCP proteins is consistent with the genetic requirement for fz and ds activity at this time to determine the axis of polarity, and it suggests that the feedback loop that organizes coupled proximal and distal domains probably acts during these early stages (Classen, 2005).

It was asked whether PCP proteins might affect packing by influencing recycling of junctional components. Therefore, it was asked whether PCP mutants enhance the hole formation caused by shi loss of function. Double mutant pupae were shifted to a subrestrictive temperature that never causes holes to form in shi mutants or in PCP mutants. When shi is combined with dgo380, stbm6, stbm153, stbmD, stan3, pk-spl1, or pk1, hole formation occurs even under these mild conditions. This raises the possibility that PCP proteins may worsen Cadherin recycling defects in dynamin mutant cells. Consistent with this, gaps in Cadherin arise more frequently in double shi;pk1 or shi;dgo380 mutant wings than in wings mutant for shi alone. This suggests that Cadherin is recycled less efficiently in the absence of PCP proteins (Classen, 2005).

Despite this enhancement, no striking abnormalities in Cadherin distribution were seen in most PCP mutants. fzP21 mutant cells sometimes show gaps in E-Cadherin that are similar to, but much less frequent than, those of shi mutants. In fmiE59 mutant cells, E-Cadherin levels are elevated, but no gaps in localization are observed. These observations suggest that PCP proteins are not required for delivery of Cadherin to cell contacts during remodeling. Nevertheless, the PCP mutants enhance Cadherin recycling defects caused by loss of Dynamin. One model consistent with this shows that PCP proteins bias Cadherin recycling to specific places on the cortex. Reducing both the rate of recycling and its elevation at a particular site could exacerbate the failure of Cadherin delivery to growing cell boundaries (Classen, 2005).

To test whether exocyst components were polarized by PCP proteins, Sec5 localization was examined during repacking of the wing epithelium. At this time, cell shapes are irregular, and Fmi polarity is not coherent between cells. Nevertheless, Fmi accumulates preferentially on specific regions of the cortex. Although Sec5 vesicles are seen throughout the cell, they are particularly enriched near Fmi-positive cell boundaries. Enrichment persists as Fmi polarity becomes aligned (Classen, 2005).

To test whether Fmi plays an active role in recruiting Sec5, Fmi was overexpressed and Sec5 localization was examined. Overexpressed Fmi is present uniformly around the cortex and in large punctate structures within the cell. Sec5 dramatically accumulates in cells overexpressing Fmi and is recruited to sites of Fmi localization. Large internal structures positive for Fmi and Sec5 also contain Cadherin. These observations indicate that Fmi can recruit Sec5-positive vesicles containing E-Cadherin, and they suggest that PCP proteins may promote hexagonal packing by polarizing membrane trafficking (Classen, 2005).

The conserved cassette of PCP proteins controls a variety of seemingly different developmental processes, and no common cell biological mechanism has ever been proposed for their action. Polarizing membrane trafficking by recruiting Sec5 is a basic function that could be utilized in many different contexts, and it may help explain the requirement of PCP proteins in a divergent set of processes. Both rotation of photoreceptor clusters and convergent extension movements depend on the ability of cells to make and break intercellular contacts, as they do during hexagonal packing in the wing. Consistent with this, Silberblick (Wnt-11) acts through the PCP pathway and appears to affect endocytic trafficking of Cadherin during zebrafish gastrulation. Recruitment of exocyst components might also be a plausible mechanism to explain the ability of PCP proteins to bias Notch Delta signaling between R3 and R4 photoreceptors, since Delta delivery is dependent on the exocyst. In the future, identifying the chain of events that leads from PCP protein localization to exocyst recruitment may increase the understanding of these important processes (Classen, 2005).

Graded maternal short gastrulation protein contributes to embryonic dorsal-ventral patterning by delayed induction

Establishment of the dorsal–ventral (DV) axis of the Drosophila embryo depends on ventral activation of the maternal Toll pathway, which creates a gradient of the NFkappaB/c-rel-related transcription factor Dorsal. Signaling through the maternal BMP pathway also alters the dorsal gradient, probably by regulating degradation of the IkB homologue Cactus. The BMP4 homologue decapentaplegic (dpp) and the BMP antagonist short gastrulation (sog) are expressed by follicle cells during mid-oogenesis, but it is unknown how they affect embryonic patterning following fertilization. This study provides evidence that maternal Sog and Dpp proteins are secreted into the perivitelline space where they remain until early embryogenesis to modulate Cactus degradation, enabling their dual function in patterning the eggshell and embryo. Metalloproteases encoded by tolloid (tld) and tolkin (tok), which cleave Sog, are expressed by follicle cells and are required to generate DV asymmetry in the Dpp signal. Expression of tld and tok is ventrally restricted by the TGF-α ligand encoded by gurken, suggesting that signaling via the EGF receptor pathway may regulate embryonic patterning through two independent mechanisms: by restricting the expression of pipe and thereby activation of Toll signaling and by spatially regulating BMP activity (Carneiro, 2006).

This study has shown that sog, dpp, and tld act during oogenesis to promote the formation of dorsal anterior structures of the eggshell and to establish the embryonic DV axis. According to a proposed model, Sog is produced in follicle cells and is processed into different forms depending on DV location and stored in the perivitelline space. These forms of Sog then persist until early stages of embryogenesis at which time they act by a delayed induction mechanism to alter signaling mediated by maternally derived Dpp. It is proposed that an asymmetric distribution of Sog peptides is produced through the action of the ventrally localized Tld and Tok metalloproteases. Different forms of Sog act locally to inhibit Dpp signaling ventrally (e.g., N-Sog) or diffuse over considerable distances to concentrate Dpp dorsally (e.g., full-length Sog or C-Sog). According to this model, a dorsal-to-ventral gradient of Dpp activity is formed in the perivitelline space that counteracts and sharpens the inverse gradient of nuclear dorsal (Carneiro, 2006).

An important finding in this study is that Sog protein produced by follicle cells is secreted into the perivitelline space where it persists until the end of oogenesis and early embryogenesis, prior to initiation of zygotic sog expression. One way maternal Sog fragments might influence DV patterning in the embryo is to modify zygotic Dpp signaling. However, maternal Dpp signaling is involved in establishing the relative positions of the ventral mesoderm versus the lateral neuroectodermal territories, while zygotic Dpp activity determines the relative positions of dorsal and lateral domains. These distinct phenotypes suggest that maternal Sog acts by modulating the maternal rather than the zygotic component of Dpp signaling (Carneiro, 2006).

This analysis also suggests that the Dpp synthesized by follicle cells is secreted into the perivitelline space and stored there until advanced stages of oogenesis. These maternally synthesized Sog and Dpp proteins may act on the embryo following fertilization when signaling through the Toll pathway is initiated. Several lines of evidence support this hypothesis. (1) Through epistatic analysis, it was shown that maternal Dpp does not act upstream of the Toll receptor. Therefore, genes expressed in the follicle cell epithelium that regulate DV patterning exclusively via the Toll pathway should not be targets of maternal Dpp signaling. Alternatively, undescribed non-Toll mediators of DV patterning could potentially be targets of maternal Dpp in the follicular epithelium. (2) Blocking Tkv receptor function or reducing maternal Dpp activity (by 8xhssog, in follicle cells has no effect on the pattern of pip expression. It has been shown that maternal dpp does not alter grk expression. Thus, no evidence was found that the embryonic effects here described in this study are due to alterations in patterning of the follicular epithelium. (3) Maternal dpp signaling increases the levels of Cactus protein in the embryo by a mechanism that is independent of Toll. Finally, inhibition of Tkv with tkvDN expressed with an early maternal driver alters the embryonic expression domains of ventral and lateral genes such as vnd and snail, which are targets of dorsal activation but not of zygotic BMP signaling. tkvDN expression also alters expression of DV genes in lateralized embryos, which lack dorsal ectoderm and early zygotic dpp expression. In aggregate, these various data support the view that maternal dpp and sog act by delayed induction on the embryo itself. The possibility cannot be ruled out, however, that the embryonic DV phenotypes described in this study result from the combined effects of direct and indirect maternal dpp signaling with the predominant effect being direct (Carneiro, 2006).

Delayed inductive activities have been proposed for a variety of proteins synthesized during oogenesis. For example, activation of the terminal system relies on delayed inductive activity of the secreted product of the torsolike gene (tsl), which is expressed by follicle cells at the two poles of the oocyte and associates with the vitelline membrane. ndl has a dual action on chorion integrity and embryonic patterning. The embryonic patterning function of ndl is thought to be mediated by Nudel protein that is secreted into the perivitelline space where it associates with the embryonic plasma membrane and initiates a proteolytic cascade. It is proposed that Sog and Dpp secreted by follicle cells also serve two roles. First, they contribute to patterning the follicle cell epithelium and chorion, and secondly, they are transferred to and stored in the perivitelline space where it is proposed that they function after fertilization to modify Toll patterning in the embryo (Carneiro, 2006).

During embryogenesis, Sog protein diffuses dorsally from the neuroectoderm and may carry Dpp dorsally in a complex with Tld, Tsg, and Scw, resulting in the generation of peak Dpp activity in the dorsal midline. The spatial distribution of maternal Sog, Dpp, Tld and Tok during oogenesis could also create asymmetric BMP activity. Since tld and tok are expressed only in ventral follicle cells, a ventral-to-dorsal gradient of Sog fragments is likely to be produced. Because cleavage of Sog by Drosophila Tld and Tok is dependent on the amount of Dpp, cleavage of Sog by Tld and Tok should be increased near the source of Dpp, generating an oblique gradient of Sog fragments in the egg chamber. The existence of such a gradient is supported by the greater staining seen in anterior ventral cells with the anti-Sog 8A antiserum during stage 10B. However, greater asymmetry may exist as a result of differential distribution of an array of Sog fragments throughout the egg chamber. Unfortunately, visualization of such asymmetry would be hard to achieve due to limitations in the ability to recognize several fragments by existing Sog antisera (Carneiro, 2006).

The analysis of marked sog− and tld− follicle cell clones suggests that the mobility of Sog fragments in the extracellular compartment may contribute to creating a maternal Dpp activity gradient. Such clones resulted in different Sog staining patterns in the perivitelline space adjacent to the clones depending on where they were located along the DV axis. The staining pattern observed with the 8A antibody suggests that ventrally generated N-Sog cleavage products may be less diffusible than intact Sog or than C-Sog and remain restricted to their site of production. In contrast, full-length Sog and C-Sog fragments appear to diffuse more readily (Carneiro, 2006).

Diffusion of Dpp may also contribute to patterning the eggshell. The expression of dpp in anterior follicle cells is consistent with its role in the formation of dorsal anterior chorionic structures. An anterior-to-posterior gradient of Dpp activity in dorsal regions of the egg chamber is suggested by the Dpp-dependent activation of the A359 enhancer trap and graded repression of bunched along the AP axis. In addition, BR-C expression is lost in mad− clones away from the source of Dpp. sog is likely to contribute to establishing this BMP gradient since ventral sog−clones act non-cell-autonomously to decrease the size of the operculum. Since ventral tld− clones also alter the extent and angle of the operculum, Tld may process Sog to generate a fragment that diffuses and carries Dpp to a dorsal anterior location, concentrating and thus enhancing Dpp activity. Further evidence that a fragment with such activity exists derives from the observation that overexpression of a C-terminal Sog fragment generates chorionic phenotypes that strongly resemble dpp overexpression (Carneiro, 2006).

A dorsally produced form of Sog also appears to participate in patterning the eggshell since sog− clones located dorsally result in fusion of dorsal appendages along the dorsal midline. DV positioning of the dorsal appendages depends on several factors, most critically on EGFR signaling. In contrast, mild overexpression of dpp generates fusion of the dorsal appendages. Considering the well-established role of Sog in modulating Dpp activity, the fused appendage phenotype generated by dorsal sog− clones most likely reflects the loss of Dpp antagonism exerted by Sog (Carneiro, 2006).

In addition to the activities described above, N-Sog fragments which remain ventrally restricted could exert Supersog-like activity, antagonizing BMPs while acquiring resistance to further cleavage and degradation by Tld. This ventrally restricted activity most likely patterns the embryo but does not affect dorsal positioning of eggshell structures, which depends on the combined activity of Dpp/BMPR signaling and dorsally generated Grk/EGFR signals (Carneiro, 2006).

The assortment of Sog fragments in egg chambers is very similar to that in the embryo. Full-length and processed forms of Sog generated by Tld during oogenesis might remain asymmetrically distributed during embryogenesis and exert distinct activities. This hypothesis is in agreement with the effect of tld− and sog− follicle cell clones on the embryo. In the majority of cases, tld− follicle cell clones result in ventralized cuticles, indicating that Tld generates some activity that synergizes with Dpp. Reciprocally, the great majority of sog− follicle cell clones result in dorsalized cuticles and embryos, indicating that Sog primarily acts by antagonizing Dpp. Since only ventral sog− clones generate cuticle defects, ventrally produced Sog presumably generates a ventralizing activity that blocks Dpp locally. In contrast, since in a minority of cases ventral shifts are observed in embryonic gene expression domains resulting from sog− clones, as well as a minority of dorsalized cuticles from tld− clones, there may also be a form of Sog that can enhance Dpp signaling. This positive BMP promoting activity could be generated ventrally, as suggested above in the case of chorion patterning (Carneiro, 2006).

A model depicting the proposed effects of different Sog forms on formation of the chorion and embryonic patterning is presented. According to this model, ventrally restricted Tld cleaves Sog near the Dpp source in ventral anterior follicle cells generating N-Sog and C-Sog. It is suggested that N-Sog fragments remain restricted near ventral anterior cells to antagonize Dpp, while C-Sog fragments diffuse dorsally concentrating Dpp in dorsal anterior cells that direct formation of the operculum. This asymmetric production of Sog molecules would generate a dorsal-to-ventral gradient of Dpp, with the highest levels dorsally near the anterior Dpp source. Although direct visualization of the predicted resulting Dpp gradient in the embryo is hard to achieve with the tools available, it is proposed that such a similarly oriented gradient persists until early embryogenesis based on the asymmetric pattern of Dpp-GFP distribution during late oogenesis and the observed alterations in embryonic gene expression domains resulting from modifications in maternal Dpp signaling (Carneiro, 2006).

The slope of the Dl nuclear gradient ultimately defines the extent of the mesoderm (Mes), neuroectoderm (NE), and dorsal ectoderm (DE). A uniform increase or decrease in nuclear Dl along the DV axis can only alter the extent of the Mes and DE and positioning of the NE, while a change in the slope of the gradient will modify the extent of NE territories such as the vnd expression domain. Under all conditions that Dpp signaling was altered, modifications were observed in the width of the vnd domain. This suggests that graded maternal Dpp signaling helps determine the slope of the dorsal gradient. Earlier studies suggested that Dpp inhibits Cactus degradation and as a consequence decreases Dl translocation into the nucleus. Increased Dpp signaling should result in more Dl retained in the cytoplasm, with consequent narrowing of the mesoderm and ventral shift in lateral and dorsal expression domains. Conversely, inhibition of Dpp signaling would result in increased levels of Dl becoming available for nuclear translocation. Considering the proposal that maternal Dpp is highest dorsally, and that Cactus may also act to prevent Dl diffusion along the DV axis, decreasing Dpp should lower Cactus levels in dorsal–lateral regions of the embryo and result in the redistribution of free Dl from ventral to lateral regions. As a consequence of this redistribution of Dl, there would be a slight decrease in Dl levels ventrally and an increase laterally that would have the net effect of flattening the gradient. Such a mechanism would require a certain degree of mobility of dorsal dimers in the syncytial blastoderm. In future studies, it will be interesting to determine the relative mobilities of Dl/Cactus complexes in the cytoplasm (Carneiro, 2006).

Maternal BMP signaling may also increase the robustness of dorsal patterning. The prevailing view of DV patterning is that signaling through the Toll pathway is sufficient to generate threshold-dependent activation of several dorsal target genes along the entire DV axis. Activation of Toll triggered by the ON/OFF pip expression pattern must be transformed into a ventrally centered gradient of Toll signaling. Several mechanisms may contribute to generate this gradient, based on autoregulatory feedback mechanisms. Although the Toll system may be internally robust, regulatory inputs from other signaling pathways could also contribute further to its stability, such as suggested for the wntD pathway and for maternal Dpp. While a significant body of evidence supports the standard view that establishment of the dorsal gradient through the Toll pathway is central to DV axis specification, the maternal Dpp pathway may constitute an important secondary mechanism that sharpens and ensures robustness and stability of the dorsal gradient in response to a rapidly changing embryonic environment (Carneiro, 2006).

The initiating event in maternal DV patterning is localized activation of the Grk/EGFR pathway in dorsal cells. Grk functions by restricting the expression of both pip and tld/tok, providing two potentially independent means for spatially regulating the activity of Toll and Dpp. This dual action of the Grk/EGFR pathway is consistent with analysis in which it was found that embryonic cuticles from gd−; grk−; Tl[3] mothers displayed a phenotype distinct from those collected from gd−; Tl[3] mothers. While cuticles from both genotypes had denticle belts surrounding the entire circumference of the embryo, cuticles from gd−; grk−; Tl[3] mothers were more elongated than those from gd−; Tl[3] mothers and exhibited a more ventral character. This suggests that grk provides an additional signal for asymmetry downstream or in parallel to gd. It is suggested that the hypothetical system proposed acts downstream of grk/EGFR and in parallel to Toll may be the Dpp pathway (Carneiro, 2006).

Effects of Mutation, Deletion or Mis-expression

What are the effects of doubling decapentaplegic gene dosage on phenotypes caused by other mutations affecting dorsal development? Like tolloid, the phenotypes of mutant embryos lacking shrew gene function are suppressed by elevated dpp, indicating that shrew also acts upstream of dpp to increase dpp activity. In contrast, increasing the number of copies of the dpp gene enhances the short gastrulation mutant phenotype, causing ventrolateral cells to adopt dorsal fates. This indicates that sog gene product normally blocks dpp activity ventrally. tolloid, shrew and sog genes are required to generate a gradient of dpp activity that directly specifies the pattern of the dorsal 40% of the embryo (Ferguson, 1992).

Mutations at the short gastrulation locus affect the timing of certain early morphogenetic events occurring during gastrulation. Specifically, the invagination and subsequent closing of the posterior midgut and the anterior midgut appear to be delayed. In addition, the germ-bands in such mutants do not extend the full distance anteriorly on the dorsal side of the embryo. The dorsal cells are abnormally thick and fall into extremely deep dorsal folds as the germ-band extends. sog embryos continue developing, but form disorganized first instar larvae. Normal sog expression is required in the zygote, but not in the mother for normal embryonic development and viability. The numbers of some types of neuroblasts are reduced in sog mutants, and there are also cuticle defects (Zusman, 1988).

Embryonic target gene activation in the absence of brinker (brk) is independent of SMAD activity. Thus brk acts either parallel to or downstream of SMADs as a specific repressor of low and intermediate level Dpp target genes. brk is expressed like another dpp antagonist, short gastrulation (sog), in ventrolateral regions of the embryo abutting the dorsal dpp domain, and in brk mutants dpp expression expands to cover the entire ectoderm. In this situation sog is largely responsible for Dpp gradient formation, since brk;sog double mutant embryos have almost no polarity information in the ectoderm. The double mutants consist mainly of mesoderm and unstructured dorsal epidermis. Thus, brk and sog together specify the neuroectoderm of Drosophila embryos (Jazwinska, 1999).

The question arises as to why uniform expression of dpp in the ectoderm is compatible with the substantial degree of DV polarity exhibited by brk mutant embryos. In wild type, Dpp activity is polarized by sog expression in the ventrolateral region of the embryo, such that ventrolateral Dpp activities are reduced and a peak of activity is established centered on the dorsal midline. Embryos mutant for sog show a reduction of ventrolateral fates, albeit to a weaker degree than brk embryos. In contrast to brk embryos, they differentiate only a small number of scattered amnioserosa cells. The lateral fate shift is not accompanied by strong expansion of dpp transcription as seen in brk embryos, and pnr expression is not as greatly expanded as in brk embryos. In brk embryos, sog is still expressed in the ventrolateral domain of the ectoderm. To test whether this expression accounts for the DV polarity in the ectoderm of brk embryos, brk embryos were constructed that were also mutant for sog. In contrast to either single mutant, the ectoderm of brk sog embryos forms only dorsal-type cuticle hairs and completely lacks ventral denticles. During germ band extension, neuroblast expression of sna cannot be detected; instead, sna expression in the peripheral nervous system (PNS) precursors, normally restricted to dorsolateral regions, expands into the ventral region of the embryo. This suggests a complete deletion of the ventral neurogenic region. Taken together, brk and sog have both overlapping and distinct roles in shaping the Dpp activity gradient of the Drosophila embryo. While sog has an important function in providing peak levels of the gradient necessary for amnioserosa development, brk and sog together are essential to limit the ventral extension of the anti-neurogenic activity of dpp (Jazwinska, 1999).

The BMP pathway patterns the dorsal region of the Drosophila embryo. Using an antibody recognizing phosphorylated Mad (pMad), signaling was followed directly. In wild-type embryos, a biphasic activation pattern is observed. At the cellular blastoderm stage, high pMad levels are detected only in the dorsal-most cell rows that give rise to amnioserosa. This accumulation of pMad requires the ligand Screw (Scw), the Short gastrulation (Sog) protein, and cleavage of their complex by Tolloid (Tld). When the inhibitory activity of Sog is removed, Mad phosphorylation is expanded. In spite of the uniform expression of Scw, pMad expansion is restricted to the dorsal domain of the embryo where Dpp is expressed. This demonstrates that Mad phosphorylation requires simultaneous activation by Scw and Dpp. Indeed, the early pMad pattern is abolished when either the Scw receptor Saxophone (Sax), the Dpp receptor Thickveins (Tkv), or Dpp are removed. After germ band extension, a uniform accumulation of pMad is observed in the entire dorsal domain of the embryo, with a sharp border at the junction with the neuroectoderm. From this stage onward, activation by Scw is no longer required, and Dpp suffices to induce high levels of pMad. In these subsequent phases pMad accumulates normally in the presence of ectopic Sog, in contrast to the early phase, indicating that Sog is only capable of blocking activation by Scw and not by Dpp (Dorfman, 2001).

Thus two distinct phases of pMad activation have been identified. The early phase requires activation by both Scw and Dpp ligands, while the second phase depends only on Dpp. Signaling is first detected in the cellular blastoderm embryo. While activation is observed within the dorsal-most 8-10 cell rows, the sensitivity of the detection method fails to monitor signaling in the rest of the dorsal domain. High signaling levels are induced by Scw, and give rise to amnioserosa. Within the domain where pMad is observed, graded activation is detected, which may have the capacity to induce more than one cell fate in the region (Dorfman, 2001).

The cardinal players in the generation of the early pMad gradient are Scw, Tld and Sog. Tld has been suggested to generate a sink for the active ligand, by cleaving the Sog/ligand complex. The similarity between the pMad pattern of scw and tld mutants suggests that Tld is primarily involved in the release of Scw from the complex with Sog. Absence of Scw, Tld or Sax abolished the early pMad pattern while retaining the second phase, indicating that the second phase relies only on Dpp signaling. Similarly, overexpression of Sog eliminated only the early but not the subsequent pMad patterns. This suggests that Sog preferentially associates with Scw, in agreement with previous biological assays of Sog activity. Generation of graded patterning in the dorsal region does not rely on restricted gene expression within this domain. Rather, expression of genes confined to the neuroectoderm may lead to graded distribution of their gene products within the dorsal domain. The essential component for generation of graded patterning appears to be Sog, which is produced only in the neuroectoderm, but is capable of diffusing to the dorsal region. Disruption of the normal distribution of Sog by uniform misexpression, abolishes the early pMad activation profile (Dorfman, 2001).

This suggests that normally Sog may form a graded distribution in the dorsal region, which is essential for patterning. When the Sog/Scw complex is cleaved by Tld, Scw is released and can bind either Sog or Sax. The data suggest that in regions closer to the neuroectoderm, the levels of Sog are high and titrate the free ligand. In the dorsal-most region however, where Sog levels are low, the released Scw has a greater probability of binding and activating the Sax receptor, rather than being trapped again by Sog. Thus, the graded distribution of Sog is critical for generating the reciprocal distribution of Scw, and the ensuing activation profile (Dorfman, 2001).

In sog mutant embryos an expansion of the early pMad pattern is observed. In the absence of Sog, a uniform distribution of Scw is expected, and hence the activation level should be lower than the maximal level in wild-type embryos. The staining levels in wild-type and sog mutant embryos have been quantitated. While the pattern of staining is reproducible in all wild-type embryos, variations in the absolute levels of up to threefold between embryos were observed in any given staining reaction. It is thus difficult to compare reliably the wild-type level to the absolute staining levels of sog mutants. Nevertheless, the impression is that the expanded pMad in sog mutant embryos is comparable in levels to the maximal signaling levels in wild-type embryos. In spite of this expanded pMad activation pattern, amnioserosa cell fates are abolished in sog mutants. This result suggests that in addition to the role of Sog in determining the graded distribution of Scw, Sog or its cleavage products may provide an additional signal facilitating the induction of amnioserosa cell fates (Dorfman, 2001).

Positional information in the dorsoventral axis of the Drosophila embryo is encoded by a BMP activity gradient formed by synergistic signaling between the BMP family members Decapentaplegic and Screw. short gastrulation, which is functionally homologous to Xenopus Chordin, is expressed in the ventrolateral regions of the embryo and has been shown to act as a local antagonist of BMP signaling. Sog has a second function, which is to promote BMP signaling on the dorsal side of the embryo. A weak, homozygous-viable sog mutant is enhanced to lethality by reduction in the activities of the Smad family members Mad or Medea, and this lethality is caused by defects in the molecular specification and subsequent cellular differentiation of the dorsal-most cell type, the amnioserosa. While previous data had suggested that the negative function of Sog is directed against Scw, data are presented that suggest that the positive activity of Sog is directed towards Dpp. Chordin shares the same apparent ligand specificity as does Sog, preferentially inhibiting Scw but not Dpp activity. However, in Drosophila assays, Chordin does not have the same capacity to elevate BMP signaling as does Sog, identifying a functional difference in the otherwise well conserved process of dorsoventral pattern formation in arthropods and chordates (Decotto, 2001).

Morphogen gradients, once a purely theoretical concept, are now viewed as central players in the establishment of cell identity in a broad range of developmental processes. However, the exact biological mechanisms used to establish and maintain a morphogen gradient vary, depending on the biological context. In the Drosophila embryo, while Dpp can act in a dose-dependent fashion to specify different cell fates along the DV axis, in vivo its activity is modulated spatially by other components of the patterning system. In particular, Sog, a diffusible BMP-binding protein, has been shown to inhibit BMP signaling ventrally by preventing ligand access to the BMP receptors. A novel aspect of Sog’s function has been characterized in this study. Specifically, Sog functions cell non-autonomously to elevate BMP signaling on the dorsal side of the embryo. Thus, the interpretation of any experiment to elucidate the role of Sog in the control of dorsoventral patterning must take into account the two apparently opposing functions of the protein (Decotto, 2001).

Loss-of-function mutations in Mad or Medea have been identified as dominant enhancers of a weak homozygous-viable sog mutation, and the enhanced embryos have been shown to have defects in amnioserosa specification. Furthermore, synthetic lethality between weak homozygous-viable alleles of sog and zen has been demonstrated, indicating that both are required for maximal production of amnioserosa. Lastly, there was a dramatic decrease in the level of zen transcription in sogP129D embryos that were derived from Mad/+ females, compared to the level of zen transcription in either genotype alone. Taken together, these results unambiguously demonstrate that the positive action of Sog is exerted before gastrulation to attain the maximal expression of a direct BMP target gene (Decotto, 2001).

Expression of Sog in dorsalized embryos causes the local inhibition of the dorsal-specific gene Race, and the upregulation of Race transcription at a distance from the site of expression, formally demonstrating the dual action of Sog upon a field of equipotent cells. However, Race is not necessary for amnioserosa specification, since sogP129D embryos, which have functional amnioserosa, lack Race expression. The positive activity of Sog is also exerted upon expression of zen, which is known to be required for amnioserosa specification. These results provide a direct link between Sog's effect on zen transcription and loss of amnioserosa in sog mutants (Decotto, 2001).

The minimal amount of sog mRNA that must be injected to observe the ectopic transcription of Race is four-fold higher than the minimal amount necessary to locally inhibit Race transcription. Thus, a small decrease in the concentration of Sog affects the positive activity of Sog to a greater extent than it affects the negative function. This could be for any of a number of reasons, including a marked decrease in the concentration of Sog (or one of its proteolytic fragments) as it diffuses away from its site of synthesis (Decotto, 2001).

These results also correlate well with phenotypic and genetic analyses of sogP129D, which causes a reduction in the level of sog transcription. Although this allele is homozygous viable, it appears to cause a preferential reduction in the positive activity of Sog, as evidenced by the loss of Race transcription in the amnioserosa. The preferential loss of positive activity in the sogP129D mutant could also explain why second site mutations were isolated that decreased the positive function of Sog, but did not recover mutations in genes such as brinker that cooperate with Sog to repress BMP signaling ventrally (Decotto, 2001).

The inhibitory function of Sog is primarily directed against the Scw ligand. Data is presented that suggest that the positive function of Sog may be directed towards Dpp. In particular, a 50% increase in dpp copy number is sufficient to restore amnioserosa to sog mutant embryos, indicating that sog is more sensitive than any other known ventralizing mutation to an increase in Mad12 gene dosage. The lack of amnioserosa in sog embryos was not rescued by injection of an amount of scw mRNA far in excess of that required to rescue a scw mutant. These results are strongly suggestive that Sog’s positive function may be directed against Dpp, not against Scw (Decotto, 2001).

These findings may allow the clarification of a series of recent results concerning the action of a second extracellular factor that modulates Dpp activity, the product of the twisted gastrulation (tsg) gene. While the phenotype of tsg embryos, a partial ventralization caused by lack of amnioserosa, is suggestive of a positive activity of Tsg upon BMP signaling, Tsg has been shown to form a complex with Sog, and coexpression of Tsg with Sog is sufficient to block Dpp activity. Similar results have been demonstrated for the vertebrate homologs of the Tsg and Sog proteins. These results have been primarily interpreted to suggest that, in vivo, Sog and Tsg cooperate to block Dpp signaling. In contrast, it has been suggested the Xenopus homolog of Tsg promotes BMP signaling primarily by antagonizing BMP binding to one of the cysteine rich domains (CR1) of Chd (Decotto, 2001).

The current data has been combined with the published results to reconcile the different interpretations of the biological functions of Sog and Tsg. Specifically, it is proposed that the positive activity shown for Sog, which is here postulated to be directed toward Dpp, is in fact mediated by a tripartite complex composed of Sog, Tsg and Dpp. It is suggested that this complex promotes BMP signaling by sequestering Dpp from its receptor, thus allowing the dorsally directed diffusion of laterally produced Dpp ligand. It is suggested that the cleavage of Sog by Tld in the dorsal-most cells releases the Dpp ligand from the tripartite complex, allowing it to signal. After cleavage of Sog by Tld, Tsg could function, antagonizing the further binding of Dpp by the proteolytic fragments of Sog (Decotto, 2001).

It is proposed that the antagonistic aspects of the complex toward Dpp signaling that were observed through overexpression studies in various developmental contexts are due to the lack of, or improper stoichiometry of, a component present in the embryo that leads to disassociation of the tripartite complex. For example, if in these developmental contexts the Tld protease was not present in sufficient quantities to release the ligand from the tripartite complex, there would be continued Dpp sequestration by the Tsg/Sog complex leading to antagonism of BMP signaling. Alternatively, there may be one or more components present in the embryo whose function is to antagonize the stability of the tripartite complex, also potentiating ligand release. In that regard, it is interesting that mutations in the shrew gene cause a phenotype (lack of amnioserosa) that is similar to that caused by sog or tsg mutations. Possibly, shrew, the sequence of which has not been reported, could encode a component that aids in the formation or dissociation of the tripartite complex (Decotto, 2001).

The conservation of the molecular mechanisms underlying the process of dorsoventral pattern formation between arthropods and chordates has allowed functional studies of vertebrate proteins to be carried out in Drosophila. Sog and Chd have been shown to be functionally interchangeable in their BMP inhibitory function. In this paper, it is demonstrated that Chd, like Sog, preferentially inhibits Scw signaling, and under the assay conditions used is not capable of blocking Dpp activity. This finding suggests that in vertebrates, as in flies, the activity of different ligands can have differential responsiveness to particular inhibitors. Although Scw does not have a known vertebrate ortholog, phylogenetic analyses have placed it in the 60A/BMP7 subgroup or in a separate clade with mouse GDF3. Furthermore, the likely receptor for Scw, Sax, has been placed in the same subfamily as the BMP7 receptor, ALK2, based in part on homology in the L45 loop that is critical in determining signaling specificity. Thus, it is proposed that in vivo, Chd alone preferentially inhibits BMP7, not BMP2/4 (Decotto, 2001).

It is of interest to enquire whether Chd, like Sog, would displays a long-range positive activity. Although injection of Chd mRNA inhibits Race expression in the anterior domain of dorsalized embryos, it does not cause activation of Race transcription in the posterior domain. Thus, in Drosophila, Chd can inhibit BMP signals locally, but does not display long-range activation of BMP signaling (Decotto, 2001).

If the model concerning the positive action of Sog is correct, Chd's inability to promote a long-range elevation of BMP signaling could be for any of a variety of reasons. For example, wild-type levels of the metalloprotease TLD are required for the positive activity of Sog. If the positive activity of Sog is mediated by a proteolytic fragment of Sog, the differences in the in vitro patterns of cleavage of Sog and Chd could be critical for determining biological function. Another possibility is that Chd is unable to form sufficiently stable tripartite complexes with Dpp and Tsg to permit long-range diffusion. Alternatively, if Chd displays a higher affinity for Dpp than does Sog, possibly sufficient Dpp remains associated with the proteolytic fragments of Chd to prohibit long-range signaling. In support of this, it has been proposed that one function of Tsg is to remove BMP ligands from the proteolytic fragments of Chd. If either of the last two explanations were correct, elevation of Tsg levels in the Drosophila embryo might permit Chd to display a positive activity. It was therefore tested whether coinjection of tsg and chd mRNAs would result in positive long-range signaling. Although a ten-fold concentration range of tsg mRNA was injected with the chd mRNA, none of the embryos in these injection experiments displayed long-range activation of Race transcription (Decotto, 2001).

Although the mechanistic difference between Sog and Chd function cannot yet be ascertained, the results do provide an opportunity to determine the domains of Sog that are important for its positive activity. Interestingly, all known zygotic-lethal sog mutations are defective in both activities of the gene, even though absence of its positive activity alone would have been sufficient to confer embryonic lethality. More generally, up to now there has been striking conservation of function of each individual component of the dorsoventral patterning system in arthropods and chordates. The identification of the basis of functional differences in apparently homologous proteins could provide insights into the degree of evolutionary divergence that can exist within the constraints of a conserved signaling system (Decotto, 2001).

Drosophila Smurf1 is a negative regulator of signaling by the BMP2/4 ortholog Decapentaplegic during embryonic dorsal-ventral patterning. Smurf1 encodes a HECT domain ubiquitin-protein ligase, homologous to vertebrate Smurf1 and Smurf2, that binds the Smad1/5 ortholog in Drosophila Mothers against dpp (Mad) and likely promotes its proteolysis. The essential function of Drosophila Smurf1 is restricted to its action on the Dpp pathway. Smurf1 has two distinct, possibly mechanistically separate, functions in controlling Dpp signaling. Prior to gastrulation, Smurf1 mutations cause a spatial increase in the Dpp gradient, as evidenced by ventrolateral expansion in expression domains of target genes representing all known signaling thresholds. After gastrulation, Smurf1 mutations cause a temporal delay in downregulation of earlier Dpp signals, resulting in a lethal defect in hindgut organogenesis. The results suggest that Smurf1 provides an important mechanism to maintain the available pool of Mad at limiting concentrations, and may have additional functions in regulating the levels of Dpp receptors (Podos, 2001).

The results suggest that Smurf1 provides an important mechanism to maintain the available pool of Mad at limiting concentrations, the necessity of which has been supported by previous genetic observations. Although not normally haploinsufficient, the Mad gene is rendered so when the activities of other components of the Dpp pathway, including dpp, zen, and sog, are reduced. More generally, limiting amounts of Smad protein might be an essential feature of all graded TGF-ß superfamily signaling systems. Cytoplasmic Smad pools are similarly limiting in Xenopus embryos, according to quantitative studies of activin signaling. Experimental elevations in Smad2 concentration cause proportionate increases in Smad activation, as represented by both nuclear Smad2 import and transcriptional readout. Therefore, it is predicted that Smurf enzymes will prove to be essential to maintain Smad proteins at limiting concentrations to ensure appropriate responses to all graded BMP and activin/TGF-ß signals (Podos, 2001).

At its lowest threshold, Dpp signaling, acting to promote its own transcription, defines the boundary between the dorsal epidermis and neurogenic ectoderm. This positive feedback of Dpp on its own transcription is opposed by the action of the negative regulators Short gastrulation (Sog) and Brinker (Brk) in the neurogenic ectoderm. Although this boundary was positioned normally in Smurf1 mutants, it was important to determine whether sog activity masked an effect of a Smurf1 mutation on this Dpp threshold (Podos, 2001).

In both Smurf115C and sog single mutant embryos, dpp is transcribed approximately within its normal dorsal domain at the onset of gastrulation. However, in sog; Smurf115C double mutant embryos, dpp transcription expands significantly, although variably, into the ventrolateral neurogenic ectoderm. Strikingly, these double mutant embryos ultimately differentiate a fully dorsalized cuticle, in which ventral denticles are replaced by dorsal hairs. These results indicate that Smurf1 and sog are genetically redundant, yet functionally distinct, in limiting the spatial extent of dpp transcription and the consequent specification of dorsal epidermis. It is concluded from this set of results that Smurf1 contributes quantitatively to the establishment of multiple Dpp signaling thresholds across the entire range of the Dpp activity gradient (Podos, 2001).

Three genetic criteria indicate that defects in Dpp signaling directly cause the hindgut phenotype in Smurf1 mutant embryos: (1) the hindgut defects are not observed in Smurf1 mutant embryos that lack one copy of dpp; (2) a complete loss of zen function substantially suppresses the Smurf115C phenotype, restoring the embryonic hindgut to a tubular morphology and an interior location. It is noted that the hindgut of Smurf115C; zen double mutant embryos often fails to adopt the normal hook-shaped trajectory, suggesting that the deregulation of other target genes also contributes to the Smurf1 phenotype. (3) The hindgut defect was also suppressed in sog; Smurf115C double mutant embryos. While Sog antagonizes BMP signaling in the ventrolateral ectoderm, a positive activity of Sog is also required at the onset of gastrulation to promote the Dpp-dependent specification of amnioserosa at the dorsal midline. It is proposed that the blastoderm-specific Dpp signaling in the dorsal-most region of sog;Smurf1 embryos is reduced to a level that, even in the absence of temporal downregulation of P-Mad, does not elicit the observed Smurf1 hindgut defect (Podos, 2001).

Transforming growth factor ß signaling mediated by Decapentaplegic and Screw is known to be involved in defining the border of the ventral neurogenic region in the fruitfly. A second phase of Decapentaplegic signaling occurs in a broad dorsal ectodermal region. The dorsolateral peripheral nervous system forms within the region where this second phase of signaling occurs. Decapentaplegic activity is required for development of many of the dorsal and lateral peripheral nervous system neurons. Double mutant analysis of the Decapentaplegic signaling mediator Schnurri and the inhibitor Brinker indicates that formation of these neurons requires Decapentaplegic signaling, and their absence in the mutant is mediated by a counteracting repression by Brinker. Interestingly, the ventral peripheral neurons that form outside the Decapentaplegic signaling domain depend on Brinker to develop. The role of Decapentaplegic signaling on dorsal and lateral peripheral neurons is strikingly similar to the known role of Transforming growth factor ß signaling in specifying dorsal cell fates of the lateral (later dorsal) nervous system in chordates (Halocythia, zebrafish, Xenopus, chicken and mouse). It points to an evolutionarily conserved mechanism specifying dorsal cell fates in the nervous system of both protostomes and deuterostomes (Rusten, 2002).

The second phase of Dpp signaling, covering most if not all the dorsal ectoderm, starts at stage 9 and lasts until stage 10/11 [3.40 to 5.20 hours after egg laying (AEL)]. Initially proneural clusters (PNCs) and later sensory organ precursors (SOPs), singled out within each PNCs, can be visualized by the expression of the proneural genes achaete (ac, 4.20-7.20 hours AEL), atonal (ato, 5-6.30 hours AEL) and amos (amo, 5.20-6 hours AEL). Thus, the second wave of Dpp signaling precedes and overlaps with the development of the PNCs and SOPs. The domain of Dpp signaling was examined using an enhancer trap lacZ line inserted in the gene daughters against dpp (dad), a target of Dpp. Double immunofluorescence staining shows that dorsally located Ac and Ato positive PNCs and SOPs originate inside the dad-lacZ positive region, suggesting that they have received, or still receive, Dpp signaling. However, a subset of PNCs and SOPs are ventral to the dad-lacZ domain. As the PNS neuronal precursors differentiate close to the position where they originate, it can be concluded that a part of the dorsal PNS forms within an active Dpp signaling region (Rusten, 2002).

A way to interfere with the second phase of Dpp signaling is to express specific inhibitors once the initial dorsoventral patterning is accomplished. Brk is a nuclear protein that negatively regulates Dpp-induced genes and is expressed ventrally in a complementary pattern to Dpp in the embryo. Sog is a secreted protein that can bind to Dpp and inhibit it from signaling, and Supersog (Ssog) is a hyperactive inhibitory fragment of Sog. In order to avoid interference with the first wave of Dpp signaling (stage 5 to 7, 2.10-3.10 hours AEL), brk and ssog were misexpressed from stage 8 (3.10 hours AEL) to interfere with the second phase (stage 9 to 10/11, 3.40-5.20 hours AEL). UAS-brk expression in segments T2-A3, which is driven by the Kr-Gal4 driver, and ubiquitous expression of ssog in the entire embryo, produced using a HS-ssog construct, leads to reduced number of neurons in the dorsal and lateral PNS. The effects are less severe for Ssog misexpression than for UAS-brk misexpression and notable for shn mutations. Approximately 20% of the embryos expressing ubiquitous ssog do not undergo dorsal closure, similar to the phenotype observed when strong alleles of shn are analyzed. The HS-ssog produces a manifest decrease in phosphorylated-Mad (p-Mad) in the dorsal region. This indicates a reduction in Dpp signaling responsible for the phenotype. The residual p-Mad staining observed in some embryos might be the reason why Ssog misexpression leads to less severe effects than UAS-brk misexpression or shn mutations (Rusten, 2002).

In all these mutant backgrounds the dorsal and lateral PNS clusters show a severe reduction in the number of neurons. No major differences are found depending on the neuronal type: the percentage of external sensory organ neurons lost is similar to the loss of neurons in the chordotonal organs. The penetrance of this effect, as measured in the differentiated PNS clusters, varies among abdominal segments. The average reduction in neuronal number ranges from 25% (HS-ssog) to 41% (shnk00401) in the dorsal cluster and 8% (HS-ssog) to 52% (shnk00401) in the lateral cluster. By contrast, the ventral cluster is less affected because it shows 2% (HS-ssog) to 18% reduction (shnk00401). The lateral pentascolopodial organ shows migration defects in these embryos, but the other sensory organs are located in their expected relative positions (Rusten, 2002).

The reduced number of neurons observed in the dorsal and lateral PNS when Dpp signaling is impeded could result from lack of proneural gene expression, which is known to be necessary for PNC and SOP formation. The expression of ato and ac was analyzed to examine the specification of progenitor cell subclasses in mutant backgrounds defective for Dpp signaling. The development of the serially homologous abdominal segments A1 to A7 is similar and very synchronous. Thus, in the wild type, whenever a specific number of PNCs and SOPs appear in one abdominal segment, a similar pattern is observed in the other abdominal segments as well. This is not true for shnk04412 mutants and for embryos expressing ubiquitous ssog, where the numbers of Ac and Ato positive SOPs and PNCs vary among the abdominal segments. This is consistent with the variably penetrant phenotypes observed in differentiated PNS among abdominal segments. In embryos expressing Kr-Gal4;UAS-brk, loss of Ato- and Ac-positive PNCs and SOPs was observed specifically in the abdominal segments A1-A3 where brk was misexpressed, when compared with abdominal segments A4-A7 that served as an internal reference. The reduced numbers of Ato- and Ac-positive neuronal progenitors appear to result from failure of PNC formation rather than an increase in cell death ratio: apoptosis does not appear to increase in segments expressing brk compared with the other abdominal segments. Taken together, these results suggest that reduction in the number of neurons is produced by failure in proneural gene expression (Rusten, 2002).

Genetic evidence suggests that the Drosophila ectoderm is patterned by a spatial gradient of bone morphogenetic protein (BMP). Patterns have been compared of two related cellular responses - signal-dependent phosphorylation of the BMP-regulated R-SMAD, MAD, and signal-dependent changes in levels and sub-cellular distribution of the co-SMAD Medea. Nuclear accumulation of Medea requires a BMP signal during blastoderm and gastrula stages. During this period, nuclear co-SMAD responses occur in three distinct patterns. At the end of blastoderm, a broad dorsal domain of weak SMAD response is detected. During early gastrulation, this domain narrows to a thin stripe of strong SMAD response at the dorsal midline. SMAD response levels continue to rise in the dorsal midline region during gastrulation, and flanking plateaus of weak responses are detected in dorsolateral cells. Thus, the thresholds for gene expression responses are implicit in the levels of SMAD responses during gastrulation. Both BMP ligands, DPP and Screw, are required for nuclear co-SMAD responses during these stages. The BMP antagonist Short gastrulation (Sog) is required to elevate peak responses at the dorsal midline as well as to depress responses in dorsolateral cells. The midline SMAD response gradient can form in embryos with reduced dpp gene dosage, but the peak level is reduced. These data support a model in which weak BMP activity during blastoderm defines the boundary between ventral neurogenic ectoderm and dorsal ectoderm. Subsequently, BMP activity creates a step gradient of SMAD responses that patterns the amnioserosa and dorsomedial ectoderm (Sutherland, 2003).

The final width of the midline peak response is sensitive to gene dosage for both dpp and sog. It is broader when dpp dosage is increased, and narrower with only one copy of dpp. Similarly, the width of the stripe is broader, but more variable, when sog levels are reduced. The response domain is broadest in sog null embryos; however, the level of response is significantly reduced. This is distinct from the effect of increased dpp dosage, in which the response domain is broader, but normal SMAD response levels are achieved or exceeded (Sutherland, 2003).

The role of Sog as both a short-range inhibitor and a long-range potentiator of dorsal patterning has led to a proposal that Sog transports BMP ligands from lateral regions to the dorsal midline. Biochemical analyses suggest mechanisms for Sog-BMP binding and release. Computational analysis have defined conditions under which transport could occur with these mechanisms. The transition from weak, broad SMAD responses to narrow, strong responses is consistent with concentration of BMP activity at the dorsal midline, and the loss of this transition with loss of Sog is consistent with a Sog-dependent transport model. However, there are significant differences between the current results and the assumptions used to develop the computational model. These include the presence of a midline SMAD response in dpp–/+ embryos and the sensitivity to reduced sog dosage. It will be important to refine future computational models to fit the complete set of BMP response data (Sutherland, 2003).

Both BMP ligands, Dpp and Scw, are required to form the dorsal-midline gradient. However, scw mutant embryos retain a small amount of dorsal ectoderm, with concomitant expansion of ventral ectoderm. Surprisingly, the weak dorsolateral Medea response is lost in scw embryos. It is concluded that the full Medea response domain encompasses the cell fates that are lost in scw mutants, amnioserosa and dorsomedial ectoderm. It appears that dorsal cells can acquire a dorsolateral fate without gastrula BMP activity (Sutherland, 2003).

Mutants with expanded ventral ectoderm show reduced SMAD responses during the first phase of BMP activity. PMad was not detected in blastoderm tld embryos. Homozygotes for moderate dpp alleles have lower PMad levels during blastoderm. Conversely, sog embryos have a slightly expanded PMad response during blastoderm, and a slight expansion of dorsal ectoderm. Thus, BMP activity during blastoderm positions the boundary between dorsal and ventral ectoderm (Sutherland, 2003).

Mutations that shift the boundary between amnioserosa and dorsal ectoderm show altered SMAD responses in the third phase of BMP activity, the dorsal-midline gradient. dpp-/+ embryos have variable reductions in midline SMAD responses and in the number of amnioserosa cells. Strikingly, sog null embryos have little amnioserosa and a strong reduction in SMAD response levels during gastrulation. Thus, SMAD response levels during gastrulation are critical for amnioserosa specification (Sutherland, 2003).

Taken together, these data suggest a multi-step model for DV patterning of the embryonic ectoderm, incorporating aspects of the two previous models. In a previous gradient model, ectodermal fates are subdivided simultaneously by a continuous BMP gradient involving Dpp and Scw. In the successive cell-fate decision model, amnioserosa is specified by dorsal-midline Dpp+Scw activity, and the dorsal ectoderm by Dpp alone at stage 9 (Sutherland, 2003).

Instead, it is proposed that the blastoderm phase of weak BMP activity establishes a dorsal ectoderm domain. Mutations that shift the boundary between dorsal and ventral ectoderm also have altered SMAD responses at this stage. It is at this stage that SMADs compete with Brinker to regulate the first phase of zen expression. Furthermore, this early signal maintains BMP activity, for the late-blastoderm domain of dpp expression is set by competition between BMPs, Sog and Brinker. BMP activity subsequently maintains the dorsal boundary for brinker expression. Thus, BMP activity at blastoderm defines a dorsal domain where dpp is expressed and brinker is not (Sutherland, 2003).

Cysteine repeat domains and adjacent sequences determine distinct Bone morphogenetic protein modulatory activities of the Drosophila Sog protein

The Drosophila short gastrulation gene encodes a large extracellular protein (Sog) that inhibits signaling by BMP-related ligands. Sog and its vertebrate counterpart Chordin contain four copies of a cysteine repeat (CR) motif defined by 10 cysteine residues spaced in a fixed pattern and a tryptophan residue situated between the first two cysteines. This study presents a structure-function analysis of the CR repeats in Sog, using a series of deletion and point mutation constructs, as well as constructs in which CR domains have been swapped. This analysis indicates that the CR domains are individually dispensable for Sog function but that they are not interchangeable. These studies reveal three different types of Sog activity: intact Sog, which inhibits signaling mediated by the ligand Glass bottom boat (Gbb), a more broadly active class of BMP antagonist referred to as Supersog, and a newly identified activity, which may promote rather than inhibit BMP signaling. Analysis of the activities of CR swap constructs indicates that the CR domains are required for full activity of the various forms of Sog but that the type of Sog activity is determined primarily by surrounding protein sequences. Cumulatively, this analysis suggests that CR domains interact physically with adjacent protein sequences to create forms of Sog with distinct BMP modulatory activities (Yu, 2004).

The Sog CR domains are defined by a set of 10 cysteine residues with a conserved spacing and a single tryptophan residue located between the first two cysteines. The function of the tryptophan residues was examined by mutating them individually or all to alanine. The finding that all four single W --> A mutants have wild-type Sog function as assayed by misexpression in the wing, either alone or when coexpressed with Tsg, indicates that none of these residues is individually essential for either Sog or Tsg + Sog (Supersog) activities. This finding is also consistent with the results of deleting the individual CR domains. When all four tryptophans were mutated to alanine, however, the Sog-like activity remained relatively unaffected but this mutant was greatly compromised in its ability to interact with Tsg to generate a Supersog-like activity. These results suggest that the tryptophan residues in two or more CRs can mediate functional interaction with Tsg and that Sog residues outside of the four conserved tryptophans are not sufficient on their own to mediate this interaction. However, deletion of the stem region also eliminates the functional Tsg interaction since a mutant lacking the stem and CR1 fails to interact with Tsg while a mutant lacking just CR1 interacts fully. The requirement for the stem region in interacting with Tsg is consistent with both CR and stem sequences being essential for Supersog activity (Yu, 2004).

Truncated forms of Sog consisting of CR1, the stem, and CR2 behave differently from either Sog or Supersog constructs when misexpressed in the wing or embryo. The strongest form of this novel Sog activity is observed when both CR3 and CR4 are deleted (e.g., SogCR1,2). Several lines of evidence suggest that Sog CR1,2 functions by promoting BMP signaling. (1) The effect of misexpressing SogCR1,2 on gene expression in wing discs is most similar to that of misexpressing an activated Sax receptor or the putative Sax ligand Gbb. This profile of gene response is quite distinct from that resulting from misexpression of activated or dominant-negative forms of the BMP receptor or EGF-receptor pathway, which is the other major signaling system regulating early vein development. (2) Misexpression of SogCR1,2 in pupal wings by heat shock results in significant ectopic expression of the rho gene, which is a good measure of BMP vs. EGF-R pathway activation during this stage. (3) Expression of SogCR1,2 in the early embryo broadens the dorsal expression domain of the BMP target gene zen (Yu, 2004).

One attractive model for a positive function of Sog such as that potentially mediated by SogCR1,2 is that in addition to binding to BMPs and preventing them from gaining access to the receptors, Sog might also act as a carrier of BMPs to either protect them from degradation or possibly transport them dorsally. Since the CR3 and CR4 domains appear to be those most critical to inhibition of Gbb/Scw activity, it is possible that the apparent positive activity of SogCR1,2 is the result of removing the Scw/Gbb inhibitory activity, while leaving a carrier function intact. The difference between the activity of SogCR1,2 and Supersog molecules, which lack the CR2 domain, might be explained if CR1 and CR2 can interact in SogCR1,2 to prevent CR1 and/or adjacent sequences from interacting with Dpp, thus neutralizing this remaining potential BMP inhibitory activity. It is currently unclear what relationship, if any, the SogCR1,2 activity has to the positive function of Sog required to sustain dorsal expression of race in the early embryo. SogCR1,2, unlike intact Sog, is unable to rescue race at a distance, but can broaden dorsal zen expression, which intact Sog does not do. The apparent differences between these activities may be the result of threshold-dependent effects in the early embryo or may reflect a fundamentally different mode of action (Yu, 2004).

An important question is whether truncated forms of Sog similar to SogCR1,2 or SogDeltaCR4 are generated and function in vivo. It is known that Tld can cleave Sog in vitro to generate products of approximately the same size as these constructs, and Sog-reactive bands of approximately the same size are observed in early embryos and pupal wings. In addition, forms of Chordin similar in structure to SogCR1,2 and Supersog are produced in humans as the result of alternative RNA splicing . Further analysis of the production and activity of SogCR1,2-like molecules will be required to determine the relevance of such forms in vivo and to determine the mechanism by which they may act on the BMP pathway (Yu, 2004).

Analysis of Sog mutants in which individual CRs are deleted or single conserved tryptophan residues are mutated to alanine suggests that the CR domains perform partially overlapping functions since none of them is absolutely essential for intact Sog activity (e.g., inhibition of Gbb/Scw) or for interaction with Tsg to create a Dpp inhibitory activity. Nonetheless, these experiments also suggest that the CR domains are not equivalent. For example, deletion of CR3 or CR4 had much greater effects in reducing the SOG-like activity than did deletion of CR1 or CR2. Moreover, the fact that Supersog, which contains only CR1, can inhibit Dpp while SogCR4 apparently interferes selectively with Gbb, suggested that CR1 might bind Dpp while CR4 bound Gbb. The results of the CR swap experiments performed in the contexts of SogCR4, Supersog1, and SogCR1,2 are quite informative in resolving this question and provide a surprising answer, namely that sequences adjacent to CR domains are the primary determinants of BMP specificity (Yu, 2004).

While replacing a CR domain in swap constructs typically resulted in greatly reduced activity or inactivity of UAS-transgenes tested as single- or multicopy insertions, those that had activity generated phenotypes similar to those of the parent constructs. These data suggest that the CRs are required in a context-specific fashion to boost the activity levels of the various forms of Sog. These findings, although limited in scope, suggest that the primary determinant of the quality of Sog activity lies within the sequences surrounding the CRs rather than within the CR domains themselves. These non-CR sequences may correspond to identified repetitive motifs such as the SR repeats or the circularly permuted SOG/CHRD domains. The simplest interpretation of this result is that CRs contribute to defining the strength and surrounding sequences determine the specificity of Sog activities. This model of Sog function is consistent with the finding that deletion of any single CR does not eliminate Sog-like activity (e.g., inhibition of Scw/Gbb) or the ability to interact with Tsg (e.g., inhibition of Dpp) and that both the CR1 domain and adjacent stem sequences are required for Supersog activity. The fact that the conserved tryptophans in the CR domains are required in aggregate, but not individually, for interaction with Tsg and that stem sequences are also required for this interaction lends additional support to the view that the CRs function in a partially redundant fashion in conjunction with non-CR sequences. One potential explanation for the results reported here is that the CR domains alone bind to BMPs, but do so with little selectivity. The surrounding sequences may provide specificity by interacting with only a subset of BMPs, thereby increasing the affinity of adjacent CRs for particular BMPs. Alternatively, surrounding sequences may alter the conformation of CR domains or sterically limit their interactions with BMPs, rendering them more selective. Further biochemical analysis will be required to determine the degree to which CRs affect affinity of the various Sog forms for particular BMPs and the mechanism by which surrounding sequences may confer specificity for binding particular BMPs (Yu, 2004).


REFERENCES

Akiyama-Oda, Y. and Oda, H. (2006). Axis specification in the spider embryo: dpp is required for radial-to-axial symmetry transformation and sog for ventral patterning. Development 133(12): 2347-57. PubMed ID: 16720876

Ambrosio, A. L., et al. (2008). Crossveinless-2 is a BMP feedback inhibitor that binds Chordin/BMP to regulate Xenopus embryonic patterning. Dev. Cell 15: 248-260. PubMed ID: 18694564

Anderson, R. M., et al. (2002). Chordin and noggin promote organizing centers of forebrain development in the mouse. Development 129: 4975-4987. PubMed ID: 12397106

Araujo, H. and Bier, E. (2000). sog and dpp exert opposing maternal functions to modify Toll signaling and pattern the dorsoventral axis of the Drosophila embryo. Development 127: 3631-3644. PubMed ID: 10903186

Araujo, H., Negreiros, E. and Bier, E. (2003). Integrins modulate Sog activity in the Drosophila wing. Development 130: 3851-3864. PubMed ID: 12835400

Ardehali, M. B. and Lis, J. T. (2009). Tracking rates of transcription and splicing in vivo. Nat. Struct. Mol. Biol. 16: 1123-1124. PubMed ID: 19888309

Ashe, H. L. and Levine, M. (1999). Local inhibition and long-range enhancement of Dpp signal transduction by Sog. Nature 398(6726): 427-31. PubMed ID: 10201373

Bachiller, D., et al. (2003). The role of chordin/Bmp signals in mammalian pharyngeal development and DiGeorge syndrome. Development 130: 3567-3578. PubMed ID: 12810603

Biehs, B., François, V. and Bier, E. (1996). The Drosophila short gastrulation gene prevents Dpp from autoactivating a suppressing neurogenesis in the neuroectoderm. Genes Dev. 10: 2922-34. PubMed ID: 8918893

Blader, P., et al. (1997). Cleavage of the BMP-4 antagonist chordin by zebrafish tolloid. Science 278(5345): 1937-1940. PubMed ID: 9395394

Bothma, J. P., Magliocco, J. and Levine, M. (2011). The Snail repressor inhibits release, not elongation, of paused Pol II in the Drosophila embryo. Curr. Biol. 21: 1571-1577. PubMed ID: 21920753

Carneiro, K., et al. (2006). Graded maternal short gastrulation protein contributes to embryonic dorsal-ventral patterning by delayed induction. Dev. Biol. 296(1): 203-18. PubMed ID: 16781701

Chang, C., et al. (2001). Twisted gastrulation can function as a BMP antagonist. Nature 410: 483-487. PubMed ID: 11260717

Classen, A.-K., Anderson, K. I., Marois, E. and Eaton, S. (2005). Hexagonal packing of Drosophila wing epithelial cells by the planar cell polarity pathway. Dev. Cell 9(6): 805-17. PubMed ID: 16326392

Collart, C., et al. (2005). The novel Smad-interacting protein Smicl regulates Chordin expression in the Xenopus embryo. Development 132: 4575-4586. PubMed ID: 16192311

da Fonseca, R. N., et al. (2008). Self-regulatory circuits in dorsoventral axis formation of the short-germ beetle Tribolium castaneum. Dev. Cell 14: 605-615. PubMed ID: 18410735

Dale, K., et al. (1999). Differential patterning of ventral midline cells by axial mesoderm is regulated by BMP7 and chordin. Development 126(2): 397-408. PubMed ID: 9847252

Decotto, E. and Ferguson, E. L. (2001). A positive role for Short gastrulation in modulating BMP signaling during dorsoventral patterning in the Drosophila embryo. Development 128: 3831-3841. PubMed ID: 11585808

De Velasco, B., Shen, J., Go, S. and Hartenstein, V. (2004). Embryonic development of the Drosophila corpus cardiacum, a neuroendocrine gland with similarity to the vertebrate pituitary, is controlled by sine oculis and glass. Dev. Biol. 274: 280-294. PubMed ID: 15385159

Dorfman, R. and Shilo, B.-Z. (2001). Biphasic activation of the BMP pathway patterns the Drosophila embryonic dorsal region. Development 128: 965-972. PubMed ID: 11222150

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

Fekany-Lee, K., et al. (2000). The homeobox gene bozozok promotes anterior neuroectoderm formation in zebrafish through negative regulation of BMP2/4 and Wnt pathways. Development 127: 2333-2345. PubMed ID: 10804176

Ferguson, E. L. and Anderson, K. V. (1992). Localized enhancement and repression of the activity of the TGF-beta family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo. Development 114: 583-97. PubMed ID: 1618130

Foo, S. M., Sun, Y., Lim, B., Ziukaite, R., O'Brien, K., Nien, C. Y., Kirov, N., Shvartsman, S. Y. and Rushlow, C. A. (2014). Zelda potentiates morphogen activity by increasing chromatin accessibility. Curr Biol 24: 1341-1346. PubMed ID: 24909324

Francois, V., Solloway, M., O'Neill, J. W., Emery J. and Bier E. (1994). Dorsal-ventral patterning of the Drosophila embryo depends on a putative negative growth factor encoded by the short gastrulation gene. Genes Dev. 8: 2602-16. PubMed ID: 7958919

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. PubMed ID: 17522157

Gonzalez, E. M., et al. (2000). Head and trunk in zebrafish arise via coinhibition of BMP signaling by bozozok and chordino. Genes Dev. 14: 3087-3092. PubMed ID: 11124801

Hashimoto, K., et al. (1999). Distinct signaling molecules control Hoxa-11 and Hoxa-13 expression in the muscle precursor and mesenchyme of the chick limb bud. Development 126(12): 2771-2783. PubMed ID: 10331987

Holley, S. A., et al. (1995). A conserved system for dorsal-ventral patterning in insects and vertebrates involving sog and chordin. Nature 376: 249-253. PubMed ID: 7617035

Holley, S. A., et al. (1996). The Xenopus dorsalizing factor noggin ventralizes Drosophila embryos by preventing DPP from activating its receptor. Cell 86: 607-617. PubMed ID: 8752215

Irion, U. and Leptin, M. (1999). Developmental and cell biological functions of the Drosophila DEAD-box protein Abstrakt. Curr. Biol. (23): 1373-81. PubMed ID: 10607561

Jazwinska, A., Rushlow, C. and Roth, S. (1999). The role of brinker in mediating the graded response to Dpp in early Drosophila embryos. Development 126(15): 3323-3334. PubMed ID: 10393112

Kalantry, S., et al. (2001). The amnionless gene, essential for mouse gastrulation, encodes a visceral-endoderm-specific protein with an extracellular cysteine-rich domain. Nat Genet. 27(4): 351-2. PubMed ID: 11279523

Kang, K. H. and Bier, E. (2011). dHIP14-dependent palmitoylation promotes secretion of the BMP antagonist Sog. Dev. Biol. 346(1): 1-10. PubMed ID: 20599894

Kim, M.-J. and Han, J.-K. (1999). The involvement of cAMP signaling pathway in axis specification in Xenopus embryos. Mech. Dev. 89: 55-64. PubMed ID: 10559480

Kireeva, M. L., et al. (1996). Cyr61, a product of a growth factor-inducible immediate-early gene, promotes cell proliferation, migration, and adhesion. Mol. Cell. Biol. 16: 1326-1334. PubMed ID: 8657105

Kotkamp, K., Klingler, M. and Schoppmeier, M. (2010). Apparent role of Tribolium orthodenticle in anteroposterior blastoderm patterning largely reflects novel functions in dorsoventral axis formation and cell survival. Development 137(11): 1853-62. PubMed ID: 20431120

Koshida, S., et al. (2002). Inhibition of BMP activity by the FGF signal promotes posterior neural development in zebrafish. Dev. Biol. 244: 9-20. PubMed ID: 11900455

Langevin, J., et al. (2005). Drosophila exocyst components Sec5, Sec6, and Sec15 regulate DE-Cadherin trafficking from recycling endosomes to the plasma membrane. Dev. Cell. 9(3): 355-76. PubMed ID: 16224820

Larrain, J., et al. (2000). BMP-binding modules in chordin: a model for signalling regulation in the extracellular space. Development 127: 821-830. PubMed ID: 10648240

Larraín, J., et al. (2001). Proteolytic cleavage of Chordin as a switch for the dual activities of Twisted gastrulation in BMP signaling. Development 128: 4439-4447. PubMed ID: 11714670

Lee, H. X., Ambrosio, A. L., Reversade, B. and De Robertis, E. M. (2006). Embryonic dorsal-ventral signaling: secreted frizzled-related proteins as inhibitors of tolloid proteinases. Cell 124(1): 147-59. PubMed ID: 16413488

Lee, H. X., Mendes, F. A., Plouhinec, J. L. and De Robertis, E. M. (2009). Enzymatic regulation of pattern: BMP4 binds CUB domains of Tolloids and inhibits proteinase activity. Genes Dev. 23(21): 2551-62. PubMed ID: 19884260

Li, L. M. and Arnosti, D. N. (2011). Long- and short-range transcriptional repressors induce distinct chromatin states on repressed genes. Curr. Biol. 21: 406-412. PubMed ID: 21353562

Liberman, L. M. and Stathopoulos, A. (2009). Design flexibility in cis-regulatory control of gene expression: synthetic and comparative evidence. Dev. Biol. 327(2): 578-89. PubMed ID: 19135437

Lock, J. G. and Stow, J. L. (2005). Rab11 in recycling endosomes regulates the sorting and basolateral transport of E-cadherin, Mol. Biol. Cell 16: 1744-1755. PubMed ID: 15689490

Londin, E. R., Niemiec, J. and Sirotkin, H. I. (2005). Chordin, FGF signaling, and mesodermal factors cooperate in zebrafish neural induction. Dev. Biol. 279(1): 1-19. PubMed ID: 15708554

Matus, D. Q., Pang, K., Marlow, H., Dunn, C. W., Thomsen, G. H. and Martindale, M. Q. (2006). Molecular evidence for deep evolutionary roots of bilaterality in animal development. Proc. Natl. Acad. Sci. 103(30): 11195-200. PubMed ID: 16837574

Markstein, M., et al. (2002). Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo Proc. Natl. Acad. Sci. 99: 763-768. PubMed ID: 11752406

Marques, G., et al. (1997). Production of a DPP activity gradient in the early Drosophila embryo through the opposing actions of the SOG and TLD proteins. Cell 91(3): 417-426. PubMed ID: 9363950

Martin-Blanco, E., et al. (1999). A temporal switch in DER signaling controls the specification and differentiation of veins and interveins in the Drosophila wing. Development 126: 5739-5747. PubMed ID: 10572049

Melby, A. E., Clements, W. K. and Kimelman, D. (1999). Regulation of dorsal gene expression in Xenopus by the ventralizing homeodomain gene Vox. Dev. Biol. 211(2): 293-305. PubMed ID: 10395789

Millet, C., et al. (2001). The human chordin gene encodes several differentially expressed spliced variants with distinct BMP opposing activities. Mech. Dev. 106: 85-96. PubMed ID: 11472837

Mizuseki, K., et al. (1998). Xenopus Zic-related-1 and Sox-2, two factors induced by chordin, have distinct activities in the initiation of neural induction. Development 125(4): 579-587. PubMed ID: 9435279

Mizutani, C. M., et al. (2005). Formation of the BMP activity gradient in the Drosophila embryo. Dev. Cell 8(6): 915-24. PubMed ID: 15935780

Nakayama, N., et al. (2001). A novel Chordin-like protein inhibitor for bone morphogenetic proteins expressed preferentially in mesenchymal cell lineages. Dev. Bio. 232: 372-387. PubMed ID: 11401399

Neul, J. L. and Ferguson, E. L. (1998). Spatially restricted activation of the SAX receptor by SCW modulates DPP/TKV signaling in Drosophila dorsal-ventral patterning. Cell 95(4):483-94. PubMed ID: 9827801

Oelgeschläger, M., et al. (2003a). Chordin is required for the Spemann organizer transplantation phenomenon in xenopus embryos. Dev. Cell 4: 219-230. PubMed ID: 12586065

Oelgeschläger, M., et al. (2003b). The pro-BMP activity of Twisted gastrulation is independent of BMP binding. Development 130: 4047-4056. PubMed ID: 12874126

Ohta, K., et al. (2004). Tsukushi functions as an organizer inducer by inhibition of BMP activity in cooperation with chordin. Dev. Cell 7: 347-358. PubMed ID: 15363410

Pappano, W. N., et al. (1998). Coding sequence and expression patterns of mouse chordin and mapping of the cognate mouse chrd and human CHRD genes. Genomics 52(2): 236-9. PubMed ID: 9782094

Peluso, C. E., Umulis, D., Kim, Y. J., O'Connor, M. B. and Serpe, M. (2011). Shaping BMP Morphogen Gradients through Enzyme-Substrate Interactions. Dev. Cell 21(2): 375-83. PubMed ID: 21839924

Petesch, S. J. and Lis, J. T. (2008). Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci. Cell 134: 74-84. PubMed ID: 18614012

Piccolo, S., et al. (1996). Dorsoventral patterning in Xenopus: Inhibition of ventral signals by direct binding of Chordin to BMP-4. Cell 86: 589-598. PubMed ID: 8752213

Piccolo, S., et al. (1997). Cleavage of Chordin by Xolloid metalloprotease suggests a role for proteolytic processing in the regulation of Spemann organizer activity. Cell 91(3): 407-16. PubMed ID: 9363949

Podos, S. D., Hanson, K. K., Wang, Y.-C. and Ferguson, E. L. (2001). The Smurf1 ubiquitin-protein ligase restricts BMP signaling spatially and temporally during Drosophila embryogenesis. Dev. Cell 1: 567-578. PubMed ID: 11703946

Rentzsch, F., Anton, R., Saina, M., Hammerschmidt, M., Holstein, T. W. and Technau, U. (2006). Asymmetric expression of the BMP antagonists chordin and gremlin in the sea anemone Nematostella vectensis: implications for the evolution of axial patterning. Dev. Biol. 296(2): 375-87. PubMed ID: 16828077

Rohr, K. B., Schulte-Merker, S. and Tautz, D. (1999). Zebrafish zic1 expression in brain and somites is affected by BMP and Hedgehog signalling. Mec. Dev. 85(1-2): 147-159. PubMed ID: 10415355

Ross, J. J., et al. (2001). Twisted gastrulation is a conserved extracellular BMP antagonist. Nature 410: 479-483. PubMed ID: 11260716

Rusten, T. E., et al. (2002). The role of TGFß signaling in the formation of the dorsal nervous system is conserved between Drosophila and chordates. Development 129: 3575-3584. PubMed ID: 12117808

Sasai, Y., Lu, B., Steinbeisser, H. and De Robertis, E. M. (1995). Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus. Nature 376: 333-336. PubMed ID: 7630399

Schmidt, J., et al. (1995). Drosophila short gastrulation induces an ectopic axis in Xenopus: evidence for conserved mechanisms of dorsal-ventral patterning. Development 121: 4319-4328. PubMed ID: 8575332

Scott, I. C., et al. (1999). Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis. Dev. Biol. 213(2): 283-300. PubMed ID: 10479448

Scott, I. C., et al. (2001). Homologues of Twisted gastrulation are extracellular cofactors in antagonism of BMP signaling. Nature 410: 475-478. PubMed ID: 11260715

Serpe, M., et al. (2005). Matching catalytic activity to developmental function: Tolloid-related processes Sog in order to help specify the posterior crossvein in the Drosophila wing. Development 132: 2645-2656. PubMed ID: 15872004

Shimmi, O. and O'Connor, M. B. (2003). Physical properties of Tld, Sog, Tsg and Dpp protein interactions are predicted to help create a sharp boundary in Bmp signals during dorsoventral patterning of the Drosophila embryo. Development 130: 4673-4682. PubMed ID: 12925593

Shimmi, O., Umulis, D., Othmer, H. and O'Connor, M. B. (2005a). Facilitated transport of a Dpp/Scw heterodimer by Sog/Tsg leads to robust patterning of the Drosophila blastoderm embryo. Cell 120(6): 873-86. PubMed ID: 15797386

Shimmi, O., Ralston, A., Blair, S. S. and O'Connor, M. B. (2005b). The crossveinless gene encodes a new member of the Twisted gastrulation family of BMP-binding proteins which, with Short gastrulation, promotes BMP signaling in the crossveins of the Drosophila wing. Dev. Biol. 282(1): 70-83. PubMed ID: 15936330

Srinivasan, S., Rashka, K. E. and Bier, E. (2002). Creation of a Sog morphogen gradient in the Drosophila embryo. Dev. Cell 2(1): 91-101. PubMed ID: 11782317

Stottmann, R. W., Anderson, R. M. and Klingensmith, J. (2001). The BMP antagonists chordin and noggin have essential but redundant roles in mouse mandibular outgrowth. Dev. Biol. 240(2): 457-473. PubMed ID: 11784076

Stathopoulos, A. and Levine, M. (2002). Linear signaling in the Toll-Dorsal pathway of Drosophila: activated Pelle kinase specifies all threshold outputs of gene expression while the bHLH protein Twist specifies a subset. Development 129: 3411-3419. PubMed ID: 12091311

Streit, A., et al. (1998). Chordin regulates primitive streak development and the stability of induced neural cells, but is not sufficient for neural induction in the chick embryo. Development 125(3): 507-519. PubMed ID: 9425145

Streit, A. and Stern, C. D. (1999). Mesoderm patterning and somite formation during node regression: differential effects of chordin and noggin, Mech. Dev. 85(1-2): 85-96. PubMed ID: 10415349

Sutherland, D. J., et al. (2003). Stepwise formation of a SMAD activity gradient during dorsal-ventral patterning of the Drosophila embryo. Development 130: 5705-5716. PubMed ID: 14534137

Taira, M., Saint-Jeannet, J.-P. and Dawid, I. B. (1997). Role of the Xlim-1 and Xbra genes in anteroposterior patterning of neural tissue by the head and trunk organizer. Proc. Natl. Acad. Sci. 94: 895-900. PubMed ID: 9023353

Varga, M., et al. (2007). Chordin expression, mediated by Nodal and FGF signaling, is restricted by redundant function of two beta-catenins in the zebrafish embryo. Mech. Dev. 124(9-10): 775-91. PubMed ID: 17686615

Wagner, D. S. and Mullins, M. C. (2002). Modulation of BMP activity in dorsal-ventral pattern formation by the Chordin and Ogon antagonists. Dev. Biol. 245: 109-123. PubMed ID: 11969259

Vilmos, P., et al. (2005). Crossveinless defines a new family of Twisted-gastrulation-like modulators of bone morphogenetic protein signalling. EMBO Rep. 6: 262-267. PubMed ID: 15711536

Wong, M., et al. (1997). Cyr61, product of a growth factor-inducible immediate-early gene, regulates chondrogenesis in mouse limb bud mesenchymal cells. Dev. Biol. 192(2): 492-508. PubMed ID: 9441684

Xie, J. and Fisher, S. (2005). Twisted gastrulation enhances BMP signaling through chordin dependent and independent mechanisms. Development 132: 383-391. PubMed ID: 15604098

Yabe, T., et al. (2003). Ogon/Secreted Frizzled functions as a negative feedback regulator of Bmp signaling. Development 130: 2705-2716. PubMed ID: 12736214

Yokota, C., Ariizumi, T. and Asashima, M. (1998). Patterns of gene expression in the core of Spemann's organizer and activin-treated ectoderm in Cynops pyrrhogaster. Dev. Growth Differ. 40(3): 335-41. PubMed ID: 9639361

Yu, K., et al. (1996). The Drosophila decapentaplegic and short gastrulation genes function antagonistically during adult wing vein development. Development 122: 4033-4044. PubMed ID: 9012523

Yu, K., et al. (2000). Processing of the Drosophila Sog protein creates a novel BMP inhibitory activity. Development 127: 2143-2154. PubMed ID: 10769238

Yu, K., et al. (2004). Cysteine repeat domains and adjacent sequences determine distinct Bone morphogenetic protein modulatory activities of the Drosophila Sog protein. Genetics 166: 1323-1336. PubMed ID: 15082551

Zakin, L., Chang, E. Y., Plouhinec, J. L. and De Robertis, E. M. (2010). Crossveinless-2 is required for the relocalization of Chordin protein within the vertebral field in mouse embryos. Dev. Biol. 347(1): 204-15. PubMed ID: 20807528

Zorn, A. M., Butler, K. and Gurdon, J. B. (1999). Anterior endomesoderm specification in xenopus by Wnt/beta-catenin and TGF-beta signalling pathways. Dev. Biol. 209(2): 282-97. PubMed ID: 10328921

Zusman, S. B., Sweeton, D. and Wieschaus, E. F. (1988). short gastrulation, a mutation causing delays instage-specific cell shape changes during gastrulation in Drosophila melanogaster. Dev. Biol. 129: 417-27. PubMed ID: 3138149


short gastrulation : Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 October 2011
    

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

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