tolloid
The Dorsal protein (DL) regulates the transcriptional activity of several genes that determine cell
fate along the dorsoventral axis of the embryo. Approximately 800 bp of 5'-flanking sequences upstream of the tld coding region
were shown to drive an expression pattern indistinguishable from the wild-type pattern. A 423-bp
fragment located within these sequences contains two DL binding sites and was shown to act as a
silencer to mediate ventral repression. Point mutations in the sites abolish not only DNA binding but
also ventral repression (Kirov, 1994).
A Dpp activity gradient specifies multiple thresholds of gene expression in the dorsal ectoderm of the early embryo. Some of these
thresholds depend on a putative repressor, Brinker, which is expressed in the neurogenic ectoderm in response to the maternal Dorsal
gradient and Dpp signaling. In this study it is shown that Brinker is a sequence-specific transcriptional repressor. It binds the consensus sequence,
TGGCGc/tc/t, and interacts with the Groucho corepressor through a conserved sequence motif, FKPY. An optimal Brinker binding site
is contained within an 800-bp enhancer from the tolloid gene, which has been identified as a genetic target of the Brinker repressor. A
tolloid-lacZ transgene containing point mutations in this site exhibits an expanded pattern of expression, suggesting that Brinker directly represses tolloid transcription (Zhang, 2001).
The interplay between extracellular signaling molecules and localized transcriptional repressors is reminiscent of the segmentation pathway in the early Drosophila
embryo. Pair-rule stripes of gene expression are established by broadly distributed transcriptional activators, such as Bicoid and Stat. The stripe borders are formed
by localized gap repressors, including Hunchback, Kruppel, and Knirps. Similarly, the activation of tolloid
and pannier might depend on broadly distributed Smad proteins, whereas the lateral limits of the expression patterns are established by the localized Brinker
repressor. It is likely that vertebrates also employ one or more transcriptional repressors to restrict TGF-beta signaling interactions (Zhang, 2001).
Embryonic target gene activation in the absence of brinker 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 cuticle of brk mutant embryos has an enlarged region
carrying dorsal hairs and a smaller region carrying ventral
denticles. The number of sna-expressing neuroblasts
in the ventral neurogenic region is reduced. This
indicates that brk mutations lead to an expansion of
dorsolateral fates and a reduction of ventrolateral fates.
However, despite these lateral fate shifts, the number of Kr-expressing
amnioserosa cells is not different from wild type. Thus, brk specifically affects cell fates depending on
low or intermediate levels of Dpp signaling, while those that
require peak levels are not altered. To identify the underlying causes of the visible changes in
cell fate, the effect of brk was examined on the expression of
two groups of dorsal/ventral (DV) patterning genes. The first group consists of
dpp, zerknullt (zen) and tolloid (tld), whose expression is initiated very early in
syncytial blastoderm stages. Since they are ventrally repressed
by Dorsal (Dl) protein, their expression domains are confined to the
dorsal 40% of the egg's circumference. In brk mutant embryos dpp, zen and tld
expression is initiated normally.
However, in contrast to wild type their expression domains
expand ventrally during mid-cellularization.
These data demonstrate that brk is not required for
the early ventrolateral repression of these genes, but is essential
to prevent their lateral expansion during cellularization. The second group of DV patterning genes includes
rhomboid (rho), u-shaped
(ush) and pannier (pnr), which
are not direct targets of repression by maternal Dl. The
initiation of their expression during cellularization requires
prior formation of the Dpp activity gradient.
Therefore, they are candidates for being direct targets of Dpp
signaling in the embryo. They are expressed in domains
straddling the dorsal midline that are 12 (rho), 14 (ush) and 32
(pnr) cells wide at cellular blastoderm (cell counts at approx.
50% egg length). The two narrowly expressed genes
rho and ush are not changed in brk mutant embryos. This is also true for late zen expression, which in brk
mutant embryos, as in wild type, refines to a narrow 5- to 6-
cell-wide stripe along the dorsal midline despite the prior
expansion. However, pnr expression expands
in brk mutant embryos and low ectopic pnr levels can be seen
in a broad lateral domain that stops about five cells short of
mesodermal sna expression. Thus, brk does not affect the
Dpp target genes that are expressed in dorsalmost regions and
supposedly depend on highest Dpp levels. However, a target
gene that is expressed in a wider domain, and is therefore
presumably activated by intermediate levels of Dpp, is
expanded. In summary, brk mutations affect the Dpp activity gradient
in the embryo by expanding the domains of expression of dpp
and one of its activators (tld) into ventrolateral regions. Despite
the uniform expression of dpp in the entire ectoderm, Dpp
activity levels appear to be only mildly increased in the
ventrolateral region since only low-level (zen) or intermediate-level
(pnr) target genes are ectopically expressed, causing a
reduction in the size of the nervous system and ventral
epidermis accompanied by an expansion of dorsal epidermis.
Peak levels of Dpp in dorsalmost positions appear to be normal,
judging from both target gene expression and cell type
differentiation (Jazwinska, 1999).
The CBP histone acetyltransferase plays important roles in development and disease by acting as a transcriptional coregulator. A small reduction in the amount of Drosophila CBP (dCBP) leads to a specific loss of signaling by the TGF-ß molecules Dpp and Screw in the early embryo. The expression of Screw itself, and that of two regulators of Dpp/Screw activity, Twisted-gastrulation and the Tolloid protease, is compromised in dCBP mutant embryos. This prevents Dpp/Screw from initiating a signal transduction event in the receiving cell. Smad proteins, the intracellular transducers of the signal, fail to become activated by phosphorylation in dCBP mutants, leading to diminished Dpp/Screw-target gene expression. At a slightly later stage of development, Dpp/Screw-signaling recovers in dCBP mutants, but without a restoration of Dpp/Screw-target gene expression. In this situation, dCBP acts downstream of Smad protein phosphorylation, presumably via direct interactions with the Drosophila Smad protein Mad. It appears that a major function of dCBP in the embryo is to regulate upstream components of the Dpp/Screw pathway by Smad-independent mechanisms, as well as acting as a Smad coactivator on downstream target genes. These results highlight the exceptional sensitivity of components in the TGF-ß signaling pathway to a decline in CBP concentration (Lilja, 2003).
These results suggest that several transcription factors that regulate expression of Dpp/Screw signaling components require the dCBP coactivator for their function in Drosophila embryos, and implicate dCBP in regulation of the Dpp/Screw pathway independently of an interaction with Smad proteins. An additional role of dCBP is to regulate Dpp-target genes, acting at a step downstream of Smad protein phosphorylation. It is likely that direct interactions between dCBP and Mad/Medea contribute to regulation of Dpp target genes). Such interactions have been observed in vitro, both in mammalian systems and using Drosophila proteins. However, a major cause of impaired Dpp/Screw signaling in dCBP mutant embryos is due to reduced tolloid expression. This prevents Dpp/Screw from initiating a signaling event in cells that would normally receive the Dpp/Screw signal, presumably by a failure to cleave the Dpp-Sog and/or Screw-Sog complexes. In fact, a majority of embryos that do not express the Dpp/Screw-target gene rhomboid in dorsal cells, also do not contain phosphorylated Smad proteins. Furthermore, the pattern of phosphorylated Smad proteins correlates closely with that of tolloid expression. For example, in many early, cellularizing dCBP mutant embryos, an anterior patch of both tolloid expression and phosphorylated Smad staining remains. At later stages, tolloid expression recovers in dCBP mutant embryos, as does Dpp/Screw signaling as revealed by Smad protein phosphorylation. This recovery of tolloid expression at later stages of development might explain the recovery of phosphorylated Smad proteins in dCBP mutant embryos, by allowing Dpp/Screw to signal. For these reasons, regulation of tolloid expression appears to be a major means of controlling Dpp/Screw signaling by dCBP (Lilja, 2003).
It is likely that reduced screw expression also contributes to the reduction of phosphorylated Smad proteins observed in dCBP mutant embryos. In both screw and tolloid mutants, phosphorylation of Mad is eliminated. Furthermore, progressive reduction in Screw activity leads to a corresponding progressive deletion of dorsal-most cell fate, the amnioserosa. Tsg is required together with Sog to generate peak Dpp activity in dorsal midline cells. Reduced tsg expression in dCBP mutants may therefore contribute to the lack of Dpp/Screw-target gene expression. However, it is not believe that this lack can explain the defects in dCBP mutants, because in tsg mutants, low levels of Dpp signaling persist in a broad dorsal domain, leading to expanded rhomboid expression in dorsal cells. By contrast, in dCBP mutant embryos, expression of genes in response to a low threshold of Dpp activity, such as U-shaped and the dorsal rhomboid pattern, is eliminated (Lilja, 2003).
These experiments do not address whether dCBP regulation of tolloid, screw, and tsg expression is direct or indirect. However, since expression of these genes begins at about the time when zygotic transcription initiates in the embryo, and the effect of dCBP is evident from the onset of expression of tolloid and screw, the notion is favored that dCBP is acting directly on the enhancers of these genes. It is not yet understood whether HATs such as CBP primarily act to acetylate large chromosomal domains, or are directed to specific genes. In the case of the tolloid gene, the results indicate that dCBP is being recruited to the enhancer by a DNA-binding protein, since the isolated enhancer removed from its normal chromosomal location requires dCBP for its activity (Lilja, 2003).
Given its central position in gene regulation and the great number of mammalian transcription factors shown to interact with CBP, relatively few genes are affected by the dCBP mutation. For example, activation and repression mediated by the Dorsal protein are unaffected in the dCBP mutant embryos, as demonstrated by the expression patterns of Dorsal target genes. Also, no defects in early segmentation gene expression could be observed in germline clone mutants. However, the nej1 mutation used in this study to create dCBP mutant germline clone embryos is a weak mutation that results in a very modest reduction in dCBP levels. Since other means of reducing the dCBP amount by approximately two-fold results in similar gene expression defects, Smad proteins and the unidentified activators of tolloid, tsg, and screw expression are particularly sensitive to a decline in dCBP concentration. It may not be a coincidence that screw, tsg, tolloid, and Dpp-target gene expression are all specifically affected by a small dCBP reduction. Perhaps components of the Dpp/Screw signal transduction pathway have evolved to be coordinately regulated by a common coactivator. Given the phylogenetic conservation of the CBP protein and the TGF-alpha signal transduction pathway, as well as the ability of CBP and Smad proteins to interact in vitro, CBP is likely to play an equally important role in TGF-ß signaling in other metazoans (Lilja, 2003).
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).
DPP's activity is modulated by Tolloid, which also has a role in the determination of dorsal cell fate. The Tolloid protein functions by forming a complex containing DPP via
protein-interacting EGF and C1r/s domains. The protease activity of Tolloid is
necessary, either directly or indirectly, for the activation of the DPP complex (Finelli, 1994).
Noggin, a protein expressed in the Spemann organizer region of the Xenopus embryo, promotes dorsal cell fate within the mesoderm and neural development within overlying ectoderm. noggin, expressed in Drosophila, promotes ventral development, specifying ventral ectoderm and CNS in the absence of all endogenous ventral-specific zygotic gene expression. Noggin blocks DPP signaling upstream of DPP receptor activation. It is proposed that, whole most or all of the DPP produced in the dorsal-most region binds to its receptors, DPP produced more laterally has an increased probability of being bound by ventrally produced Short gastrulation, and that DPP can be released from this diffusible complex by the action of a third dorsal-specific gene, perhaps tolloid (Holley, 1996).
Extracellular gradients of signaling molecules can specify different thresholds of gene activity in development. A gradient of Decapentaplegic (Dpp) activity
subdivides the dorsal ectoderm of the Drosophila embryo into amnioserosa and dorsal epidermis. The proteins Short gastrulation (Sog) and Tolloid (Tld) are
required to shape this gradient. Sog has been proposed to form an inhibitory complex with either Dpp or the related ligand Screw, and is subsequently processed by
the protease Tld. Paradoxically, Sog appears to be required for amnioserosa formation, which is specified by peak Dpp signaling. Sog appears to be required for peak Dpp/Screw activity, since sog mutants lack amnioserosa. SOG transcripts are detected in two ventrolateral stripes within the presumptive neurogenic ectoderm. Several amnioserosa marker genes, including Kruppel, rhomboid and hindsight exhibit broadened patterns of expression that gradually diminish in older embryos. In contrast, the Race (Related to angiotensin converting enzyme) pattern is not transiently expanded in sog mutants; instead, by the onset of gastrulation, expression is nearly lost in central regions. Race may represent a more definitive marker for the presumptive amnioserosa than the genes used in previous studies (Ashe, 1999).
The
misexpression of sog using the even-skipped stripe-2 enhancer redistributes Dpp signalling in a mutant background in which dpp is expressed throughout the
embryo. Dpp activity is diminished near the Sog stripe and peak Dpp signaling is detected far from this stripe. However, a tethered form of Sog suppresses local
Dpp activity without augmenting Dpp activity at a distance, indicating that diffusion of Sog may be required for enhanced Dpp activity and consequent amnioserosa
formation. The long-distance stimulation of Dpp activity by Sog requires Tld, whereas Sog-mediated inhibition of Dpp does not. The heterologous Dpp inhibitor
Noggin inhibits Dpp signaling but fails to augment Dpp activity. These results suggest an unusual strategy for generating a gradient threshold of growth-factor activity,
whereby Sog and its protease specify peak Dpp signaling far from a localized source of Sog. Different models have been proposed to explain the requirement of Sog in generating peak Dpp activity. One invokes the diffusion of Sog-Dpp or Sog-Screw complexes away from the ventrolateral Sog stripes, thereby focusing Dpp and/or Screw at the dorsal midline. An alternative model suggests that a product resulting from the cleavage of Sog directly signals formation of the amnioserosa, possibly by augmenting the binding of Dpp or Screw to the receptors Thick veins and Saxophone (Ashe, 1999).
Structurally unrelated neural inducers in vertebrate and
invertebrate embryos have been proposed to function by
binding to BMP4 or Dpp, respectively, and preventing these
homologous signals from activating their receptor(s). The functions of various forms
of the Drosophila Sog protein were examined using the discriminating
assay of Drosophila wing development. Misexpression of Drosophila Sog, or its vertebrate
counterpart Chordin, generates a very limited vein-loss
phenotype. This sog misexpression phenotype is very
similar to that of viable mutants of glass-bottom boat (gbb),
which encodes a BMP family member. Consistent with Sog
selectively interfering with Gbb signaling, Sog can block
the effect of misexpressing Gbb, but not Dpp in the wing.
In contrast to the limited BMP inhibitory activity of Sog,
carboxy-truncated forms of Sog,
referred to as Supersog, have been identified which when misexpressed cause a
broad range of dpp minus mutant phenotypes. Evidence is provided that Twisted gastrulation (Tsg) functions in the embryo to generate a
Supersog-like activity, perhaps by modifying the enzymic activity of Tolloid, the enzyme that processes Sog (Yu, 2000).
The predicted Sog protein is 1038
amino acids in length and contains four cysteine-rich (CR) domains
in the extracellular domain. The
metalloprotease Tld cleaves Sog at three major sites. Supersog1 is
an N-terminal fragment of Sog including CR1 plus another 114
amino acids, and contains an additional 33 amino acids derived from
vector sequences at its C terminus. Supersog2, which
contains the same amino acids as Supersog1 but terminates abruptly
at the end of Sog sequences, also generates Supersog phenotypes,
albeit slightly weaker than those observed with Supersog1. Supersog4 is an N-terminal fragment of Sog ending 80
amino acids before CR2 and includes 130 sog 3' UTR derived amino
acids (Yu, 2000).
In line with its
phenotypic effects, Supersog can block the effects of both
misexpressing Dpp and Gbb in the wing. Vertebrate
Noggin, in contrast, acts as a general inhibitor of
Dpp signaling, which can interfere with the effect of
overexpressing Dpp, but not Gbb. Evidence suggests that
Sog processing occurs in vivo and is biologically relevant.
Overexpression of intact Sog in embryos and adult wing
primordia leads to the developmentally regulated
processing of Sog. This in vivo processing of Sog can be
duplicated in vitro by treating Sog with a combination of
the metalloprotease Tolloid (Tld) plus Twisted Gastrulation
(Tsg), another extracellular factor involved in Dpp
signaling. In accord with this result, coexpression of intact
Sog and Tsg in developing wings generates a phenotype
very similar to that of Supersog. Evidence is provided that tsg functions in the embryo to generate a
Supersog-like activity, since Supersog can partially rescue
tsg minus mutants. Consistent with this finding, sog minus and tsg minus
mutants exhibit similar dorsal patterning defects during
early gastrulation. These results indicate that differential
processing of Sog generates a novel BMP inhibitory activity
during development and, more generally, that BMP
antagonists play distinct roles in regulating the quality as
well as the magnitude of BMP signaling (Yu, 2000).
To determine whether Sog might be processed in vivo to
generate Supersog-like molecules, an anti-Sog
antibody directed against an amino fragment of Sog was used on
immunoblots to analyze protein extracts from different stages
and tissues of developing Drosophila. This antibody
recognizes an epitope present in the stem portion of Supersog. An examination was made of the nature of Sog products
produced both in wild-type individuals as well as in flies
overexpressing Sog. This analysis reveals that Sog is
processed in vivo, and that this processing is developmentally
regulated. For example, in heat shocked early embryos carrying
eight copies of an HS-sog construct, a 76 kDa band, a doublet of bands migrating at 42/40
kDa, and a 28 kDa band were observed, in addition to a 120
kDa band corresponding to full-length Sog. These bands are likely to represent various forms of Sog
since they are strongly induced only in heat shocked HS-sog
blastoderm stage embryos. Heat induction of HS-sog pupae results in the
elevated production of prominent Sog fragments migrating at
76, 60, 50 and 42 kDa.
In pupal wings, the same pattern of Sog fragments is present in
overloaded extracts of wild-type pupal wings as observed in heat induced HS-sog wings, albeit at
lower levels. This significant level of endogenous processing is
not surprising given that wild-type pupal wings express high
levels of Sog throughout intervein regions, which account for
approximately 90% of cells in the wing (Yu, 2000).
Processing of exogenously provided Sog is developmentally
regulated. During embryonic and pupal stages, when Sog is
expressed in a significant fraction of cells and plays important
developmental roles, distinct patterns of Sog fragments are
produced. For example, during pupal development, 60, 50 and
42 kDa fragments are induced in heat shocked HS-sog
wings, while in early embryos, a pair of
induced bands migrating at 42/40 kDa is most prominent. In contrast, during late embryonic or third larval
instar stages, only the full length Sog band is observed upon
induction of HS-sog larvae. During
these latter stages of development, sog is expressed in only a
small percentage of cells and is not known to have any
significant developmental function. Thus, Sog is processed
in vivo at developmentally
relevant times and in different
patterns to generate fragments
that are likely to have distinct
activities from Sog in addition
to being degraded into inert
products (Yu, 2000).
The fact that pulses of Supersog1 expression delivered during
the late blastoderm stage of development can partially rescue
the tsg minus mutant embryos suggests that a Supersog-like activity
might mediate part of tsg function in vivo. In addition, late
blastoderm stage tsg minus mutant embryos display defects similar
to those of sog mutants, suggesting that tsg is involved in a late
function of Sog. Consistent with the view that tsg acts during
early gastrulation, tsg minus mutants
can not be rescued by driving expression of a tsg transgene
under the control of the tld promoter, which is expressed only
early during the blastoderm stage. In contrast, it is possible to rescue tsg minus mutants by driving tsg
expression with promoters that continue to be expressed into
early gastrulation. Several possible ways in
which Supersog-like activities could contribute to this stage of
development can be imagined, given that they have different ligand specificities
from intact Sog and are stable to further proteolysis by Tld.
Since Sog has been proposed to block the activity of Scw in
embryos, it is likely that some other BMP is the preferred target
of Supersog molecules. In addition, since Scw is only
expressed transiently during the blastoderm stage of
development, intact Sog would have no obvious target to
inhibit beyond this stage. Perhaps a stable broad-spectrum
BMP antagonist such as Supersog could inhibit the action of
other BMPs expressed in the dorsal ectoderm during early
stages of gastrulation (possibly Dpp itself) and thereby provide
a form of molecular memory, which helps maintain the
distinction between neural and non-neural ectoderm (Yu, 2000).
The observation that Supersog is less effective than Sog in
blocking BMP signaling in the early embryo is consistent with
the view that Supersog is not just a higher affinity version of Sog
and suggests that Supersog is actually less effective than Sog at
blocking the effect of Scw. The fact that Supersog does not
inhibit Dpp itself during early blastoderm stages is likely to be
the result of insufficient levels of Supersog being expressed by
the heat shock vector. It is possible, however, that an
endogenously produced Supersog activity (e.g. generated upon
Tsg binding to Sog) has a higher affinity for Dpp than the
artificially created Supersog1 construct. In any case, it is proposed
that Supersog acts in the late blastoderm embryo or during early
gastrulation stages rather than in the early blastoderm embryo,
and that during this latter period, it is able to block the activity
of a BMP (e.g. Dpp?) not recognized by Sog.
It is tempting to consider a two step temporal model for the
action of Sog and Supersog during embryonic dorsal-ventral
patterning to account for the fact that sog mutants display a
dorsal-ventral phenotype earlier than tsg minus mutants. According to
one such scenario, the labile Tld-sensitive form of full-length
Sog is produced from a localized source (i.e. the neuroectoderm)
and diffuses dorsally to be degraded by Tld. Tld acts as a sink
to create a transiently stable gradient of Sog, which creates a
reciprocal gradient of Dpp activity. The Sog gradient created by
this classic source/sink configuration would only be short-lived,
however, since cells begin migrating when gastrulation begins.
At this stage, the embryo elongates and the Dorsal gradient
collapses, leading to loss of gene expression in early zygotic D/V
domains. Following the establishment of the short-lived
hypothetical Sog gradient, tsg expression is initiated in dorsal
cells and leads to the production of stable Supersog-like
molecules by switching the activity of Tld from degrading to
activating Sog. Supersog-like molecules then could provide a
stable record of high versus low BMP signaling domains during
a subsequent step of development (Yu, 2000).
A variety of genetic evidence suggests that a gradient of Decapentaplegic (Dpp) activity determines distinct cell fates in the dorsal region of the Drosophila embryo, and that this gradient may be generated indirectly by an
inverse gradient of the BMP antagonist Short gastrulation (Sog). It has been proposed that Sog diffuses dorsally from the lateral neuroectoderm where it is produced, and is cleaved and degraded dorsally by the metalloprotease Tolloid (Tld). This study shows directly that Sog is distributed in a graded fashion in dorsal cells and that Tld degradation limits the levels of Sog dorsally. In addition, Dynamin-dependent retrieval of Sog acts in parallel with degradation by Tld as a dorsal sink for active Sog (Srinivasan, 2002).
The finding that Tld collaborates with the Tolkin (Tok) protease to limit Sog diffusion indicates that these two closely related proteases are likely to share at least this one important substrate. Consistent with this possibility, in vitro studies indicate that Tok can cleave Sog in vitro, but with significantly reduced activity relative to Tld. Since Tld cleaves Sog in only a limited number of specific sites in vitro, it is likely that another class of extracellular protease degrades the products of Tld/Tok cleavage to peptide fragments, which may be too small to be recognized by either the 8A or 8B Sog antibodies. It is noteworthy that Tld degradation of Sog occurs on a much more rapid time scale in vivo (e.g., 30 min) than in vitro (e.g., several hours). This finding is consistent with the developmental timescale of Tld activity and suggests that additional factors present in vivo accelerate the action of Tld (Srinivasan, 2002).
The observation that Sog degradation fails to take place in dorsal cells of dpp- mutants is consistent with in vitro experiments in which Dpp is required as a cofactor for Tld-dependent cleavage of Sog. In contrast to in vitro studies in which either Dpp or Scw can act as cofactors, only Dpp serves as a critical cofactor function for in vivo degradation of Sog. An interesting difference between the ectopic Sog observed in dpp- versus Df(tld) embryos is that the staining is uniform in dpp- mutants but retains some degree of gradation in Df(tld) mutants. It is possible that another yet uncharacterized metalloprotease collaborates with Tld and Tok to degrade Sog in the early embryo. Alternatively, Sog might bind to a complex containing Dpp that is still present in Df(tld) mutant and limits Sog diffusion dorsally. The formation of this complex, or the ability of Sog to bind to it, may be strictly dependent on Dpp, so that in its absence, there is no restraint on Sog diffusion dorsally (Srinivasan, 2002).
An additional aspect of this study is the finding that Dynamin (shi) functions in parallel with Tld/Tok to limit active Sog levels in dorsal cells, which is required to generate a peak response to BMP signaling in dorsal-most cells. The fact that shi was not picked up previously as a D/V mutant in systematic screens for embryonic patterning mutants presumably reflects the pleiotropic requirement for Dynamin function, which is also required for Hh, Wg, Notch, and EGF-R signaling as well as various other cell biological processes involving membrane trafficking. While Dynamin function is not required for diffusion of Sog dorsally, it does appear to be required for the maintenance of the Sog gradient by removing Sog from the extracellular space. It is also possible that Dynamin plays other roles in promoting BMP signaling and that removing Sog from shits; sog RNAi embryos compensates for this reduced function. One argument against this latter possibility is that elimination of Dynamin function prior to the production and secretion of Sog does not compromise BMP signaling at that earlier stage. In any case, it is clear that an active form of Sog mediates the reduction of BMP signaling associated with loss of Dynamin function (Srinivasan, 2002).
In addition to inhibiting the activity of Scw and thereby reducing BMP signaling, there is evidence that Sog can exert other activities. For example, in the presence of the secreted protein Twisted gastrulation, Sog is cleaved in a different pattern by Tld in vitro to generate a truncated form of Sog consisting of CR1 and part of the stem. This truncated molecule, called Supersog, can inhibit Dpp as well as the auxiliary BMPs Scw and Glass bottom boat (Gbb). A major function of Tsg is to generate a Supersog-like activity in vivo, since expression of Supersog, but not intact Sog, can partially rescue tsg- mutant embryos. Supersog may play a persistent role in inhibiting BMP signaling following the transient expression of Scw, since it is refractory to degradation by Tld. There is also indirect genetic evidence that Sog acts at a long range to promote BMP signaling as judged by activation of the target gene RACE. Since Tld plays a dose-dependent role in generating this putative positive Sog activity, it too may be a processed form of Sog. It has also been proposed that some form of Sog might carry Dpp to the dorsal midline and thereby concentrate BMP along the dorsal midline. One line of evidence supporting this model is that the pattern of phosphorylation and activation of Mad observed in situ by staining with an anti-pMAD antibody reveals a narrow dorsal band of peak BMP activity with little evidence for a gradient diminishing ventrally. However, there is also a wealth of indirect genetic evidence that there are several intermediate levels of BMP activity that activate several dorsally expressed BMP target genes at different levels (Srinivasan, 2002).
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