short gastrulation

Promoter

Genome-wide analysis of clustered Dorsal binding sites was used to examine the distribution of Dorsal recognition sequences in the Drosophila genome. The homeobox gene zerknullt (zen) is repressed directly by Dorsal, and this repression is mediated by a 600-bp silencer, the ventral repression element (VRE), which contains four optimal Dorsal binding sites. The arrangement and sequence of the Dorsal recognition sequences in the VRE were used to develop a computational algorithm to search the Drosophila genome for clusters of optimal Dorsal binding sites. There are 15 regions in the genome that contain three or more optimal sites within a span of 400 bp or less. Three of these regions are associated with known Dorsal target genes: sog, zen, and Brinker. The Dorsal binding cluster in sog is shown to mediate lateral stripes of gene expression in response to low levels of the Dorsal gradient. Two of the remaining 12 clusters associated with genes that exhibit asymmetric patterns of expression across the dorsoventral axis. These results suggest that bioinformatics can be used to identify novel target genes and associated regulatory DNAs in a gene network (Markstein, 2002).

zen is an immediate target of the maternal Dl gradient. The gene is activated initially at nuclear cleavage cycle 11-12 within 1 h after the Dl gradient is formed. zen initially exhibits a broad pattern of expression in the presumptive dorsal ectoderm and at the termini. High and low levels of the Dl gradient keep zen off in ventral and lateral regions. sog exhibits a complementary pattern of expression because it is activated by Dl, whereas zen is repressed. As seen for zen, sog expression is detected shortly after the formation of the Dl gradient (Markstein, 2002).

The zen VRE contains four optimal Dl recognition sequences within a span of 400 bp. Three of the four Dl binding sites contained within the zen VRE conform to the following consensus sequence for high-affinity Dl binding sites: GGG(W)_nCCM (where W = A or T, M = C or A, and n corresponds to either four or five W residues). The fourth recognition sequence (binding site 3 within the VRE) contains a G residue in the AT-rich central region and is represented by the optimal consensus sequence GGGWDWWWCCM (where D = A, T, or G). To determine whether a similar density of optimal Dl sites might account for the regulation of sog, the entire Drosophila genome was scanned for clusters of any of the 208 unique Dl sequences that conform (either directly or by reverse complement) to two degenerate sequences: GGG(W)₄CCM and GGGWDWWWCCM (Markstein, 2002).

The genome was scanned for clusters of Dl binding sites in windows of 400 bp, the interval within which the sites are clustered in the zen VRE, and also for clustering in windows of 1,000 bp because the operational size of enhancers can generally be thought of as about 1,000 bp. Although the genome-wide occurrence of 676 clusters of two or more optimal Dl sites in 1,000 bp is not statistically significant, the occurrence of 55 clusters with at least three sites and of eight clusters containing four sites is enriched beyond what one would expect from random chance. However, none of the clusters within 1,000 bp identified known Dl targets that were missed by the more stringent screen for clustering within 400 bp. Therefore, this study focussed on the results from the more stringent screen (Markstein, 2002).

As expected, the occurrence of 400-bp windows containing at least two sites (327 clusters) is much greater than the occurrence of 400-bp windows containing at least three sites (15 clusters) or four sites (3 clusters). However, the statistical significance of the clusters increases with their rarity. For example, the occurrence of 15 clusters with three or more Dl sites is 6 standard deviations from expected, making the probability of finding 15 clusters by random chance less than one in a million. The probability of finding three 400-bp clusters with at least four Dl sites is less than 10^-49. Remarkably, two of the clusters in this rarest class are associated with the sog and zen genes, which exhibit the most sensitive response to the Dl gradient. Of the remaining 13 clusters containing three or more Dl sites, one is associated with the Brinker gene, which is expressed in lateral stripes and probably is a direct target of the Dl gradient. The Brinker site is located ~10 kb 5' of the transcription start site. Brinker probably is a direct target of the Dl gradient in that it exhibits lateral stripes of expression that are similar to those observed for rhomboid. The other remaining 12 clusters were found to neighbor genes that were not known previously to be involved in dorsoventral patterning (Markstein, 2002).

sog is expressed initially in broad lateral stripes that encompass the entire presumptive neurogenic ectoderm. Staining persists in these lateral stripes during cellularization and the onset of gastrulation but quickly refines within the mesectoderm at the ventral midline of elongating embryos. There are four optimal Dl binding sites located within a 263-bp region of sog intron 1. Three different DNA fragments that encompass this region of the sog gene were placed 5' of a lacZ reporter gene and expressed in transgenic embryos. The largest fragment is 6 kb in length and includes two-thirds of intron 1, whereas the smallest is just 393 bp and centered around the cluster of Dl binding sites. A 1.5-kb fragment that extends into the 5'-flanking region was also tested. All three fragments direct lateral stripes of lacZ expression that are similar to those seen for the endogenous gene. These broad stripes persist until gastrulation and then refine within the mesectoderm. Midline staining becomes weak and erratic in older embryos. The 393-bp sog fragment directs essentially the same staining pattern as those obtained with the 6-kb DNA fragment as well as the 1.5-kb fragment. These results suggest that the 393-bp fragment (hereafter called the sog lateral stripe enhancer) contains most of the cis elements responsible for regulating the early sog pattern (Markstein, 2002).

The sog lateral stripe enhancer shares a number of similarities with the previously characterized rhomboid NEE, which also mediates gene expression in the neurogenic ectoderm. However, the NEE stripes are narrower than those generated by the sog enhancer, suggesting that the sog enhancer responds to lower levels of the Dl gradient than does the NEE. This difference might be due, at least in part, to the quality or organization of the Dl binding sites in the two enhancers. For example, only two of the four Dl binding sites contained in the NEE are optimal sites, whereas all four sites are optimal in the sog enhancer. The NEE contains four binding sites for the zinc finger snail repressor, which is expressed selectively in the ventral mesoderm and thereby restricts rhomboid expression to lateral regions. The sog lateral stripe enhancer contains two potential snail repressor sites (CACCT) that might be responsible for attenuated staining in ventral regions (Markstein, 2002).

Design flexibility in cis-regulatory control of gene expression: synthetic and comparative evidence

In early Drosophila embryos, the transcription factor Dorsal regulates patterns of gene expression and cell fate specification along the dorsal-ventral axis. How gene expression is produced within the broad lateral domain of the presumptive neurogenic ectoderm is not understood. To investigate transcriptional control during neurogenic ectoderm specification, divergence and function were examined of an embryonic cis-regulatory element controlling the gene short gastrulation (sog). While transcription factor binding sites are not completely conserved, it has been demonstrated that these sequences are bona fide regulatory elements, despite variable regulatory architecture. Mutation of conserved sequences revealed that putative transcription factor binding sites for Dorsal and Vielfaltig, also known as Zelda, a ubiquitous maternal transcription factor, are required for proper sog expression. When Zelda and Dorsal sites are paired in a synthetic regulatory element, broad lateral expression results. However, synthetic regulatory elements that contain Dorsal and an additional activator also drive expression throughout the neurogenic ectoderm. These results suggest that interaction between Dorsal and Zelda drives expression within the presumptive neurogenic ectoderm, but they also demonstrate that regulatory architecture directing expression in this domain is flexible. A model for neurogenic ectoderm specification is proposed in which gene regulation occurs at the intersection of temporal and spatial transcription factor inputs (Liberman, 2009).

Through a comparative analysis of orthologous sog cis-regulatory modules from twelve Drosophilid species, core regulatory elements conserved in these sequences were identified. Considerable binding site turnover has occurred during the approximately 40 million years of evolution, yet some sequences are conserved. This observation supported the hypotheses that were investigated in this work which are, 1) that conserved sequences are functionally required and, 2) that variable architectures might generate the same or similar patterns of expression. Surprisingly, despite the opportunity for binding site turnover during the course of evolution, the sog regulatory regions from D. virilis can still be interpreted faithfully when used to drive reporter expression in D. melanogaster. It is concluded from these experiments, despite flexibility in the cis-regulatory element structure, regulatory logic has been conserved during evolution of the cis-regulatory module sequences to support sog expression (Liberman, 2009).

Though this comparative analysis identified limited sequence homology, what sequence conservation that was present facilitated efforts to examine the core regulatory elements required for patterning the neurogenic ectoderm. Using site-directed mutagenesis to eliminate sites within the sog cis-regulatory sequence, results were obtained that suggest that Dorsal functions together with the ubiquitous activator Zelda to control sog expression within the neurogenic ectoderm. Furthermore, synthetic cis-regulatory elements were constructed, consisting of Dorsal and Zelda or Dorsal and D-STAT sites, which are both able to support expression in the broad lateral domain of Drosophila early embryo. From these results it is concluded that broad lateral expression is achieved by a combination of Dorsal sites and sites for the ubiquitous activator Zelda, which suggests that a more general mechanism to create broad expression may involve interactions between Dorsal and other broadly expressed transcription factors (Liberman, 2009).

Mutagenesis and mutant analysis results demonstrate that Dorsal and Zelda support expression of sog along the dorsal-ventral axis. In the absence of Dorsal protein, expression of sog is gone; however when Dorsal binding sites were mutagenized, weak ventral-lateral reporter expression remains that could be due to unknown Dorsal binding sites that were not detected by PWM searches or due to input from another transcription factor. In the absence of Zelda binding sites or in Zelda mutants, expression is slightly broader than when Dorsal sites are eliminated. This residual expression could be due to Dorsal and/or another transcription factor (e.g. bHLH) functioning to direct expression, in a Zelda-independent manner, within the ventral-neurogenic ectoderm; however, the data suggests that Twist is not likely involved, as the domain of sog expression along the dorsal-ventral axis is not severely affected in twist mutants (Liberman, 2009).

Previous genetic studies have demonstrated that Dorsal is required for specification of the presumptive neurogenic ectoderm, but binding sites for Dorsal alone are not sufficient to generate expression within the broad lateral domain of embryos. Dorsal has been shown to function synergistically with Twist to pattern the presumptive mesoderm and ventral neurogenic ectoderm. This study presents evidence that Dorsal and Zelda function synergistically to regulate expression that is able to encompass the entire presumptive neurogenic ectoderm domain. Some method of cooperativity likely exists between Dorsal and Zelda, at the level of DNA binding or downstream, and is responsible for extending the expression domain into dorsal-lateral regions of the embryos, where the levels of nuclear Dorsal are low (Liberman, 2009).

It is proposed that Dorsal functions as a spatial regulator in the neurogenic ectoderm and that additional transcription factors like Zelda, act as co-activators to regulate the precise onset of expression. Furthermore, it is suggested that multiple ubiquitous or broadly expressed activators may function with Dorsal to support expression in a broad lateral domain (e.g. Zelda, STAT, and bHLH transcription factors such as Daughterless (Da). This study has demonstrated that STAT binding sites can also function together with Dorsal to drive expression in a broad lateral domain. Further support for this idea includes the observation that sog as well as ths exhibit broad expression early. Sites for Zelda are also present in the ths cis-regulatory module, and these sites likely direct the almost-ubiquitous early expression of ths observed. Interaction of Dorsal with distinct co-activators may not only regulate the spatial domain of expression supported, but also the temporal output. Zelda along with Dorsal or a Dorsal target initiates the earliest zygotic expression detected; perhaps interactions between Dorsal and other activators facilitate expression within a broad lateral domain (or other defined pattern) at later time-points. It is asserted that gene expression is achieved at the intersection of the Dorsal nuclear gradient and the additional activator which could either be ubiquitous in the case of Zelda or localized in the case of Twist (Liberman, 2009).

Even equipped with this new knowledge, other cis-regulatory modules that support co-expression of genes SoxN, pyramus and Neu3 have proven difficult to identify. To date, SoxN and pyramus regulatory elements remain unidentified. Flexible regulatory structures could account for some of the obscurity that has been encountered in the identification of cis-regulatory modules that support expression of genes within Drosophila early embryos. Flexibility in binding site composition, orientation and number of sites has also been demonstrated in the regulation of co-expressed genes in Ciona by extensive co-expression analyses. Possibly the observed flux in binding site composition and arrangement provides a mechanism that facilitates the introduction of mutations, which may be selected when a fitness advantage is provided to the developing embryo (Liberman, 2009).

Recently, a second regulatory element for sog located upstream of the gene was identified which also drives expression in a broad lateral stripe in the presumptive neurogenic ectoderm of cellularized embryos. This novel regulatory element as well as the known regulatory element, the intronic enhancer examined in this study, probably function together to control the full expression pattern of sog in the developing embryo. While both cis-regulatory sequences contain Dorsal and Zelda binding sites, the novel enhancer contains many more bHLH sites (L. Liberman, unpub. obs.), which is in stark contrast to the intronic sog regulatory element, which contains only one bHLH site and exhibits very little change of expression in twist mutant embryos. This new regulatory element presents further evidence that there exist multiple solutions for the developmental problem of producing spatially and temporally regulated expression. Future experiments will address whether these early embryonic enhancers controlling the expression of the sog gene within similar domains use the same mechanism (i.e. Dorsal + Zelda cooperativity) to support expression in a broad lateral stripe or whether different mechanisms are used (Liberman, 2009).

Evolutionary comparisons of sequences from diverged species can be very useful for the dissection of underlying cis-regulatory logic, as has been shown in this study; yet the important variable is that the proper comparisons of sequences must be made (i.e. species of appropriate evolutionary distance) and this is not always easy to define. In vertebrate systems, analyses of cis-regulatory modules usually focus on modules identified by methods that select for high degrees of conservation, which inherently have a low amount of flexibility. Arguments have been made that deciphering the underlying regulatory logic from evolutionary comparisons of sequences, when conservation is too high, is hard to interpret. However, it is contended that the relevant comparisons that provide insights into cis-regulatory logic are context-dependent. In analysis of the sog and Neu3 cis-regulatory modules, only limited sequence conservation was identified in comparisons of homologous sequences isolated from D. melanogaster and other Drosophilids. In the sog early embryonic regulatory element that was analyzed in this study, 71 (of 395) base-pairs of non-contiguous sequence exhibits conservation. The degree of conservation that was retained however was useful for dissecting the underlying regulatory logic (Liberman, 2009).

Identifying regulatory regions with flexible structure is more challenging than scanning for a stringent set of binding sites, but it may also reveal alternative mechanisms for specification that were not previously considered. It is predicted that studies that dissect the flexibility of cis-regulatory modules may one day provide insights to facilitate dissection of vertebrate regulatory elements in general, including ones that exhibit flexibility of sequence. It seems plausible that stringently conserved regulatory elements control gene expression of certain classes of genes, like those required for certain essential processes. Flexible regulatory architectures may provide a mechanism for generating variability throughout evolution. Ultimately it will prove useful to make evolutionary comparisons with both highly conserved sites and flexible architectures to determine how each contributes to establishment or maintenance of gene regulation (Liberman, 2009).

The Snail repressor inhibits release, not elongation, of paused Pol II in the Drosophila embryo

The development of the precellular Drosophila embryo is characterized by exceptionally rapid transitions in gene activity, with broadly distributed maternal regulatory gradients giving way to precise on/off patterns of gene expression within a one-hour window, between two and three hours after fertilization. Transcriptional repression plays a pivotal role in this process, delineating sharp expression patterns (e.g., pair-rule stripes) within broad domains of gene activation. As many as 20 different sequence-specific repressors have been implicated in this process, yet the mechanisms by which they silence gene expression have remained elusive. This study reports the development of a method for the quantitative visualization of transcriptional repression. The focus of this study was the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm. Elongating Pol II complexes were found to complete transcription after the onset of Snail repression. As a result, moderately sized genes (e.g., the 22 kb sog locus) are fully silenced only after tens of minutes of repression. It is proposed that this 'repression lag' imposes a severe constraint on the regulatory dynamics of embryonic patterning and further suggest that posttranscriptional regulators, like microRNAs, are required to inhibit unwanted transcripts produced during protracted periods of gene silencing (Bothma, 2011).

Snail typically binds to repressor sites located near upstream activation elements within distal enhancers. Repression might result from the passive inhibition of upstream activators, such as the failure of the activators to mediate looping to the core promoter. Alternatively, Snail might alter the chromatin state of the promoter region, resulting in diminished access of the Pol II transcription complex. Such repression mechanisms might cause a lag in gene silencing due to the continued elongation of Pol II complexes that were released from the promoter prior to the onset of repression. As in the case of the delay in the production of mature mRNAs after initiation, the lag in repression would be commensurate with the size of the gene, with large genes taking longer to silence than small genes. This can take a significant amount of time due to the surprisingly slow rate of Pol II elongation, only ∼1 kb/min (Ardehali, 2009; Bothma, 2011 and references therein).

Alternatively, elongating Pol II complexes might be arrested or released from the DNA template due to changes in chromatin structure and/or attenuation of Pol II processivity. Such mechanisms could lead to the immediate silencing of all genes regardless of size. Recent studies have documented rapid changes in the chromatin structure across the entire length of genes, exceeding the rate of Pol II processivity (Petesch, 2008). Certain corepressors in the Drosophila embryo (e.g., Groucho) are thought to mediate repression by a 'spreading' mechanism that modifies chromatin over extensive regions. Indeed, this type of mechanism has been invoked to account for the repression of the pair-rule gene even-skipped (eve) by the gap repressor Knirps. The attenuation of Pol II elongation has been implicated in a variety of processes. For example, Pol II attenuation has been documented for the transcriptional repression of MYC. Moreover, the activation of the HIV genome is regulated by Pol II processivity. In an effort to distinguish these potential mechanisms, the repression dynamics of several Snail target genes were visualized, because they are silenced in the presumptive mesoderm of precellular embryos (Bothma, 2011).

short gastrulation (sog) encodes an inhibitor of BMP/Dpp signaling that restricts peak Dpp signaling to the dorsal midline of cellularizing embryos. The sog locus is ∼22 kb in length and contains three large introns, including a 5′ intron that is ∼10 kb in length and a 3′ intron that is ∼5 kb in length. The use of separate intronic hybridization probes permits independent detection of 5′ and 3′ sequences within nascent sog transcripts. Individual nuclei are then false colored according to the probe combination they contain (Bothma, 2011).

sog exhibits synchronous activation at the onset of cell cycle 13 (cc13), ∼2 hr after fertilization. There is a lag between the time when nascent transcripts are first detected with the 5′ probe and subsequently cross-hybridize with both the 5′ and 3′ intronic probes. This lag is consistent with the established rates of Pol II elongation in flies, ∼1.1–1.5 kb/min. cc13 persists for ∼20 min, and by the completion of this time window, most of the nuclei in ventral and lateral regions exhibit yellow staining, indicating the presence of multiple nascent transcripts containing 5′ and 3′ intronic sequences within each nucleus. There is little or no repression in ventral regions, presumably due to insufficient levels of the Snail repressor prior to cc14 (Bothma, 2011).

As shown previously, nascent transcripts are aborted during mitosis. Consequently, only the 5′ hybridization probe detects nascent sog transcripts at the onset of cc14. Moreover, a small number of nuclei (at the ventral midline) fail to exhibit nascent transcripts with either the 5′ or 3′ probe, suggesting repression by Snail. This repression becomes progressively more pronounced during cc14 (Bothma, 2011).

Within about 10 min of the first detection of nascent sog transcripts at the onset of cc14, most of the nuclei exhibiting sog expression stain yellow, indicating expression of both 5′ (green) and 3′ (red) intronic sequences. During the next several minutes, progressively more nuclei exhibit only 3′ (red) hybridization signals in ventral regions. This transition from yellow to red continues and culminates in a 'red flash' where the majority of the ventral nuclei that contain nascent transcripts express only the 3′ (red) probe. As cc14 continues, there is a progressive loss of staining in the presumptive mesoderm, and eventually, nascent sog transcripts are lost entirely in the presumptive mesoderm (Bothma, 2011).

These results suggest that after its release from the promoter, Pol II continues to elongate along the length of the sog transcription unit, even as Snail actively represses its expression in the mesoderm. The red flash observed during mid-cc14 represents partially processed nascent sog transcripts that have lost the 5′ intron (hence no green signals with the 5′ hybridization probe) but retain 3′ sequences. Previous studies are consistent with sequential processing of nascent transcripts, beginning with the removal of 5′ intronic sequences and concluding with the removal of 3′ introns. As a control, two separate hybridization probes were used to label opposite ends of sog intron 1. As expected, there was no red flash, because both hybridization signals were simultaneously lost when intron 1 was spliced (Bothma, 2011).

There is an ∼20 min lag between the onset of repression at early cc14 and the complete silencing of sog expression in the presumptive mesoderm during mid- to late cc14. To determine whether this repression lag is a common feature of Snail-mediated gene silencing, additional target genes, including ASPP, Delta, canoe, and scabrous (sca) were examined. ASPP encodes a putative inhibitor of apoptosis, whereas Delta encodes the canonical ligand that induces Notch signaling. All four of these genes exhibit repression lag as they are silenced in the presumptive mesoderm of cc14 embryos (Bothma, 2011).

With the notable exception of Delta, the genes examined in this study contain promoter-proximal paused Pol II, as do most developmental patterning genes active in the precellular embryo. Moreover, results from whole-genome Pol II binding assays indicate that these genes maintain promoter-proximal paused Pol II in the presumptive mesoderm as they are actively repressed by Snail. These findings are consistent with the observation that the segmentation gene sloppy paired 1 retains promoter-proximal paused Pol II even after being silenced by the ectopic expression of Runt and Ftz. Thus, the Snail repressor does not appear to affect Pol II recruitment but rather inhibits the release of Pol II from the promoter-proximal regions of paused genes. At every round of de novo transcription, each Pol II complex at the pause site must receive an activation signal for its release into the transcription unit. It is proposed that the Snail repressor interferes with this signal, resulting in the retention of Pol II at the pause site (Bothma, 2011).

It is currently unclear whether repression lag is a general feature of transcriptional silencing. A recent study suggests that the gap repressor Knirps reduces the processivity of Pol II complexes across the eve transcription unit (Li, 2011). Snail and Knirps might employ distinctive modes of transcriptional repression. Snail recruits the short-range corepressor CtBP, whereas Knirps recruits either CtBP or the long-range corepressor Groucho. When bound to certain cis-regulatory elements within the eve locus, Knirps recruits Groucho, which might propagate a repressive chromatin structure. In contrast, Snail-CtBP might interfere with the release of Pol II from the proximal promoter, as discussed above. There is a considerable difference in the lengths of the genes examined in the two studies. The eve transcription unit is only 1.5 kb in length, less than one-tenth the size of sog. In fact, many patterning genes active in the early fly embryo contain small transcription units only a few kilobases in length. Small transcription units offer dual advantages in rapid patterning processes: essentially no lag in activation or repression (Bothma, 2011).

All five Snail target genes examined in this study exhibit Pol II elongation after the onset of repression. The number of transcripts produced during repression lag depends on the Pol II density across the transcription unit at the onset of repression. Whole-genome Pol II binding assays suggest that there are at least several Pol II complexes per kilobase. This estimate is based on comparing the total amount of Pol II within these genes to that present at the promoter of the uninduced hsp70 gene, for which there are accurate measurements. As a point of reference, the Pol II density on induced heat-shock genes is one complex per 75-100 bp, which is comparable to the footprint size, ∼50 bp, of an elongating Pol II complex. Thus, somewhere in the vicinity of ∼50 (or more) sog transcripts may be produced in a diploid cell after the onset of Snail repression. This represents a significant fraction of the steady-state expression of a typical patterning gene (∼200 transcripts per cell (Bothma, 2011).

Repression lag could impinge on a number of patterning processes, such as Notch signaling. The specification of the ventral midline of the central nervous system depends on the activation of Notch signaling in the ventralmost regions of the neurogenic ectoderm. Sca products somehow facilitate the activation of the Notch receptor, and repression lag could potentially disrupt this process by producing high steady-state levels of Sca in the mesoderm where Notch is normally inactive. Similar arguments might apply to the unwanted accumulation of Delta products in the mesoderm. Perhaps microRNAs are required to inhibit these transcripts and thereby facilitate localized activation of Notch signaling. Indeed, miR-1 is expressed in the presumptive mesoderm, at the right time and place to regulate Sca and/or Delta, and is known to be able to target Delta transcripts. Repression lag is potentially quite severe for Hox genes, particularly Antp and Ubx, which contain large transcription units (75–100 kb) that could take over an hour to silence after the onset of repression. It is conceivable that miRNAs encoded by the miR-iab4 gene, which are known to target Antp and Ubx transcripts, might inhibit postrepression transcripts (Bothma, 2011).

The precellular Drosophila embryo possesses a number of inherently elegant features for the detailed visualization of differential gene activity in development. Indeed, such studies were among the first to highlight the importance of transcriptional repression in the delineation of precise on/off patterns of gene expression. This study extends this rich tradition of visualization by providing the first dynamic view of gene silencing. The key feature of this method is the use of sequential 5′ and 3′ intronic probes to distinguish nascent transcripts produced by Pol II complexes shortly after their release from the promoter versus mature Pol II elongation complexes that have already transcribed 5′ intronic sequences. Elongating Pol II complexes have been shown to complete transcription after the onset of Snail repression and, as a result, moderately sized genes are fully silenced only after a significant lag. It is suggested that this repression lag represents a previously unrecognized constraint on the regulatory dynamics of the precellular embryo (Bothma, 2011).

Zelda potentiates morphogen activity by increasing chromatin accessibility

Zygotic genome activation (ZGA) is a major genome programming event whereby the cells of the embryo begin to adopt specified fates. Experiments in Drosophila and zebrafish have revealed that ZGA depends on transcription factors that provide large-scale control of gene expression by direct and specific binding to gene regulatory sequences. Zelda (Zld) plays such a role in the Drosophila embryo, where it has been shown to control the action of patterning signals; however, the mechanisms underlying this effect remain largely unclear. A recent model proposed that Zld binding sites act as quantitative regulators of the spatiotemporal expression of genes activated by Dorsal (Dl), the morphogen that patterns the dorsoventral axis. This study tested this model experimentally, using enhancers of brinker (brk) and short gastrulation (sog), both of which are directly activated by Dl, but at different concentration thresholds. In agreement with the model, it was shown that there is a clear positive correlation between the number of Zld binding sites and the spatial domain of enhancer activity. Likewise, the timing of expression could be advanced or delayed. Evidence is presented that Zld facilitates binding of Dl to regulatory DNA, and that this is associated with increased chromatin accessibility. Importantly, the change in chromatin accessibility is strongly correlated with the change in Zld binding, but not Dl. It is proposed that the ability of genome activators to facilitate readout of transcriptional input is key to widespread transcriptional induction during ZGA (Foo, 2014).

In blastoderm embryos, brinker (brk) is activated in an eight- to ten-cell-wide domain that develops into the ventral neurogenic ectoderm (NE), whereas short gastrulation (sog) is expressed in a broader band of 16-18 cells encompassing the entire NE. Both genes have the same ventral expression boundary due to repression by Snail (Sna) in the presumptive mesoderm. The dorsal borders of their domains lie in regions of the Dorsal (Dl) gradient where amounts are low and change little, raising the question of how their enhancers can interpret small differences in Dl concentrations (Foo, 2014).

sog and brk each have two reported cis-regulatory modules (enhancers) that are active in early embryos. The sog intronic lateral stripe enhancer (LSE) is less well conserved and drives a slightly narrower stripe of expression relative to the sog shadow enhancer, also known as the neurogenic ectoderm enhancer (NEE), which recapitulates the broad endogenous sog pattern. The brk 5′ and 3′ enhancers both support lateral stripes similar to endogenous brk; however, the brk 3′ enhancer drives a more dynamic pattern that broadens at cellularization. Thus, this study focused on the brk 5′ enhancer to avoid confounding dynamic change of width (Foo, 2014).

The sog 426 bp NEE contains three CAGGTAG heptamer sites for optimal Zelda (Zld) binding. However, the brk 498 bp 5′ enhancer does not have any canonical Zld binding sites (also known as TAGteam sites). To explain its Zld dependence, electrophoretic mobility shift assays were used to look for Zld binding sites in the brk 5′ enhancer. Three CAGGTCA sequences and a tandem GAGGCACAGGCAC sequence were identified that promote very weak Zld binding, which was abolished upon mutation of the sites (Foo, 2014).

To test whether altering the number of Zld binding sites in the NE enhancers can affect the expression they drive, mutant forms of the brk and sog enhancers were created. The sog NEE drives a lacZ reporter expression pattern identical to endogenous sog. Mutation of all three CAGGTAG sites dramatically reduced the expression width (sog). Similar changes were also observed by a previous study when the CAGGTAG sites were mutated in the sog LSE. Costaining of lacZ and endogenous sog illustrates that the narrowed lacZ domain resulted from a collapse of the dorsal, not the ventral, border. It is inferred that without Zld, sog is unable to be activated by the lower levels of Dl in the dorsal neuroectoderm region. In embryos lacking maternal Zld (referred to as zld^-), both the endogenous sog and sog wt domains shrink and become sporadic. This is not due to an indirect effect on the Dl concentration gradient because it is unchanged in zld^-. Thus, loss of Zld in trans, or Zld binding sites in cis, has the same effect on NEE activity, indicating a direct modulation of sog by Zld (Foo, 2014).

Next the opposite experiment was performed by introducing three CAGGTAG sites into the brk 5′ enhancer. This modified enhancer (brk^+3a) drives a considerably expanded expression domain compared to brk^wt. A second form of the brk enhancer with CAGGTAG sites added to different locations (brk^+3b) also drives the same expanded expression domain, arguing against the requirement of precise motif grammar in Zld’s regulation of NE genes (Foo, 2014).

To rule out the possibility that the expansion in domain width of brk^+3a is caused by inadvertent disruption of a repressor binding site rather than addition of Zld binding sites, the three added CAGGTAG sequences were mutated in brk^+3a into 7-mers that are neither the original sequence nor Zld binding sites. Mutation of these sites reduced the expanded domain of brk^+3a back to a width similar to brk wt. When each of the brk^+3a , brk^+3b , and brk^+3m transgenic enhancers was placed into a zld^- background, narrow and sporadic expression resulted resembling that of endogenous brk in zld^-, again supporting that the CAGGTAG-driven broadened expression is Zld dependent. Moreover, mutation of the newly found weak Zld binding sites led to a narrowed and weakened stripe of expression, identical to the pattern of brk wt in zld^- (Foo, 2014).

To better correlate the number of Zld sites with the extent of reporter expression, six different forms of the sog NEE were constructed containing either one or two of the three CAGGTAG sites). The width of expression correlated moderately to the number of Zld sites in the enhancer. However, some sites appear to be more important than others in contributing to the expression width, indicating a context dependency for Zld binding sites. From these results and others’ work demonstrating weakened NE gene expression upon removal of Zld or Zld sites, it is evident that Zld is indispensable for the proper expression of NE genes (Foo, 2014).

It was next asked whether the number of Zld binding sites also influences the timing of Dl target expression, since previous reports have implicated Zld as a developmental timer. A correlation has been observed between the onset of zygotic gene expression and strength of Zld binding at nuclear cycle 8. Besides that, when the enhancer region of zen, which contains four Zld binding sites, was multimerized, it drove precocious activation of reporter expression. And finally, it has been shown that the expression of many patterning genes is delayed in zld^- embryos, including sog and brk. It was reasoned that since Dl nuclear concentrations increase from nuclear cycles 10 to 14, the lower levels of Dl present in earlier cycles would no longer be adequate to activate target genes without Zld’s input, resulting in delayed activation of sog and brk (Foo, 2014).

To measure the onset of transcription, it was determined when the four transgenic enhancers (sog^wt, sog⁰, brk^wt, and brk^+3a) could activate an intron-containing yellow reporter gene, which allows detection of nascent transcripts. Reporter expression driven by the sog wt enhancer was first detectable in nuclear cycle 10 embryos, whereas no reporter activity was observed for the sog⁰ enhancer until nuclear cycle 11. Even in nuclear cycle 12, the expression driven by sog⁰ is more sporadic compared to sog wt. Unlike in nuclear cycle 14 embryos, reporter expression can be seen in ventral nuclei of nuclear cycle 11 and nuclear cycle 12 embryos because the Sna repressor has not yet accumulated to high levels. Adding three Zld sites to the brk enhancer resulted in advanced initiation of reporter activity from nuclear cycle 11 to nuclear cycle 10, and reporter expression also became more robust, in terms of both the proportion of nuclei showing expression and the ratio of embryos with expressing nuclei. These results clearly illustrate that by manipulating Zld binding sites, the timing of NE gene activation can be altered. Temporal regulation by transcription factor binding sites has also been shown in Ciona where the number of Brachyury binding sites governs the timing of notochord gene expression (Foo, 2014).

It is believed that Zld regulates the temporal and spatial expression of NE genes by promoting Dl activity, rather than acting independently, because nuclear Dl is absolutely required for the activation of brk and sog, which exhibit no expression in genetic backgrounds lacking nuclear Dl. One possible mechanism may involve cooperativity at the level of DNA binding. To test the hypothesis that the extent of Zld binding impacts Dl binding at target enhancers, chromatin immunoprecipitation was performed followed by quantitative PCR (ChIP-qPCR) to measure Zld and Dl binding to the different transgenic enhancers (Foo, 2014).

The sog⁰ enhancer without Zld sites has diminished Zld binding when compared to sog wt. Dl binding is also much reduced. As an internal control, Zld and Dl binding to the endogenous sog locus showed no significant difference between the lines. On the other hand, introduction of Zld sites into the brk transgenic enhancer led to higher Zld binding and Dl binding, while Zld and Dl binding to the endogenous locus remained similar between lines. These results illustrate that changing the number of Zld sites, and therefore changing the amount of Zld binding to the NE enhancers, influences the level of Dl binding to its target sites in vivo (Foo, 2014).

The results from reporter expression analyses and ChIP experiments suggest that Zld promotes transcriptional output by facilitating Dl DNA binding. Zld might directly interact with Dl, leading to cooperative DNA binding as in the Dl-Twist (Twi) interaction. Alternatively, Zld might assist factor binding by interacting with common coactivators or by changing the local chromatin accessibility. The latter possibility is favored for several reasons: (1) Zld binding greatly overlaps with that of many other transcription factors such as Bcd, Hunchback, Dl, Twi, Sna, and Mothers against Dpp (Mad); (2) Zld helps the binding of Twi and Bcd to target DNA; (3) the presence of Zld binding sites is associated with high levels of transcription factor binding; and (4) the Zld site (CAGGTA; [2]) is the most enriched motif in transcription factor binding 'HOT regions,' which were seen to correlate with decreased nucleosome density. Hence, it is more likely that Zld plays a more general role, such as 'opening' the underlying chromatin, than that it interacts specifically with multiple other factors (Foo, 2014).

The hypothesis was addressed that Zld facilitates the binding of Dl by making the local chromatin more accessible. DNase I’s preferential digestion of nucleosome-depleted DNA in the genome can be used to map active regulatory regions accessible for transcription factor binding. DNase I hypersensitivity assays followed by qPCR (DNase I-qPCR) were performed to measure the chromatin 'openness' of transgenic enhancers carrying varying numbers of Zld sites. The sog transgenic enhancer region had significant reduction of chromatin accessibility when Zld sites were mutated, while adding Zld sites to the brk transgenic enhancer increased sensitivity to DNase I digestion. The DNase I hypersensitivity assessed on endogenous brk and sog loci were comparable between transgenic lines, serving as a control for embryo staging between transgenic lines and the DNase I digestion procedure (Foo, 2014).

These results suggest that the presence of Zld sites, and thus Zld binding, makes the local chromatin more accessible for Dl, and potentially other transcription factors. However, it is feasible that the total number of factor binding sites influences chromatin accessibility rather than the number of Zld sites in particular. Therefore, the DNase I hypersensitivity of a transgenic brk enhancer was assayed that lacks all Dl binding sites and shows no reporter expression. Dl binding decreased nearly to background levels compared to brk wt, but the Zld binding and DNase I hypersensitivity showed only slight decreases, which is not comparable to the effects seen upon manipulation of Zld sites on the brk and sog enhancers. It was reasoned that the binding of each transcription factor may contribute to the DNase I hypersensitivity to a certain extent but that the major influence comes from Zld binding. To further evaluate the contribution of Zld versus Dl sites to chromatin accessibility, the fold change in Zld and Dl binding for sog⁰, brk^+3a , and brk^0Dl was calculated relative to their corresponding wt transgenic enhancers and then correlated the fold change in factor binding with the change in DNase I hypersensitivity. A strong correlation was found between the change in Zld binding and DNase I hypersensitivity, whereas the change in Dl binding and DNase I hypersensitivity do not correlate. These results support the idea that the number of Zld sites rather than Dl sites is important in determining chromatin accessibility (Foo, 2014).

Using Zld's coregulation of NE genes as a case in point, this study has shed light on how Zld functions as a zygotic genome activator. The data reveal that Zld works in combination with Dl and regulates Dl target genes by binding differentially to their regulatory sequences. Changing the number of Zld sites on Dl target gene enhancers has a pronounced effect on their expression both temporally and spatially. As a uniformly distributed factor, Zld supplies positional information by promoting Dl binding to target enhancers, thereby increasing the 'apparent dosage' of Dl. Zld's input is especially important where the level of morphogen is low and likely plays a similar role for other key factors in the blastoderm embryo, such as Twi, Bcd, and Mad. Uniform factors have been found to act in combination with Sonic Hedgehog in neural tube differentiation, and the current findings on how Zld potentiates morphogen activity will be relevant to vertebrate systems (Foo, 2014).

Although the results do not rule out other possible mechanisms, they strongly support the idea that Zld binding increases chromatin accessibility, which is thought to contribute greatly to how it activates such a wide range of targets. In this model, the amount of Zld binding on a region would determine how open and therefore how active it is. At the center of this property is Zld’s ability to occupy a large fraction of its recognition sites in early embryos. Besides that, Zld is present in nuclei as early as nuclear cycle 2, which is considerably earlier than other factors. Therefore, Zld may act as a pioneer factor as previously suggested, but whether Zld binds to its sites in nucleosomes and repositions them, or whether it recruits histone modifiers that in turn affect binding of other factors like Dl, awaits further investigation. Interestingly, this idea may extend beyond flies, since newly discovered genome activators in zebrafish zygotic genome activation have been seen to cooperate with developmental regulators and prime the genome for subsequent activation. Thus, it seems that developmental control of zygotic genome activation is highly similar in flies and fish (Foo, 2014).

Transcriptional Regulation

Decapentaplegic, in addition to its role as a morphogen in structuring gene expression and positioning of veins in the larval wing disc, is expressed in vein primordia during pupal wing development and functions to promote vein formation. In contrast, sog is expressed in complementary intervein cells and suppresses vein formation. sog and dpp function during the same phenocritical periods (i.e. 16-28 hours after pupariation) to influence the vein versus intervein cell fate choice. The conflicting activities of dpp and sog are also revealed by antagonistic dosage-sensitive interactions between these two genes during vein development. Analysis of vein and intervein marker expression in dpp and sog mutant wings suggests that dpp promotes vein fates indirectly by activating the vein gene rhomboid (rho), and that sog functions by blocking an autoactivating DPP feedback loop. Ectopic expression of dpp activates rho and suppresses sog expression. It is thought that the dpp suppression of sog is indirect, acting through rhomboid. A network of gene interactions promote vein fates as EGF-R ligands Vein and Spitz are also involved in intervein and vein fates respectively. These data support the view that SOG is a dedicated DPP antagonist (Yu, 1997).

Differential activation of the Toll receptor leads to the formation of a broad Dorsal nuclear gradient that specifies at least three patterning thresholds of gene activity along the dorsoventral axis of precellular embryos. The activities of the Pelle kinase and Twist basic helix-loop-helix (bHLH) transcription factor in transducing Toll signaling have been investigated. Pelle functions downstream of Toll to release Dorsal from the Cactus inhibitor. Twist is an immediate-early gene that is activated upon entry of Dorsal into nuclei. Transgenes misexpressing Pelle and Twist were introduced into different mutant backgrounds and the patterning activities were visualized using various target genes that respond to different thresholds of Toll-Dorsal signaling. These studies suggest that an anteroposterior gradient of Pelle kinase activity is sufficient to generate all known Toll-Dorsal patterning thresholds and that Twist can function as a gradient morphogen to establish at least two distinct dorsoventral patterning thresholds. How the Dorsal gradient system can be modified during metazoan evolution is discussed and it is concluded that Dorsal-Twist interactions are distinct from the interplay between Bicoid and Hunchback, which pattern the anteroposterior axis (Stathopoulos, 2002).

The snail, sim, vnd and sog expression patterns represent four different Toll-Dorsal signaling thresholds. snail is activated only by peak levels of the Dorsal gradient; sim and vnd are activated by intermediate levels, and sog is activated by the lowest levels of the gradient. These expression patterns were visualized in mutant and transgenic embryos via in situ hybridization using digoxigenin-labeled antisense RNA probes (Stathopoulos, 2002).

Dorsal target genes are essentially silent in mutant embryos that lack an endogenous dorsoventral Dorsal nuclear gradient. Mutant embryos were collected from females that are homozygous for a null mutation in the gastrulation defective (gd) gene, which blocks the processing of the Spätzle ligand and the activation of the Toll receptor. These mutants permit the analysis of ectopic, anteroposterior Dorsal and Twist gradients in 'apolar' embryos that lack dorsoventral polarity. snail, vnd, and sog are sequentially expressed along the anteroposterior axis of mutant embryos that contain a constitutively activated form of the Toll receptor (Toll^10b) misexpressed at the anterior pole using the bicoid (bcd) promoter and 3' UTR. These expression patterns depend on an ectopic anteroposterior Dorsal nuclear gradient. The repression of the vnd and sog patterns at the anterior pole is probably mediated by Snail, which normally excludes expression of these genes in the ventral mesoderm of wild-type embryos (Stathopoulos, 2002).

The activated Pelle-Tor⁴⁰²¹ kinase also directs sequential anteroposterior patterns of snail, vnd, and sog expression in gd^–/gd^– mutant embryos. As in the case of Toll^10b, the activated Pelle kinase was misexpressed at the pole using the bcd 3' UTR. The snail, vnd and sog expression patterns are similar to those obtained with the Toll^10b transgene. The vnd and sog expression patterns are probably repressed at the anterior pole by Snail. These results suggest that the levels of Pelle kinase activity are sufficient to determine different Dorsal transcription thresholds (Stathopoulos, 2002).

sog is normally activated throughout the neurogenic ectoderm by the lowest levels of the Dorsal gradient. The low levels of Dorsal present in Toll^rm9/Toll^rm10 mutant embryos are sufficient to activate sog everywhere except the extreme termini. The twist-bcd transgene leads to the loss of sog expression in anterior regions, probably because of repression by Snail. Snail also appears to repress vnd and sog expression in anterior regions of transgenic embryos that contain the Toll^10b or Pelle-Tor⁴⁰²¹ transgenes (Stathopoulos, 2002).

The twist-bcd transgene was introduced into mutant embryos that completely lack Dorsal. Without the transgene these mutants do not express twist, snail, sim, vnd or sog. Introduction of the twist-bcd transgene causes intense expression of twist in the anterior 40% of the embryo. This broad Twist gradient fails to activate snail, but succeeds in inducing weak expression of sim and somewhat stronger staining of vnd at the anterior pole. The activation of vnd in mutant embryos is comparable with the expression seen in wild-type and Toll^rm9/Toll^rm10 embryos. However, in both wild-type and mutant embryos the vnd pattern is transient, and lost after the completion of cellularization. These results indicate that Twist can activate dorsoventral patterning genes in the absence of Dorsal (Stathopoulos, 2002).

Mitosis-associated repression in development

Transcriptional repression is a pervasive feature of animal development. This study employed live-imaging methods to visualize the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm of early Drosophila embryos. Snail target enhancers were attached to an MS2 reporter gene, permitting detection of nascent transcripts in living embryos. The transgenes exhibit initially broad patterns of transcription but are refined by repression in the mesoderm following mitosis. These observations reveal a correlation between mitotic silencing and Snail repression. The study proposes that mitosis and other inherent discontinuities in transcription boost the activities of sequence-specific repressors, such as Snail (Esposito, 2016).

The enhancers tested in this study were derived from two different dorsal-ventral patterning genes: brk and sog; both encode inhibitors of BMP signaling. The brk enhancer is located ~10 kb upstream of the transcription start site, while the sog enhancer is located within the first intron of the transcription unit, ~1.5 kb downstream from the start site. Each enhancer was placed immediately upstream of its cognate promoter and attached to a yellow reporter gene containing 24 MS2 stem-loops within the 5' untranslated region (UTR). Nascent transcripts were visualized in living embryos using a maternally expressed MCP-GFP fusion protein (Esposito, 2016).

Both transgenes recapitulate the expression profiles of the endogenous genes; namely, they are activated throughout the presumptive mesoderm and neurogenic ectoderm and then repressed in the mesoderm. Prior studies with fixed embryos suggest that these enhancers respond to different levels of the Snail repressor (e.g., see Bothma et al. 2011). The brk 5' enhancer appears to be more efficiently repressed by Snail as compared with the sog intronic enhancer. Both transgenes were examined in living embryos to determine whether they exhibit distinctive repression dynamics (Esposito, 2016).

The btk>MS2 transgene exhibits an expression profile that is similar to that seen for the endogenous locus based on conventional in situ hybridization methods. The main difference is that the transgene produces a slightly narrower pattern due to the absence of the 3' 'shadow' enhancer (Esposito, 2016).

There is broad activation of the btk>MS2 transgene in both ventral and lateral regions during nuclear cleavage cycles 10-13 (nc10-nc13). btk>MS2 nascent transcripts are lost during the general silencing of transcription at each mitosis. Interestingly, upon reactivation of the transgene at the onset of nc14, a sudden loss of de novo transcription was observed in the mesoderm. Transcripts are restricted to the neurogenic ectoderm, suggesting that the mature brk expression pattern is established immediately following mitosis (Esposito, 2016).

In an effort to quantify the dynamics of this repression, individual nuclei were partitioned within the presumptive mesoderm and ectoderm and the fraction of active nuclei was calculated in these regions throughout nc13-nc14. Active nuclei are defined as those exhibiting nascent RNA signals in at least one z-series (20 sec). No significant variation was observed in the fraction of active nuclei between the mesoderm and lateral ectoderm during nc13. However, at the onset of nc14, ~90% of the nuclei in the mesoderm are silenced, while expression persists in the lateral ectoderm (Esposito, 2016).

To obtain more detailed information on the dynamics of repression in the ventral mesoderm, the fluorescence of individual transcription foci was quantified, since previous studies have shown that it scales with the number of RNA polymerase II (Pol II) complexes engaged in active transcription. Quantitative analysis of the btk>MS2 transgene reveals that the ventral-most nuclei in the mesoderm exhibit ~25% reduction in signal intensity during nc13 as compared with active nuclei in the lateral ectoderm. The majority of nuclei that display nascent transcripts at the onset of nc14 is located in the ectoderm, and the few active nuclei in the mesoderm show a substantial reduction in signal intensity (~60% reduction). Thus, Snail begins to attenuate the brk 5' enhancer during nc13, and this repression appears to be strongly reinforced during mitosis. It is suggested that mitotic silencing augments the activity of the localized Snail repressor (Esposito, 2016).

sog is regulated by two enhancers with overlapping activities: a distal enhancer located ~20 kb upstream of the sog promoter and an intronic enhancer located ~1.5 kb downstream from the transcription start site. The distal 5' enhancer contains high-affinity Snail-binding sites and exhibits repression dynamics similar to that of the btk>MS2 transgene following mitosis. The intronic enhancer contains weak Snail-binding sites and shows modest repression in the ventral mesoderm of wild-type embryos. Most nuclei are reactivated following mitotic silencing but display a significant reduction (more than twofold) in expression (Esposito, 2016).

To explore the dynamics of sog repression, embryos carrying three copies of the snail locus were examined. They exhibit significantly more complete repression of the sog>MS2 transgene during the onset of nc14 as compared with wild-type embryos. Fewer than half the nuclei in the mesoderm reactivate sog>MS2 expression at the onset of nc14 (Esposito, 2016).

The preceding results suggest a clear correlation between mitotic silencing and repression of the btk>MS2 transgene and attenuation of sog>MS2. There is an approximately twofold reduction in the levels of sog>MS2 expression in the mesoderm following mitotic silencing in wild-type embryos and a substantial reduction in the number of nuclei that reactivate the transgene at the onset of nc14 in embryos containing three copies of snail. Nonetheless, it is not clear whether the ~8-min interval during mitosis is a more effective period of Snail-mediated repression than a comparable interphase period (Esposito, 2016).

Additional support stems from the analysis of a rare haploid embryo. Following fertilization, the paternal and maternal haploid pronuclei sometimes fail to fuse, and development proceeds with successive divisions of the maternal pronucleus. Due to the reduced amount of DNA, these haploid nuclei undergo an additional, 14th mitotic division. As expected, the resulting nc15 nuclei are half the volume of normal nc14 diploid nuclei due to the extra division cycle. Strikingly, the sog>MS2 transgene exhibits a dramatic loss of expression in mesoderm nuclei at the onset of nc15. There is only a modest loss in the number of nuclei that exhibit repression in the mesoderm during nc14 (~10%). This is followed by an ~10-fold reduction in the number of nuclei that reactivate the transgene at the onset of nc15. This loss in expression following mitosis is similar to that seen for the btk>MS2 transgene in wild-type embryos. Unfortunately, given the scarcity of spontaneous haploid embryos, it was not possible to perform these measurements on only one embryo (Esposito, 2016).

These observations reinforce the correlation between mitosis and repression. Additional evidence was obtained by extending the normal period of mitosis by lowering the temperature of developing embryos. A temperature-controlled microfluidic chamber was used to produce a transient reduction in temperature-from 22°C to 17°C during the 13th mitosis while maintaining the temperature at 22°C during interphases 13 and 14. This treatment diminishes the rate of embryonic development and extends the time of mitosis from ~8 to ~15 min. There is a more pervasive loss of expression in the mesoderm upon reactivation of the sog>MS2 transgene at the onset of nc14. Less than half the mesoderm nuclei exhibit expression of the sog>MS2 transgene upon reactivation at the onset of nc14 (Esposito, 2016).

It is not believed that the correlation between mitosis and repression is a peculiarity of the Snail repressor. Mitotic silencing might influence gap repressors such as Kruppel, since there is a substantial refinement in the eve stripe-2 expression pattern at the onset of nc14 (Esposito, 2016).

The preceding analyses employed heterozygous embryos carrying a single copy of the brk or sog transgenes. Next homozygous embryos were examined to determine whether the two alleles of a locus display coordinated or uncoupled patterns of repression. The btk>MS2 transgene exhibits a sharp transition from active to inactive mesoderm nuclei following mitosis. This study therefore focused analysis on the regulation of sog>MS2 homozygotes (Esposito, 2016).

There is no detectable repression of either sog>MS2 allele during nc13. At the onset of nc14, most of the daughter nuclei reactivate expression following mitotic silencing but produce fewer transcripts than the nuclei in the ectoderm. Moreover, mesoderm nuclei exhibit asymmetric reactivation of the two alleles. One of the alleles exhibits expression that is comparable with each allele in the ectoderm, whereas the other allele is either silent or exhibits very weak and transient expression. These observations suggest that the diminished levels of expression that are observed for sog>MS2 at the onset of nc14 are mainly due to the repression of one of the alleles. Thus, Snail represses sog expression in the mesoderm one allele at a time. Instead of diminishing the levels of both alleles, there is a clear trend to silence one of the alleles. The basis for this 'digital' mode of repression is uncertain, but it is possible that it reflects the exact time when each homolog is silenced and then decondensed following mitosis (Esposito, 2016).

This study presents evidence that transcriptional repression is intimately linked to the cell cycle, and it is proposed that Snail exploits the general silencing of transcription that occurs during mitosis. Mitotic silencing offers an opportunity to reset the balance between transcriptional activators and repressors. It is possible that Snail outcompetes the Dorsal activator during mitosis. Indeed, immunohistochemical localization assays suggest that the Snail repressor remains associated with the apical cytoplasm during mitosis, whereas Dorsal becomes distributed throughout the cytoplasm. This might give Snail 'the jump' on Dorsal after the completion of mitosis. It is also possible that the balance between the Dorsal activator and the Snail repressor is influenced by 'titration' of Dorsal due to increases in chromosomal templates arising from replication (Esposito, 2016).

It is proposed that Snail and other developmental repressors exploit natural discontinuities in transcription. In addition to mitotic silencing, many genes exhibit transcriptional bursts. It is possible that repressors like Snail get the upper hand during the refractory periods between bursts. Indeed, inhibition between successive bursts of sog>MS2 expression was observed in the mesoderm following mitotic silencing. Approximately 80% of mesoderm nuclei exhibit a single burst of expression before falling silent, and a similar trend in the repression of the btk>MS2 transgene was observed. The Snail repressor may be more effective in maintaining the off state following a burst (or mitotic silencing) than inhibiting a gene at the peak of its activity. Mitotic silencing and transcriptional bursting might represent intrinsic mechanisms that foster dynamic repression of gene expression during development (Esposito, 2016).

Targets of Activity

SOG acts non-cell autonomously to antagonize the activity of dorsally active genes, such as decapentaplegic and tolloid (Francois, 1994).

SOG counteracts the antineurogenic effects of Decapentaplegic. To assess the role of dpp in regulating neurogenesis, the effect of this signaling pathway on neurogenesis in dorsal cells was examined. The dorsal cuticle of embryos lacking dpp activity appears to be ventralized. However, this inference is tenuous, as the number of differentiated neurons is reduced, not expanded, in late dpp mutant embryos. In contrast, for early gastrulating mutants, dorsal expression is observed of neuroectodermal markers such as thick veins and lethal of scute. Similarly, neuroblasts visualized with markers such as scratch and snail subsequently form ectopically in the dorsal region of dpp mutants. Consistent with dpp acting early to suppress initiation of neurogenesis, ectopic expression of the proneural gene lethal of scute is first detectable in dorsal cells of late blastoderm stage dpp mutants. Paradoxically, the increased number of neuroblasts in dpp mutants does not generate a hypertrophied differentiated nervous system. Thus, dpp mutants may lack a late positive role for dpp in neuronal maturation or may hyperactivate pathways functioning to inhibit subsequent steps in neurogenesis (Biehs, 1996).

Short gastrulation prevents DPP from suppressing neurogenesis laterally in the blastoderm embryo. It is possible to exacerbate defects in sog mutants by increasing the level of DPP. The earliest neuroectodermal marker affected in sog mutants with a double dose of dpp is rhomboid, which is normally expressed in lateral stripes 8-10 cells wide in wild-type embryos but rapidly narrows to stripes 4-6 cells across in sog mutants with elevated DPP. Similarly l'sc expression is reduced in sog mutants with elevated DPP. Surprisingly, dpp itself is induced throughout the neuroectoderm in this genetic combination. This provides the first evidence that dpp is capable of autoactivating its own expression during early embryogenesis. Ubiquitous dpp expression results in zerknüllt expression throughout the entire trunk neuroectoderm and mesoderm (Biehs, 1996).

A striking feature of the effects of DPP on neural suppression and dorsalization is that neuronal suppression is induced by a lower threshold of DPP activity than is dorsalization. Much less DPP is required to suppress expression of neuroectodermal genes than is required to activate dorsal markers. For example, brief submaximal heat induction of heat shock dpp in a wild type sog background leads to nearly maximal suppression of lethal of scute, scratch and snail expression during germ band extension, but there is no detectable ectopic expression of zerknüllt in the neuroectoderm (Biehs, 1996).

Gene dosage experiments are consistent with SOG diffusing dorsally 12 to 15 cell diameters from the lateral source of SOG mRNA to determine the limit of dorsal rhomboid expression. Thus SOG diffuses from the neuroectoderm into the presumptive mesoderm to interfere with DPP signaling. Since the effect of SOG is highly dosage dependent, it is likely that there is a gradient of SOG activity in both dorsal and ventral regions of the embryo creating a reciprocal gradient of DPP activity in the dorsal region of the embryo. In this respect, SOG displays many features of a classic morphogen (Biehs, 1996).

The short gastrulation (sog) and decapentaplegic (dpp) genes function antagonistically in the early Drosophila zygote to pattern the dorsoventral (DV) axis of the embryo. This interplay between sog and dpp determines the extent of the neuroectoderm and subdivides the dorsal ectoderm into two territories. Evidence exists that sog and dpp also play opposing roles during oogenesis in patterning the DV axis of the embryo. Maternally produced Dpp increases levels of the IkappaB-related protein Cactus and reduces the magnitude of the nuclear concentration gradient of the NFkappaB-related Dorsal protein, and Sog limits this effect. Evidence is presented suggesting that Dpp signaling increases Cactus levels by reducing a signal-independent component of Cactus degradation. Epistasis experiments reveal that sog and dpp act downstream of, or in parallel to, the Toll receptor to reduce translocation of Dorsal protein into the nucleus. These results broaden the role previously defined for sog and dpp in establishing the embryonic DV axis and reveal a novel form of crossregulation between the NFkappaB and TGFbeta signaling pathways in pattern formation (Araujo, 2000).

In aggregate, the results support models in which Sog and Dpp proteins are produced by the follicle cells and then are delivered to the embryo. These proteins could be deposited in the vitelline membrane or in the oocyte plasma membrane, or might be sequestered in the perivitelline space and remain there protected until early embryogenesis. The fact that sog and dpp are expressed in follicle cells of stage 10 egg chambers, around the time that follicle cells are secreting major structural proteins of the vitelline envelope, is consistent with their products being delivered to the vitelline membrane or perivitelline space. Since sog and dpp are secreted proteins, they could be exported like components of the vitelline membrane to the extracellular compartment between the follicle cells and the oocyte. After stage 13, the vitelline membrane is thought to be an impermeant barrier separating the oocyte from follicle cells making it unlikely that sog and dpp products are transferred after this time. A similar model has been proposed to explain the functions of the dorsal group gene nudel and of the maternal terminal system gene torsolike (tsl). Both of these genes are expressed during midoogenesis, long before their activity is required during early embryogenesis. According to this model, the Sog and Dpp proteins would remain in the perivitelline space until early embryogenesis, when the Tl pathway is activated by Spatzle. In the early embryo, maternal Dpp would decrease the level of Tl-mediated nuclear translocation of Dorsal by decreasing Cactus signal-independent degradation through a pathway acting in parallel to Tl. Presumably, Sog antagonizes the action of Dpp, resulting in maximal nuclear Dorsal translocation (Araujo, 2000).

Consistent with the view that Sog and Dpp proteins are made early (e.g. midoogenesis), but act later in the early embryo, induction of sog expression during midoogenesis by use of a heat-shock sog construct increases levels of a Sog fragment in the early embryo detected by a specific anti-Sog antibody. Thus, Sog protein produced during midoogenesis can be stably stored for a protracted period until the onset of embryogenesis. In contrast to Sog protein, SOG mRNA does not perdure at detectable levels in early pre-blastoderm embryos in these experiments. The fragment of Sog generated in these experiments is the same size (60 kDa) as one that may have activity during pupal development (Araujo, 2000).

There are several unanswered questions regarding how maternal Dpp signaling contributes to embryonic DV patterning. An important remaining question is how maternal Dpp signaling contributes to defining discrete zones of gene expression along the DV axis? Two leading possibilities, which are not necessarily mutually exclusive are: (1) sog and dpp function to determine the relative proportions and positions of the different primary DV domains, and (2) Dpp signaling is necessary to sharpen borders between embryonic DV territories. There is good evidence in support of the first possibility, since the extents of DV expression domains can be altered by increasing maternal Dpp activity. As mentioned above, maternally produced Dpp results in a ventral shift of all DV domains, presumably by lowering the amount of nuclear Dorsal in cells along the entire DV axis. These results also support a role for maternal sog and dpp in refining the normally sharp borders between different territories, since altering the maternal dose of sog or dpp generates overlapping expression of mesodermal and neuroectodermal genes (Araujo, 2000).

Another question is by what mechanism does maternal Sog oppose Dpp in patterning the embryo? Perhaps Sog is necessary to inhibit Dpp signaling through a specific receptor subtype such as the Sax receptor or to restrict Dpp signaling to a specific type of Dpp receptor (e.g. mediated only by Tkv). Alternatively, Sog could be involved in antagonizing another BMP molecule in addition to Dpp, which also functions in embryonic DV patterning. In summary, the results presented in this study indicate that maternal components of Dpp signaling modify elements that converge with signaling downstream of the Tl receptor by regulating Cactus levels and nuclear translocation of Dorsal. This analysis suggests that maternal sog and dpp function to define the relative proportions of embryonic DV domains and may play a role in creating sharp borders between these domains. Further experiments will be necessary to determine the mechanism by which maternal sog and dpp function and how interactions between the Tl and Dpp pathways collaborate to pattern the DV axis of the Drosophila embryo (Araujo. 2000).

Protein Interactions

Although a direct physical interaction between DPP and SOG has not been established, such an interaction is implied by the known physical interaction of BMP4 and Chordin, DPP and SOG homologs in Xenopus (Piccolo, 1996).

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).

Processing of the Drosophila Sog protein creates a novel BMP inhibitory activity

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 (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).

Creation of a Sog morphogen gradient in the Drosophila embryo

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).

As a first step in determining whether Sog diffusion contributes to formation of a Dpp activity gradient, the distribution of Sog protein was directly examined in wild-type Drosophila embryos using two anti-Sog antibodies raised to different regions of the protein. In wild-type cellular blastoderm stage embryos, high levels of Sog protein are recognized by both the 8B and 8A anti-Sog antibodies, which colocalize with SOG RNA in broad ventrolateral stripes corresponding to the neuroectoderm. In addition to the high levels of Sog protein present in the lateral neuroectoderm, lower levels of staining are also observed in dorsal epidermal and ventral mesodermal cells far from the source of Sog. It is notable that the staining observed with either anti-Sog antiserum is consistently stronger in the ventral mesoderm than in the dorsal ectoderm, consistent with there being a mechanism(s) to limit accumulation of Sog dorsally. In the dorsal ectoderm, Sog staining appears graded, with the highest levels present immediately adjacent to the neuroectoderm and progressively lower levels observed dorsally. The dorsal gradient of Sog protein is best revealed by the 8A anti-Sog antibody and becomes most pronounced in late blastoderm stage embryos. Sog immunolabeling is present in two parallel tracks of punctate staining in dorsal ectodermal and ventral mesodermal cells. These observations indicate that an early gradient of Sog is present in wild-type embryos at a time when Sog is known to function in a dose-dependent fashion as a Dpp antagonist (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 shi^ts; 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).

The findings in this study, which reveal a continuous Sog gradient that diminishes progressively in dorsal cells with antibodies recognizing two different portions of Sog, do not provide any direct support for the existence of processed forms of Sog accumulating dorsally in a pattern that presages the profile of pMAD staining. These differences in assessing the shape of the BMP activity gradient may reflect nonlinear properties of the anti-pMAD antibody or the existence of distinct forms of pMAD, only one of which is efficiently recognized by the currently used anti-pMAD reagent. Alternatively, an intrinsically nonlinear pMAD transducing mechanism may be integrated with other spatially regulated information to create graded and distributed activation of various BMP target genes (Srinivasan, 2002)

Although the final BMP activity gradients generated in the early embryo and wing imaginal discs have strikingly similar shapes, the mechanisms for creating them are very different. In the wing disc, the spread of Dpp from its narrow localized source in the center of the disc is limited by sequestration by means of the BMP receptor Tkv. In contrast, in the embryo, a BMP activity gradient forms within a broad domain of uniform dpp expression in response to the creation of an inverse gradient of the BMP antagonist Sog, which diffuses into the dorsal domain from the adjacent neuroectoderm. This Sog gradient in dorsal cells is created by a combination of specific proteolytic degradation by Tld and Tok and Dynamin-mediated retrieval of Sog. It is noteworthy that Dynamin exerts opposite effects in the early embryo and wing disc. In the precellular embryo, where extracellular molecules may diffuse in a passive ink-in-water fashion in the surrounding perivitelline fluid, a Dynamin-dependent mechanism limits extracellular accumulation of Sog dorsally. In contrast, in the cellularized context of the wing imaginal disc, an active Dynamin-dependent transport process is required for Dpp movement between cells. The fact that completely different mechanisms can ultimately create similarly shaped BMP activity gradients highlights the flexibility of evolutionary processes, which can arrive at more than one type of solution to the same basic problem (Srinivasan, 2002).

Robustness of the BMP morphogen gradient in Drosophila embryonic patterning

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Dorsal cell fate in Drosophila embryos is specified by an activity gradient of Decapentaplegic. Genetic and biochemical studies have revealed that the Sog, Tsg and Tld proteins modify Dpp activity at the post-transcriptional level. The predominant view is that Sog and Tsg form a strong ternary complex with Dpp that prevents it from binding to its cognate receptors in lateral regions of the embryo, while in the dorsalmost cells Tld is proposed to process Sog and thereby liberate Dpp for signaling. In this model, it is not readily apparent how Tld activity is restricted to the dorsal-most cells, since it is expressed throughout the entire dorsal domain. In this study, additional genetic and biochemical assays were developed to further probe the relationships between the Sog, Tsg, Tld and Dpp proteins. Using cell based assays, it has been found that the dynamic range over which Dpp functions for signaling is the same range in which Dpp stimulates the cleavage of Sog by Tld. In addition, the data support a role for Tsg in sensitizing the patterning mechanism to low levels of Dpp. It is proposed that the strong Dpp concentration dependence exhibited by the processing reaction, together with movement of Dpp by Sog and Tsg protein can help explain how Tld activity is confined to the dorsal-most region of the embryo through formation of a spatially dependent positive and negative reinforcement loop. Such a mechanism also explains how a sharp rather than smooth signaling boundary is formed (Shimmi, 2003).

According to the prevailing view, Sog, Tsg and Tld act to create a transport mechanism that helps promote Dpp diffusion from lateral regions of the embryos towards the dorsal side. According to this model, Sog would diffuse into the dorsal domain from its ventral lateral site of synthesis and capture Dpp, thereby preventing Dpp from binding to receptor. Net flux of Sog towards the dorsal side is envisioned to help transport Dpp and thereby increase its concentration in the dorsalmost tissue, which is destined to become the amnioserosa. Tld acts to liberate Dpp by cleaving Sog, and Dpp once released, will either be recaptured by another Sog molecule or bound to its receptors (Shimmi, 2003).

In order for the transport model to produce a Dpp concentration peak, the proper balance between binding affinities, diffusion rates and proteolytic processing is needed. Tsg has been suggested to have several activities that could influence this balance. In one model, Tsg would act to slow down the intrinsic rate of Sog cleavage by Tld. In this case, loss of Tsg is predicted to result in elevated processing of Sog. This should produce a sog loss-of-function phenotype, as is observed when molecular markers are examined. That data argues strongly against this possibility. First, it has been demonstrated that Tsg function is epistatic to Tld. If the tsg mutant phenotype is caused by excess Tld activity, then eliminating Tld should produce a tld loss-of-function phenotype. However, a tsg-like phenotype is observed where there is a general lowering and flattening of the Dpp activity gradient, as assayed by marker gene expression. In addition, biochemical studies reveal that Tsg actually enhances the ability of Tld to cleave Sog. Taken together, it is concluded that Tsg does not function during DV patterning to retard Tld proteolytic activity (Shimmi, 2003).

A second property has been attributed to Tsg: it alters the selection of Tld cleavage sites in Sog thereby producing novel Sog fragments with unique properties. In particular, a Sog fragment termed Supersog containing the first CR domain and a region of the spacer between CR1 and CR2 appears to be produced in vitro by the action of Tsg and Tld. Although the production of Supersog-like fragments are seen under the present reaction conditions described in this study, no enhancement in their production is seen upon Tsg addition. This may reflect loss of an unidentified component during purification or differences in the sensitivities of the CR1 antibodies used in the two studies. These issues are presently under examination. Whether Supersog-type molecules contribute to DV patterning in vivo is unclear. The fact that overexpression of Supersog can partially rescue tsg mutant embryos suggests that they could be important. A full resolution of the role of Supersog will need to await the results of in vivo rescue experiments employing mutants of the different Sog cleavage sites, especially those that lead to the production of Supersog-like fragments (Shimmi, 2003).

One of the primary findings in this report is that the rate of Sog cleavage is very sensitive to the level of the Dpp protein and varies substantially over a 10-fold range. Interestingly, this is the same Dpp concentration range within which low to maximal signaling occurs in S2 cell culture. Tsg sensitizes the system such that both the binding of Dpp to Sog as well as the rate of cleavage of Sog by Tld is stimulated by Tsg protein. Because in the invertebrate system, the binding of ligand to Sog is required for efficient processing of Sog, it is not surprising that the rate of Sog processing goes up in the presence of Tsg. This follows because, at a given concentration of Sog and Dpp, more complex will be formed in the presence of Tsg leading to a higher substrate concentration for the Tld protease. It is speculated that this system evolved in part to enable the embryo to produce a patterning mechanism that functions within the context of a very short developmental window. In Drosophila, the time between initial transcription of dpp during the early blastoderm stage and assignment of fate required for proper gastrulation is only about 40 minutes. In this short time-window, Dpp concentration must reach an effective signaling level. However, using a genomic Dpp-HA construct, it has been possible to visualize Dpp in the early embryo and it is present at much lower levels than in other tissues, such as the epidermis, at later stages of embryogenesis. It is proposed that under these conditions of low Dpp concentration, the presence of Tsg is required to enable Sog to bind to Dpp and to stimulate Sog cleavage in order to create a cyclic binding and release process that enables Dpp to be carried towards the dorsal midline. Furthermore, it is proposed that the intrinsic sensitivity of the cleavage reaction to the Dpp concentration is crucial for formation of a sharp signaling boundary. Thus, as the Dpp concentration drops in the lateral regions as a consequence of Dpp movement towards the dorsal side, the rate of Sog cleavage drops, allowing more Sog to enter this region and further reducing signaling in lateral regions. The movement of Dpp will simultaneously raise Dpp concentration in the dorsal region, further stimulating cleavage and clearance of Sog and thereby reinforcing Dpp signaling at the dorsal midline. This built-in positive and negative reinforcement mechanism should help establish sharp signaling boundaries by formation of steep ligand gradients, instead of the more gradual gradients that would form if Sog cleavage was not sensitive to the Dpp concentration (Shimmi, 2003).

In some vertebrate systems, DV patterning mechanisms have been conserved with respect to the molecules employed, but the polarity of axis over which they act has been inverted. Thus, in both amphibians and zebrafish, Bmp ligands specify ventral cell fates, whereas Bmp inhibitors, such as Chordin, are secreted from dorsal cells. In each of these systems, Tsg- and Tld-like proteins also contribute to axis formation, but the biochemical details of their associations appear different from those found in Drosophila. Two distinctions are most apparent and these probably have biological significance with respect to the patterning mechanism employed by these organisms. In Xenopus, the affinity of chordin for Bmps is significantly higher than Sog for Dpp; Bmps can be coimmunoprecipitated by chordin alone whereas this is not the case for the Drosophila components. In addition, once cleaved by Xolloid, at least some of the CR1 containing fragments of chordin continue to have significant affinity for the Bmp ligand preventing it from signaling (Shimmi, 2003 and references therein).

The second major difference between the Drosophila and Xenopus systems is that the Drosophila processing of Sog is dependant on prior binding of Sog to Dpp, while in Xenopus this is not the case. Rather, Chordin cleavage by Xolloid appears to be constitutive and is not enhanced by any tested ligand. Without ligand dependent cleavage, net movement of Bmps by Chordin diffusion may not readily occur nor would there be a mechanism to both positively and negatively reinforce the processing reaction. Indeed, recent studies have demonstrated that in the Drosophila embryo, Chordin does not have the ability to promote Dpp signaling at a distance, whereas Sog does. As a result, spatially enhanced Bmp concentrations and sharp signaling boundaries that result from net ligand movement by the activities of the Chordin, Xolloid and Tsg proteins may not occur in Xenopus. In fact there is no evidence in Xenopus that loss of Chordin activity actually results in a reduction in Bmp signaling in select regions of the embryo as occurs in Drosophila (Shimmi, 2003).

Despite these differences, Tsg may, nevertheless, play both positive and negative roles in modulating Bmp signaling; however, its mechanism is somewhat different. As processed fragments of Chordin still have reasonable affinity for ligand, they may need to be dislodged to allow for signaling. Tsg binding to Bmps appears to help promote this dislodgment and their ultimate degradation. In Drosophila, since Sog binds poorly to ligand in the absence of Tsg there is no need for Tsg to help promote dissociation of Sog fragments. Rather, it is its ability to help promote association of Sog with Dpp that is key to understanding its function. Tsg appears also to alter the rate of chordin proteolysis. Thus, at a high Tsg-to-chordin ratio, Chordin may be degraded and in this way Tsg might help promote signaling. It is possible that some combination of these properties is used in other vertebrates. For example, in zebrafish it has recently been shown that loss of chordin can enhance a phenotype that results from haplo-insufficiency for swirl, a gene that encodes Bmp2b. This paradoxical observation, that loss of an inhibitor exacerbates a phenotype resulting from loss of a ligand, is exactly analogous to the case of amnioserosa development in Drosophila where loss of Sog (an inhibitor) leads to less Dpp signaling in the dorsal domain. Detailed studies examining the ligand dependence of Chordin cleavage in zebrafish by minifin, the gene encoding a Tld homolog, have not been reported. It is possible therefore, that like Drosophila, this system may also employ a transport mechanism involving Tsg, Chordin and Tld that acts to boost Bmp signaling in specific tissues. It is interesting to note that the mouse homologs of Tsg, Chordin and Tld also exhibit their own distinct biochemical properties. Thus, a new Tld processing site in Chordin is induced by the presence of Tsg but this is not seen when the Xenopus components are used. Thus, it seems probable that the inherent complexity of this multi-component regulatory mechanism has provided numerous targets for evolutionary change. It is speculated that these changes account for the remarkable diversity that this mechanism exhibits with respect to the actual details by which it regulates Bmp signaling in different organisms (Shimmi, 2003).

A model for formation of the BMP activity gradient in the Drosophila embryo

The dorsoventral axis of the Drosophila embryo is patterned by a gradient of bone morphogenetic protein (BMP) ligands. In a process requiring at least three additional extracellular proteins, a broad domain of weak signaling forms and then it abruptly sharpens into a narrow dorsal midline peak. Using experimental and computational approaches, how the interactions of a multiprotein network create the unusual shape and dynamics of formation of this gradient was investigated. Starting from observations suggesting that receptor-mediated BMP degradation is an important driving force in gradient dynamics, a general model is developed that is capable of capturing both subtle aspects of gradient behavior and a level of robustness that agrees with in vivo results (Mizutani, 2005).

This study began by showing that robustness with respect to variations in the expression of single genes is not a characteristic of this system. This is an important observation, given that considerable attention has been focused lately on the robustness of morphogen-patterning systems, as well as biological signaling in general. The fact that sog^-/+ embryos eventually develop normally underscores the ability of embryos to compensate at later stages for early errors. It is not clear why marked effects of sog heterozygosity were not seen previously in previous experiments (Mizutani, 2005).

The diffusibility of BMPs in the embryo in the presence and absence of Sog was examined. By examining embryos in which Dpp is ectopically expressed, it was observed that the range of Dpp action is reduced in the absence of Sog, but still substantial, and consistent with unhindered diffusion. By observing the rate at which continuous ectopic Dpp expression gives rise to an unchanging response profile, it was also possible to infer that Dpp must undergo rapid degradation, presumably through receptor-dependent means. In these experiments, levels of expression of ectopic Dpp were not high (2.5-fold above normal when two copies of st2-dpp were present; presumably only slightly above normal when one copy was present (Mizutani, 2005).

The above observations were used to produce a simplified model of gradient formation. The goal was not necessarily to reproduce all aspects of the in vivo gradient, but rather to begin with a minimum number of elements -- and as few assumptions as possible -- and then ask which of the behaviors of the in vivo gradient could be captured. Interestingly, a great many of those behaviors emerge from a model in which a single ligand (e.g., Dpp or a Dpp/Scw heterodimer) diffuses freely, is degraded by receptors, forms a complex with Sog and Tsg, and is released from that complex when Tld cleaves Sog. These behaviors include rapid dynamics, formation of a broad domain of weak dorsal signaling that abruptly refines to a sharp midline peak, and peak narrowing or broadening when sog dosage is either increased or decreased, respectively. These behaviors depend upon the combined presence of Sog, Tsg, and Tld and are also highly sensitive to dpp dosage. Interestingly, highly localized expression of Tld and an absolute dependence of Sog cleavage on Dpp are not essential. Also not critical is the order of assembly of Dpp-Sog-Tld complexes (Mizutani, 2005).

Although the ability of the model to form a midline peak of BMP activity exemplifies the Sog/Tld-dependent 'shuttling', that mechanism does not give a complete picture of events for two reasons: (1) the abrupt onset of midline peak growth after a substantial plateau phase reflects a BMP-catalyzed chain reaction of Sog destruction that is independent of BMP transport per se; (2) calculations show that any soluble inhibitor has the ability to expand the range of action of a morphogen simply by protecting it from receptor-mediated destruction. Indeed, this effect alone could underlie some of the greater range of action of ectopically expressed Dpp in wild-type versus sog^- embryos (Mizutani, 2005).

At least one feature of the model that does not match in vivo observations, even when investigated over a wide range of parameter values, is the magnitude of the effect of sog heterozygosity on PMad peak width. The results suggest a near doubling of peak width, whereas calculations predict a more modest increase. Even accounting for the nonlinearity of immunohistochemistry and the fact that PMad may not be an instantaneous read-out of BMP receptor occupancy, the data suggest that other processes, not captured in the simple model, regulate the shape of PMad peaks. For example, it might be necessary to include the effects of a novel truncated form of Sog that promotes, rather than inhibits, BMP signaling (Mizutani, 2005).

One process that seems especially likely to shape PMad peaks is a BMP-driven, transcription-dependent feedback loop that has very recently been shown to markedly amplify high and depress low levels of BMP signaling in the Drosophila embryo. Such feedback could not only modify the shapes of PMad peaks, but also potentially explain another peculiarity of the model, which is that its peak heights and widths best fit mutant data when they are looked at up to the 30-45 min period, but not much later (i.e., not in the mathematical steady state). Since positive-feedback regulation of BMP signaling can be expected to both sharpen and maintain patterns that might otherwise have continued to evolve, it is perhaps not surprising that, at long enough times, in vivo behavior diverges from predictions of the model. Put another way, this issue serves as a reminder that, unlike BMP gradients at larval stages of Drosophila development (e.g., in the imaginal discs), the embryonic BMP gradient forms and acts so rapidly that there is little justification for assuming that steady-state calculations should reproduce in vivo observations. Indeed, it is only by considering the dynamics of gradient formation that the model presented here is able to explain the seemingly paradoxical result that decreased dorsal midline PMad staining in dpp^-/+ embryos can be rescued by lowered sog dosage, when loss of sog function, by itself, is associated with decreased dorsal midline PMad staining (Mizutani, 2005).

In summary, the results presented here indicate that known properties of the molecules required for formation of the Drosophila embryonic BMP gradient are sufficient to account for many aspects of gradient dynamics, shape, and robustness, with no need for assumptions such as lack of diffusion of free BMP, transient BMP synthesis, removal of BMP from its receptors by Sog, or attainment of a steady state. Although computational data indicate that a Sog/Tld-dependent shuttling mechanism plays a key role in shaping and timing this BMP gradient, other dynamic processes appear to participate as well (Mizutani, 2005).

Tolloid-related processes Sog in order to help specify the posterior crossvein in the Drosophila wing

Tolloid (Tld) and Tolloid related (Tlr) belong to a family of developmentally important proteases that includes Bone Morphogenetic Protein 1 (Bmp1). Tld is required early in Drosophila development for proper patterning of dorsal embryonic structures, whereas Tlr is required later during larval and pupal stages of development. The major function of Tld is to augment the activity of Decapentaplegic (Dpp) and Screw (Scw), two members of the Bmp subgroup of the Tgfߟ superfamily, by cleaving the Bmp inhibitor Short gastrulation (Sog). Evidence is presented that Tlr also contributes to Sog processing. Tlr cleaves Sog in vitro in a Bmp-dependent manner at the same three major sites as does Tld. However, Tlr shows different site selection preferences and cleaves Sog with slower kinetics. To test whether these differences are important in vivo, the role of Tlr and Tld during development of the posterior crossvein (PCV) in the pupal wing was investigated. tlr mutants lack the PCV as a result of too little Bmp signaling. This is probably caused by excess Sog activity, since the phenotype can be suppressed by lowering Sog levels. However, Tld cannot substitute for Tlr in the PCV; in fact, misexpressed Tld can cause loss of the PCV. Reducing levels of Sog can also cause loss of the PCV, indicating that Sog has not only an inhibitory but also a positive effect on signaling in the PCV. It is proposed that the specific catalytic properties of Tlr and Tld have evolved to achieve the proper balance between the inhibitory and positive activities of Sog in the PCV and early embryo, respectively. It is further suggested that, as in the embryo, the positive effect of Sog upon Bmp signaling probably stems from its role in a ligand transport process (Serpe, 2005).

The major distinction between the two Drosophila proteases in terms of their Sog processing function is the time and tissue in which they act. Tld activity is primarily confined to the early embryo, while Tlr is required during pupal wing development. To some extent, the functional differences between them can be attributed simply to differences in expression pattern. In the pupal wing Tlr is far more abundantly expressed than Tld, and this alone might account for the lack of redundancy. In the early embryo, however, the situation is more complex. Both enzymes are expressed with similar profiles, but Tlr does not seem to be capable of providing sufficient Sog processing activity, even when several extra copies are provided as transgenes (Serpe, 2005).

It has been speculated that this difference in activity might reflect differences in activation of the proteases at the level of pro-peptide removal. Like all the members of the Bmp1 family, Tld and Tlr are secreted as pro-enzymes; the processing of the pro-peptide is necessary for the activation of proteolytic activity, since the N-terminal end of the astacin motif is buried inside the catalytic domain forming an internal salt bridge. Mutations at the processing site render the enzymes inactive, whereas removal of the pro-peptides produce activated forms of Tld and Tlr. Tlr has a much longer pro-peptide that could either aid or inhibit activation in a tissue-specific manner. However, the inability of Tlr to rescue Tld mutants does not appear to result from an inefficient activation step. Tld activation, both in the embryo and in S2 cells, is very inefficient with most of the protein found in the pro-enzyme state. By contrast, pro-peptide removal from Tlr is very efficient in S2 cells, and the same is true when Tlr is ectopically expressed in the embryo (Serpe, 2005).

Instead, it seems likely that the difference in kinetics of Sog processing by Tlr is the reason behind the inability of Tlr to rescue tld mutants. Tlr is much less efficient at cleaving Sog in vitro than Tld. Given the rapid developmental time of early embryogenesis, where patterning by Bmps during cellularization occurs within approximately a 30 minute time window, the slower kinetics of Sog processing by Tlr may not support proper patterning. Indeed, computational work has shown that a three to fourfold reduction in kinetic properties of Tld will completely disrupt the patterning process (Serpe, 2005).

Although the slow processing kinetics of Tlr towards Sog may prevent it from functioning effectively in early embryonic patterning, this property may be exactly what is required to achieve proper formation of the PCV. Unlike patterning in the early embryo, formation of the PCV, as assessed by profile of pMad accumulation, occurs over at least a 7 hour time frame. The slower processing rate of Tlr towards Sog may be required to achieve the appropriate balance of Sog destruction and diffusion that is necessary for proper patterning to occur. Consistent with this view, overexpression of various UAS-tld lines under the control of the A9-Gal4 driver in a tlr mutant background does not rescue PCV formation. In fact, in many cases overexpression of an activated Tld, or co-expression of wild type tld and tlr together produce loss of the crossvein tissue in a wild-type background. It is envisioned that, under these conditions, the increased level of enzymatic activity results in over-digestion of Sog, a situation that would phenocopy sog hypomorphs. Consistent with this view, hypomorphic sog allelic combinations also result in the loss of the PCV. In addition, large sog null clones can also result in loss of the PCV (Serpe, 2005).

In a Xenopus assay, it was found that Tlr is only slightly less efficient than Tld at reverting secondary axis induction caused by Sog. Although it is not known how well each enzyme is activated in this animal, it should be noted that the developmental time period over which the patterning process functions in Xenopus is long compared with early Drosophila development. The longer time frame may enable the less efficient protease to produce a similar biological response. Protease domain swap experiments suggest that the reduced processing rate does not involve evolution of intrinsic differences in the catalytic abilities of the protease domain itself, but rather changes occur in the way that the Sog substrate initially interacts with the enzyme. In summary, it is proposed that during evolution there was selection for particular biophysical properties of these two enzymes to properly match the developmental time frame over which the patterning mechanisms operate. It cannot be exclude however, that other differences besides kinetic activity might also play a role in providing functional specificity. For example, it is possible that variation in cleavage site selection might also contribute to the different biological activities of Tlr and Tld. It is worth noting in this regard that different fragments of Sog have been shown to have both positive and negative effects when overexpressed in the wing. However the in vivo roles of endogenous Sog fragments have not been defined (Serpe, 2005).

The results suggest that a proper balance of Sog and protease activity is necessary to pattern the PCV. Interestingly, the same situation holds true in the early embryo. In this case, Sog plays both positive and negative roles in patterning the dorsal domain. It is required in the dorsolateral regions to block Bmp signaling, but it also acts as an agonist to achieve peak levels of Bmp signals at the dorsal midline. Two types of models have been proposed to account for these dual activities. In one model, the different cleavage fragments of Sog are thought to provide either agonist or antagonist function, but the details of the mechanism are unclear. In the other model, both functions are proposed to come about as a result of Sog providing a transport mechanism that spatially redistributes Bmp ligands from the lateral region to the dorsal most cells. This transport mechanism also requires the activity of Tsg, a small cysteine-rich secreted protein which has been shown to form a tripartite complex with Sog and Dpp. The prevailing view is that as Sog diffuses into the dorsal domain it forms a high affinity complex with Tsg and Dpp. This complex is unable to bind to receptors and is responsible for the antagonistic activity of Sog. At the same time, the complex protects Dpp from degradation and receptor binding allowing it to diffuse and accumulate dorsally where it is released by Tld processing. The ability of Sog to redistribute the Bmp ligands accounts for the agonist function of Sog. Computational analyses have provided additional support for this model (Serpe, 2005).

It is proposed that the same type of mechanism may be responsible for patterning the PCV. Recent analysis has provided evidence that the longitudinal veins act as the source of Dpp for PCV specification. Dpp is thought to diffuse from these veins into a PCV competent zone. The exact mechanism by which the competent zone is specified is not clear, but low levels of Sog expression are required. tlr is expressed within the PCV competent zone during the initial stages of crossvein development, suggesting the Tlr:Sog ratio will be higher in this region. Furthermore, because processing of the Sog/Dpp complex by Tld-like enzymes is dependent on the Dpp concentration, the complex will be most efficiently processed in the center of the competent zone (Serpe, 2005).

According to this model, there is limited processing of Sog and therefore limited release of Dpp from its inhibitor in tlr mutants. Conversely, Sog also supplies a positive function for PCV formation, probably by providing a transport mechanism for Dpp, accounting for the partial loss of the PCV in hypomorphic sog mutants and complete loss of the PCV in large sog-null clones. The partial reversion of the tlr mutant phenotype by introduction of hypomorphic sog alleles is also consistent with the view that it is the balance between these two factors that is crucial for proper patterning. Interestingly, this is the way in which Sog was originally identified as an inhibitor of Dpp signaling in the embryo: weak sog alleles were isolated as partial suppressors of tld mutations. One difference is that, in the case of this partial reversion, lowering Sog levels is able to revert a null mutation instead of a hypomorphic condition, as was the case in the embryo. There are at least two possibilities that can explain this suppression effect. First, although these animals may be null for tlr, there could be some low level tld expression in the pupal wing. If so, then these wings would not be devoid of all Sog-processing activity and therefore lowering Sog levels might enable the limited amount of Tld to provide the proper production-destruction balance. Alternatively, neither Sog nor Tlr may be absolutely required for PCV formation. Instead, their functions may be simply to ensure that the patterning occurs reproducibly. Thus, in the absence of both Sog and Tlr, partial PCV formation may occur as a result of some Bmp ligand accumulating in the correct position. However, under these circumstances, the patterning mechanism would be unreliable and would produce different results on case-by-case basis. To prevent this from occurring, it is posited that evolution has selected for supplementary regulatory controls involving Sog and Tlr to ensure that the PCV always forms completely and reliably at the correct position (Serpe, 2005).

Two additional observations make the comparison between formation of the PCV and establishing the high point in embryonic Bmp signaling even more compelling. (1) In the embryo, Tsg is required to enable Sog to bind to Dpp and Scw to achieve peak levels of Bmp signaling. Although Tsg and Scw are not transcribed in the pupal wing, a Tsg-related gene, encoded by the crossveinless (cv) locus, is expressed in the pupal wing. Since cv mutants exhibit a crossveinless phenotype, it seems likely that Cv functionally substitutes for Tsg during PCV formation. (2) Gbb, another Bmp-like ligand, may functionally replace Scw, since gbb hypomorphic mutations lack the PCV and associated Bmp signaling (Serpe, 2005).

A major distinction between embryonic amnioserosa development and PCV formation is that PCV specification also requires the activity of Cv2, a protein that contains cysteine-rich repeats similar to those found in Sog, while amnioserosa specification does not, despite the expression of Cv2 in those cells. Vertebrate homologs of Cv2 can bind Bmps, and act variously as agonists or antagonists of Bmp signaling in different assays. It is not clear by what mechanism Cv2 promotes Bmp signaling during PCV formation. It is also not clear why Cv2 is not required in the early embryo, even though it is expressed in dorsal blastoderm cells (Serpe, 2005).

Finally, Tlr plays additional roles during development, besides processing Sog for specification of the PCV. In contrast to cv and cv2 null mutations, which result in homozygous viable and fertile flies, most tlr mutant animals die during larval stages when there is no known requirement for Sog. In addition, although reducing Sog levels does suppress the PCV defect observed in the tlr mutant escaper flies, it does not increase the frequency of eclosing animals. Therefore Tlr may be required for processing of some other essential component(s) during Drosophila development (Serpe, 2005).

Facilitated transport of a Dpp/Scw heterodimer by Sog/Tsg leads to robust patterning of the Drosophila blastoderm embryo

Patterning the dorsal surface of the Drosophila blastoderm embryo requires Decapentaplegic (Dpp) and Screw (Scw), two BMP family members. Signaling by these ligands is regulated at the extracellular level by the BMP binding proteins Sog and Tsg. Tsg and Sog play essential roles in transporting Dpp to the dorsal-most cells. Furthermore, biochemical and genetic evidence is presented that a heterodimer of Dpp and Scw, but not the Dpp homodimer, is the primary transported ligand and that the heterodimer signals synergistically through the two type I BMP receptors Tkv and Sax. It is proposed that the use of broadly distributed Dpp homodimers and spatially restricted Dpp/Scw heterodimers produces the biphasic signal that is responsible for specifying the two dorsal tissue types. Finally, it is demonstrated mathematically that heterodimer levels can be less sensitive to changes in gene dosage than homodimers, thereby providing further selective advantage for using heterodimers as morphogens (Shimmi, 2005a).

The suggestion that the facilitated transport of a BMP signaling molecule might be the primary mechanism that generates pattern within the dorsal domain of the Drosophila blastoderm embryo (Holley, 1996) was a conceptual breakthrough, since it could account for the paradoxical abilities of Sog and Tsg to have both positive and negative effects on patterning. However, there was no direct evidence that either Dpp or Scw actually concentrated to the midline. In addition, it did not explain the roles of Dpp and Scw in producing the restricted high-level signaling output at the midline, as measured by p-Mad accumulation, nor did it explain how a lower level of signal was maintained in the more lateral regions to help fate the future dorsal ectoderm. Lastly, it was not apparent how the system achieves resiliency to changes in gene dosages of certain components. The experimental and computational observations described in this study have addressed these issues (Shimmi, 2005a).

One of the primary findings is that Dpp and Scw form heterodimers both in tissue culture and in vivo and that these heterodimers are able to synergistically stimulate phosphorylation of Mad in cell culture. Since the Dpp/Scw heterodimers have highest affinity for Sog and Tsg, it is inferred that the heterodimer is the primary ligand transported dorsally by Sog and Tsg, resulting in high levels of p-Mad accumulation at the dorsal midline just prior to gastrulation. Consistent with this view, it was found that Dpp localization to the midline depends on Scw (Shimmi, 2005a).

In addition to heterodimers being the preferred translocated species, the heterodimer model also explains the mechanism by which Scw contributes to dorsal patterning. This issue has been enigmatic since scw and its receptor, Sax, are expressed ubiquitously in the early embryo, yet signal output is limited to dorsal cells. In addition, misexpression of Scw or activated Sax produces very limited effects in most tissues, while misexpression of Dpp or activated Tkv results in very dramatic consequences. A partial resolution to this issue was suggested by the finding that coexpression of activated Sax and activated Tkv in embryos or imaginal discs produces a synergistic signal, implying that both the Sax and Tkv signals are necessary for a robust output. However, it has remained unclear whether endogenous, nonactivated receptors can produce a synergistic signal in response to ligands. As described in this study, the formation of a heterodimer between Dpp and Scw resolves these issues. In tissue culture assays, Scw homodimers produce very limited signal, while Dpp homodimers produce a moderate signal requiring only the Tkv receptor. The differential signaling ability of each homodimer explains their nonequivalence in producing patterning abnormalities when misexpressed during development. In contrast, the Dpp/Scw heterodimer is able to produce a synergistic phosphorylation of Mad that requires both the Tkv and Sax receptors; simply mixing homodimers is not sufficient. These observations demonstrate that synergistic signaling occurs at the level of receptor-mediated Mad phosphorylation and not through integration of separate signals at downstream targets. The molecular mechanism by which the Tkv and Sax receptors produce a synergistic output remains unclear (Shimmi, 2005a).

Although the original role for Scw in dorsal patterning invoked formation of a heterodimer as the primary signaling species, this model fell into disfavor because ventral injection of scw mRNA or ventral expression of scw from the twist promoter can partially rescue amnioserosa formation. Since disulfide-linked heterodimer formation of TGF-β type ligands is known to occur in the Golgi during the secretion process, ventral expression of Scw without Dpp should preclude formation of heterodimers, and, therefore, any rescuing activity should be brought about by homodimers. Although some rescue was observed in these experiments, it is important to note that even multiple copies of ventrally expressed Scw do not lead to viability. In contrast, a single copy of Scw expressed in the dorsal domain using the tld promoter gives complete viability and fertility. In addition, these experiments assume that there is no internalization within the dorsal domain of Scw homodimers followed by isomerization with Dpp and resecretion. This possibility is mechanistically very similar to models in which Dpp is proposed to undergo transcytosis. Therefore, while ventral overexpression of Scw homodimers may have some ability to compensate for loss of Scw dorsally, normal patterning is most efficiently achieved when Scw is expressed in a domain in which heterodimers can form (Shimmi, 2005a).

BMP-directed patterning of dorsal blastoderm cells ultimately results in the specification of two tissues, amnioserosa and dorsal ectoderm. In general, these tissues derive from cells receiving high and low BMP signal, respectively. Whether there are additional cell fate subdivisions specified within the steep signaling transition zone is not clear, although cells can discriminate subtle signaling differences as evidenced by the slightly wider expression pattern of the BMP target genes rho and usp compared to zen and race. Although both Dpp and Scw are required to establish the high point of signaling necessary to specify amnioserosa, only Dpp is needed to specify dorsal ectoderm. This is consistent with observations that the Dpp/Scw heterodimer will be preferentially concentrated at the midline because of its high affinity for Sog and Tsg. In contrast, Dpp and Scw homodimers will be more broadly distributed because of their lower affinities for Sog and Tsg. Although the different species cannot be directly distinguished in vivo, analysis of downstream target genes in a scw mutant embryo revealed that there is sufficient BMP activity to activate pnr transcription, but its pattern is very wide, consistent with the observed broad distribution of Dpp homodimers. In the wild-type case, Dpp and Scw homodimers, together with a small number of heterodimers that escape from Sog and Tsg, may contribute to signaling in the lateral ectoderm, since the pnr signal is stronger in wild-type than in scw mutants. These homodimers also likely signal in a repressive manner to prevent ectopic transcription of neurogenic genes within the dorsal domain. Thus, patterning of dorsal tissue appears to take advantage of the differing properties of homo- and hetero-dimers to establish a biphasic signaling state. Specifically, selective transport of the heterodimer and synergistic receptor signaling produce a restricted high point and amnioserosa cell fate, while Dpp and Scw homodimers generate a broad low level of signal that help fate the future dorsal ectoderm and restrict neurogenic activity to more lateral regions. It is likely that the full specification of dorsal ectoderm does not occur until a second round of dpp transcription takes place after germ band extension. It is also likely that additional components help reinforce the formation of the biphasic state, since recent genetic data indicate that tight localization of Dpp to the midline requires an initial phase of low-level Dpp signal reception (E.L. Ferguson, personal communication). The suggestion is that this initial low-level Dpp signal induces expression of an additional component that participates in the localization process. The identity of this component remains elusive (Shimmi, 2005a).

Lastly, it is noted that employment of heterodimers in early embryonic patterning may be a common theme. In zebrafish, both BMP2b and BMP7 are required for dorsal-ventral patterning, and loss-of-function mutations in each gene exhibit identical severely dorsalized phenotypes. Since this phenotype is not enhanced in double mutants and overexpression of these two gene products reveals synergy in the ventralization of wild-type embryos, it has been suggested that BMP2a/BMP7 heterodimers are the primary molecules that specify ventral cell fates in this organism. These observations further highlight the overall similarity in the molecular components used to pattern the early zebrafish and Drosophila (Shimmi, 2005a).

Use of the Dpp/Scw heterodimer provides the patterning system with an effective buffer at a very early step in dorsal cell fate specification. Buffering for reductions of Scw or Dpp is predominantly determined by the relative monomer production rates, and if Scw is in slight excess with respect to Dpp, reductions in the levels of Scw will have little effect on the output Dpp/Scw heterodimer, regardless of the specific choices of parameters (Shimmi, 2005a).

Patterning is also resilient to reductions of Sog and Tsg. Sog and Tsg have synergistic BMP binding activity and the concentration of Sog/Tsg in the PV space is governed by the interaction of reaction and diffusion. The Sog/Tsg ratio can be computed as described for Dpp/Scw to determine the compensation in this subsystem, and the results are different from those for Dpp/Scw. Now there are two distinct solution regions, one for small β (β is the ratio of the wild-type production rates for monomer Dpp to monomer Scw) where many choices of parameters provide significant compensation for reductions of gene dosage, and one for large β where there is virtually no compensation. Because the behavior for large β and small β is very different, this analysis can explain the compensation for reductions in either Sog or Tsg but not both. This suggests that other mechanisms must be involved to explain the experimentally observed resilience in both sog and tsg heterozygous embryos. These could include the following: (1) the spatial separation of Sog and Tsg expression, (2) downstream kinetic mechanisms that compensate after Sog/Tsg formation, or (3) both. Both may contribute, but the following focuses on the possible effects of compensation in downstream kinetic interactions (Shimmi, 2005a).

After Sog/Tsg formation, the next step downstream is the binding of the inhibitor Sog/Tsg to Dpp/Scw. Experimentally, it is observed that Tolloid cleavage of Sog is greatly enhanced when bound to Dpp/Scw and is enhanced in the presence of Tsg. In addition, a previous mathematical model of BMP patterning suggested that cleavage of Sog (only when bound in the complex Sog/BMP) is a requirement for the system to exhibit resilience to changes in gene dose of sog, tsg, or scw. These data support the idea that Dpp/Scw transported from the broad dorsal region must be released from the Sog/Tsg/Dpp/Scw complex. Interestingly, the local dynamics of Sog/Tsg + Dpp/Scw complex formation are completely analogous to the local dynamics for Sog + Tsg complex formation. This suggests that, if the level of Dpp/Scw or Sog/Tsg is decreased from the original wt levels, the output complex Sog/Tsg/Dpp/Scw would be less affected. Taken together, the Sog/Tsg and Sog/Tsg/Dpp/Scw steps lead to a cascade in which the compensation in the first step is enhanced in the second step. In effect, the output from one complex formation stage becomes the input substrate for the next stage. Of course, the level of buffering achieved depends on the system parameters. The output suggests that patterning would be most compensated for reduction of Scw, followed by Tsg, then Sog, and lastly Dpp. Of course, other downstream steps may also contribute to compensation (Shimmi, 2005a).

In reality, patterning involves diffusive transport as well, but the analysis shows how a cascade of stages can produce compensation in the kinetic steps. When the full BMP patterning model that incorporates transport is compared to a previous model mediated by homodimers and monomers, there are approximately 100 times more 'robust' hits when scw^+/−, sog^+/−, tsg^+/−, and tld^+/− cases are considered. In principle, the binding cascade analysis extends to other systems and can be used to explore other changes of input, including overexpression of a protein (Shimmi, 2005a).

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

In the early Drosophila embryo, Bone morphogenetic protein (BMP) activity is positively and negatively regulated by the BMP-binding proteins Short gastrulation (Sog) and Twisted gastrulation (Tsg). A similar mechanism operates during crossvein formation, utilizing Sog and a new member of the tsg gene family, encoded by the crossveinless (cv) locus. The initial specification of crossvein fate in the Drosophila wing requires signaling mediated by Dpp and Gbb, two members of the BMP family. cv is required for the promotion of BMP signaling in the crossveins. Large sog clones disrupt posterior crossvein formation, suggesting that Sog and Cv act together in this context. sog and cv can have both positive and negative effects on BMP signaling in the wing. Moreover, Cv is functionally equivalent to Tsg, since Tsg and Cv can substitute for each other's activity. It is also confirmed that Tsg and Cv have similar biochemical activities: Sog/Cv complex binds a Dpp/Gbb heterodimer with high affinity. Taken together, these studies suggest that Sog and Cv promote BMP signaling by transporting a BMP heterodimer from the longitudinal veins into the crossvein regions (Shimmi, 2005b).

One interesting aspect of BMP signaling in many developmental contexts is that its activity can be regulated at the extracellular level by a number of secreted factors. In Drosophila, these include the products of the short gastrulation (sog), twisted gastrulation (tsg), and tolloid (tld) genes. All three genes were identified as modulators of BMP signaling in the early embryo, and their developmental functions have been well characterized. At the blastoderm stage, BMP signals provided by the dorsally expressed Decapentaplegic (Dpp), and by the generally expressed Screw (Scw), a second ligand that forms a heterodimer with Dpp (Shimmi, 2005a), instruct cells to adopt either amnioserosa or dorsal ectoderm fate. Proper subdivision into these two cell types requires the action of Sog, Tsg, and Tld. Sog and Tsg are BMP-binding proteins that make a high-affinity complex with the Dpp/Scw heterodimer. This complex reduces BMP signaling in the dorsal-lateral regions by blocking the ability of the heterodimer to bind to receptors. Thus, a major role of the Sog/Tsg complex is to antagonize signaling, and similar activity has been found for the vertebrate homologs Chordin and Tsg (Shimmi, 2005b and references therein).

However, the Sog/Tsg complex also stimulates BMP signaling in the dorsal-most cells of Drosophila embryo; it is thought to do so by protecting the ligand from degradation and enabling it to diffuse over long distances. Since sog is expressed in ventral-lateral cells adjacent to the dorsal cells that express dpp, scw, and tsg, the net flux of Sog towards the dorsal side provides a driving force that concentrates BMP heterodimer in the dorsal-most region of the embryo. The ligand is then released for signaling by Tld, an extracellular metalloprotease that cleaves Sog in a BMP-dependent manner. Concentration of the heterodimer to the dorsal-most cells by this facilitated transport mechanism provides a high level signal that specifies amnioserosa cell fate, while dorsal-lateral cells receive less BMP signal and become dorsal ectoderm (Shimmi, 2005b and references therein).

The ability of Tsg to stimulate BMP signaling is apparently not limited to the early Drosophila embryo. Vertebrate Tsg can stimulate BMP signaling in some circumstances, and is required to stimulate high levels of BMP signaling during axis formation in the zebrafish embryo. However, in these cases, Tsg may act, not via a transport mechanism, but by antagonizing Chordin's ability to inhibit BMP signaling. Tsg increases the rate at which Chordin and Sog are cleaved and thus inactivated by Tolloid-like protease. Nonetheless, zebrafish Chordin can also apparently stimulate BMP signaling in some circumstances (Shimmi, 2005b and references therein).

This paper reports another context in which both Sog and a novel Tsg family member stimulate BMP signaling at the developing crossveins in the Drosophila pupal wing. The Drosophila wing has proven to be an attractive model system for elucidating molecular mechanisms that regulate growth and patterning. A major attribute of this system is the stereotypical array of veins that develop along the wing surfaces. These thickenings of the ectodermal cuticle serve both structural support roles for flight and act as channels for the supply of nutrients to the wing cells. For the geneticist, they provide a key set of morphological landmarks for identification of genes that affect the patterning process. Analysis of many classical mutations that alter vein cell fate and patterning have revealed the fundamental roles played by three highly conserved growth factor signaling pathways. For the five longitudinal veins (L1-L5) that form along the proximal-distal axis, a key initiating event is the localized expression of Epidermal growth factor (EGF) signaling components in the vein primordial cells during late imaginal disc development. In response to EGF receptor signaling, Delta is expressed along the veins and induces Notch to inhibit vein formation in neighboring cells. Subsequently, during pupal stages, EGF receptor signaling induces expression of dpp within the developing longitudinal veins. Expression of dpp in the longitudinal veins is required for maintenance of EGF receptor signaling and final vein differentiation, especially at the distal tips (Shimmi, 2005b and references therein).

In addition to the five longitudinal veins, two other shorter veins form perpendicular to the longitudinal veins; these are the anterior crossvein (ACV), which forms between L3 and L4, and the posterior crossvein (PCV), which forms between L4 and L5. Unlike the longitudinal veins, the crossveins do not rely on early EGF signaling for their initial specification. Instead, their formation is initiated during pupal stage by localized BMP signaling, which requires Dpp. However, in the case of the PCV, Dpp is not initially produced in the crossvein, but instead diffuses into the PCV region from the longitudinal veins. Dpp does not act alone during this process since mutations in glass bottom boat (gbb), a member of the BMP5/6/7 subfamily, also eliminate the PCV. Gbb is widely expressed during pupal wing development, but an analysis of gbb mutant clones has suggested that the active BMP component for PCV specification might be a heterodimer of Dpp and Gbb formed in the longitudinal veins, since the PCV is lost only when the clone includes cells of the longitudinal veins where Dpp is produced (Shimmi, 2005b and references therein).

Like the embryo, modulation of BMP signaling in the PCV also appears to involve several additional secreted proteins. For example, mutations in tolloid-related (tlr; also known as tolkin) and crossveinless 2 (cv-2) eliminate PCV formation by preventing BMP signals in the primordial PCV cells. The tlr gene encodes a Tolloid-like metalloprotease that is able to cleave Sog, while cv-2 encodes a protein containing 5 cysteine-rich (CR) domains, similar to the BMP-binding modules found in Sog (Shimmi, 2005b).

The similarity of these proteins to those involved in patterning the early Drosophila embryo suggests that correct specification of the PCV likely involves establishing a spatially regulated distribution of BMP ligand(s) through the activity of extracellular modulatory factors. This report provides additional evidence supporting this hypothesis by cloning the crossveinless (cv) gene and analyzing its function. cv encodes a new member of the tsg gene family. Like mutations in cv-2 and tlr, loss of cv prevents accumulation of phosphorylated Mad (pMad), the active form of the major transcription effector of BMP signaling, in the crossvein cells. In addition, ectopic expression studies were used to show that Cv and Tsg have functionally related activities, since each can substitute for the other in vivo, and ectopic expression of Cv and Sog phenocopies co-expression of Tsg and Sog. These observations led to a re-examination of the role of Sog during wing vein development. Previous clonal analyses suggested that Sog acts as a dedicated antagonist of BMP signaling and helps maintain longitudinal vein integrity. However, this study shows that Sog is in fact required for BMP signaling in the PCV, since large sog clones inhibit PCV formation. These data suggest that, as in the early embryo, Sog plays a dual role in promoting and inhibiting BMP signaling. In light of these results, the biochemical properties of Cv were examined, and it was found that, like Tsg, it can form a high-affinity oligomeric complex with Sog and BMP heterodimers (Shimmi, 2005b).

Taken together, these results suggest that similar mechanisms govern PCV development and early embryonic development. In the embryo, a Dpp/Scw heterodimer specifies amnioserosa fate following ligand transport from lateral to dorsal-most regions through the action of Sog and Tsg. Processing of the complex by Tld then enables signaling in a restricted spatial domain. It is speculated that formation of the PCV likely requires selective transport of a Dpp/Gbb heterodimer from the longitudinal veins to the PCV competent zone through the action of Sog and the Tsg-like protein Cv. As in the embryo, the Tolloid-related enzyme may release the ligand through processing of the Sog/Cv/BMP complex to generate a spatially restricted pattern of signaling in the PCV. This example illustrates how, in different developmental contexts, related molecules and common mechanistic processes can achieve new patterning outcomes (Shimmi, 2005b).

The formation of Drosophila wing veins is a very sensitive system for examining the activity of BMP signaling within the context of a developmental patterning process. Two distinct aspects of the BMP signaling process in veins have been recognized. (1) BMP signals are produced by the developing longitudinal veins where they act locally to help maintain the vein fate earlier specified by EGF signaling. (2) BMP signals produced in the longitudinal veins act at longer range to initiate BMP signaling in the crossveins. This study shows evidence that this long-range signaling requires the activity of both Sog and a Tsg-like molecule encoded by the cv gene. Thus, within the context of the crossveins, Cv and Sog play positive roles in BMP signaling. Based on analogy to the embryonic patterning system, these results suggest that Cv and Sog aid in the transport of BMP ligands from producing cells to receiving cells in the posterior crossvein competent zone (Shimmi, 2005b).

Since Tsg and Cv showed a similar domain structure, attempts were made to determine if they were functionally equivalent by expressing one in place of the other during either embryonic or pupal development. These experiments showed that these two products are, to some extent, genetically interchangeable. However, Tsg and Cv may have been optimized for a particular developmental function that likely represents interactions with a particular ligand, i.e., Dpp/Scw heterodimers in the case of Tsg and Dpp/Gbb heterodimers in the case of Cv. A recent phylogenetic comparison of the Cv and Tsg proteins from different insect species suggests that these two proteins fall into distinct families, one Cv-like and one Tsg-like). In addition, under conditions of overexpression, cv and tsg exhibit enhanced genetic interactions with different BMP ligands. For instance, cv interacts better with gbb than with dpp. While no difference was seen in the ability of Sog and Tsg versus Sog and Cv to bind to Dpp/Gbb heterodimers, these data are qualitative. Thus, it is possible that these two protein complexes could have different affinities for different ligands that are optimal for their particular developmental function (Shimmi, 2005b).

A similar observation has recently been made for Tld and Tlr proteins. These two metalloproteases show very similar overall structure and both cleave Sog in the same positions, but with different kinetics and site preferences. In this case, the two proteins cannot substitute for the other and it has been proposed that this represents optimization of catalytic activity for a fast (Tld in the embryo) or slow (Tlr in pupal wing vein) developmental function (Shimmi, 2005b).

In the early embryo, Tsg and Sog function together to help redistribute BMP signals from their broad initial distribution profiles throughout the dorsal half of the embryo into a narrow stripe of cells centered on the dorsal midline. In this model, Tsg and Sog play both positive and negative roles. The positive role comes via transport and the resulting increase in BMP concentration at the dorsal midline. The negative role comes from blocking access of the ligand to receptors in the lateral regions during BMP transport (Shimmi, 2005b).

The process of PCV formation appears remarkably similar, at least in terms of the BMP signaling components employed. Both Sog and the Tsg-like molecule Cv are required for BMP signaling, as indicated by the accumulation of pMad, in the developing crossveins. Thus, both Cv and Sog play positive roles in augmenting BMP signaling during crossvein formation. This positive role may also come from facilitating transport of BMPs, since co-expression of Cv and Sog in the posterior compartment resulted in ectopic vein formation and BMP signaling gains in the anterior compartment. Similar, although less penetrant, effects on anterior venation have been observed when Sog alone is misexpressed (Shimmi, 2005b).

In addition, Cv and Sog may also inhibit BMP signaling around the longitudinal veins. A sog mutant clonal analysis has demonstrated a requirement for Sog in keeping longitudinal veins straight and narrow. In the absence of Sog, the veins meandered. A similar effect on the longitudinal veins is seen when cv is lost, with an expansion in pMad accumulation around the longitudinal veins. Thus, Cv and Sog may function together to restrict the range of Dpp signaling along the longitudinal veins (Shimmi, 2005b).

Two other similarities between embryonic patterning and PCV formation are worth noting. In the embryo, the Tld metalloprotease is required to release ligand from the inhibitor complex of Sog and Tsg. Likewise, the Tolloid-related protease Tlr is required for PCV formation. Tlr is expressed in the pupal wings, when it is required for crossvein pMad, and has recently been shown to process Sog at the same three sites as does Tld (Serpe, 2005). Thus, it seems likely that Tlr is needed to release a BMP ligand for signaling in the PCV competent zone (Shimmi, 2005b).

There may also be strong similarities between the embryo and crossvein patterning in their use of ligands. In the embryo, both Dpp and Scw are needed to specify the amnioserosa, while for PCV specification, both Dpp and Gbb are required. Sog and Tsg show the highest affinity for the heterodimer of Dpp/Scw in the embryo, suggesting that this is the primary transported ligand. Similarly, the ligand with highest affinity for Cv and Sog is a heterodimer of Dpp and Gbb. Interestingly, gbb mutant clonal analysis has shown that the PCV is lost only when a gbb clone encompasses adjacent longitudinal vein material. Since the longitudinal veins serve as the source of Dpp during PCV specification, these observations are consistent with the notion that a heterodimer of Dpp and Gbb is the primary ligand that specifies PCV formation (Shimmi, 2005b).

Although similarities between embryonic dorsoventral patterning and PCV formation are striking, there are clear differences. The most notable is the geometry of the system. Why is the long-range signaling from the longitudinal veins limited to the crossvein regions? Examination of the expression patterns of several components may provide some clues. Tkv expression is reduced in the crossveins, and since binding to receptor is a major impediment to diffusion in wing discs, this might enhance net flux of ligand into the area of reduced Tkv expression. However, down-regulation of tkv in the PCV actually depends on high levels of BMP signal (Ralston, 2005). Therefore, it is not likely that reduced Tkv expression provides a channel for ligand flow; rather, it may reinforce a flux direction that is initiated by other means (Shimmi, 2005b).

In this regard, it is notable that sog expression is also reduced in the crossvein regions and this is independent of BMP signaling (Ralston, 2005). As in the embryo, Sog flux from areas of high expression, i.e., intervein regions in the wing, into areas of low expression, the crossvein zones, might provide the proper positional information. Consistent with this view is the observation that uniform expression of Sog eliminates the initial stages of crossvein development. However, there are inconsistencies in this simple model. While misexpression of Sog can lead to loss of the crossvein, normally positioned crossveins appear when Sog misexpression is coupled with ubiquitous expression of Cv-2 (Ralston, 2005), suggesting that crossvein positioning can be independent of the sog expression pattern. Similarly, loss of sog from clones does not induce ectopic crossveins (this study) (Shimmi, 2005b).

Another possibility is that the cleavage of Sog is spatially regulated. In the embryo, Tld is expressed in the dorsal domain, and since its ability to process ligand is dependent on the Dpp concentration, the processing rate will be highest at the dorsal midline. However, in the pupal wing, tlr is not expressed at higher levels in the crossvein zones; instead, it is high in the entire intervein. Therefore, it is not clear how the ligand would be released from a complex of Cv and Sog specifically in the crossveins. Moreover, uniform expression of tlr causes only mild expansion of the crossveins (Shimmi, 2005b).

Perhaps the key to understanding the differences between the embryonic patterning process and that of the crossveins will be determining the mechanism of action of other gene products that are required for crossvein formation. These include cv-c, cv-d, detached, and the cv-2 gene products. Among these genes, only the cv-2 product has been identified; it is a large secreted factor that contains CR domains, similar to those found in Sog, and is expressed in the developing crossveins. The major distinction between Sog and Cv-2 is that Cv-2 contains a Von Willebrand type D domain found on many blood-clotting proteins that is not present in Sog. In Chordin, the CR modules are responsible for BMP binding and vertebrate Cv-2 homologs have also been shown to bind BMPs. Depending on the assay used, vertebrate Cv-2 homologs can either inhibit or promote signaling. Cv-2 does not seem to be required in the early embryo, yet it is essential for crossvein formation. Drosophila Cv-2, like its vertebrate counterparts, can also bind BMPs and, although it is a secreted protein, Cv-2 can associate with the cell surface. One possibility is that it captures BMPs, perhaps from a Sog/Cv complex, and keeps them close to the cell surface and in this way promotes BMP signaling by keeping the local BMP concentration high. It may also play a more direct role as a coreceptor (Shimmi, 2005b).

Nonetheless, while cv-2 is expressed in the crossveins, and is required for BMP signaling there, ubiquitous expression of cv-2 does not disrupt the positioning of the crossveins, even when coupled with ubiquitous expression of sog. Thus, other genes must act in conjunction with or upstream of these BMP modulators to help establish the crossvein competent zone. It is interesting to note in this regard that mutations in CDC42 induce ectopic crossveins, suggesting that it might be involved in the process that selects the site of crossvein formation (Shimmi, 2005b).

Normally, tsg is expressed only in the early blastoderm embryo. However, sog is expressed at several other developmental stages. In fact, this was one of the motivations to look for additional Tsg homologs, so that it might be determined if Sog always utilizes a Tsg-like partner or whether in some developmental processes it might act alone. One late embryonic process in which Sog has been implicated is to regulate tracheal morphogenesis. As in vein formation, tracheal patterning requires input from the EGFR and Dpp pathways. However, in this case, each pathway is antagonistic to the other. Normally, sog is expressed as a dorsal stripe abutting the tracheal pits, and in sog mutant embryos, hyper-activation of Dpp leads to a loss of dorsal trunk and a reduction in visceral branches. It was therefore interesting that cv is also expressed in and around the tracheal pits, but tsg is not expressed at this stage. However, no alteration was observed in tracheal development in cv mutants. Indeed, these embryos appear fully viable and the resulting adults are fertile. These results suggest that Cv has no other essential role in development. The pattern of cv expression around the tracheal pits may reflect a prior evolutionary involvement in tracheal development that is now provided by Sog alone or perhaps by Sog in conjunction with some other unknown BMP modulatory factor (Shimmi, 2005b).

Crossveinless defines a new family of Twisted-gastrulation-like modulators of bone morphogenetic protein signalling

The Twisted gastrulation (Tsg) proteins are modulators of bone morphogenetic protein (BMP) activity in both vertebrates and insects. The crossveinless (cv) gene of Drosophila encodes a new tsg-like gene. Genetic experiments show that cv, similarly to tsg, interacts with short gastrulation (sog) to modulate BMP signalling. Despite this common property, Cv shows a different BMP ligand specificity as compared with Tsg, and its expression is limited to the developing wing. These findings and the presence of two types of Tsg-like protein in several insects suggest that Cv represents a subgroup of the Tsg-like BMP-modulating proteins (Vilmos, 2005).

A tsg-like gene (CG12410) was found on the first chromosome between CG3160 and CG3149. It encodes a protein with about 50% homology to the Tsg protein and the same molecular topology: two cysteine-rich (CR) domains connected by a variable hinge domain. A comparison of tsg-like genes in insects suggests two subgroups in the tsg-like family typified by cv and tsg. Of the five insects for which complete genomes are available, Drosophila melanogaster, Drosophila pseudoobscura and Drosophila simulans have both a tsg-like and a cv-like gene, whereas the mosquito and bee seem to have only a cv-like gene (Vilmos, 2005).

To determine the function of CG12410, the element EP1349 that shows no mutant phenotype was excised; it is located about 700 base pairs (bp) 5' of the exon containing the predicted start codon. Four strains were recovered that delete portions of CG12410 and all showed a recessive visible crossveinless phenotype with loss of the anterior crossveins (ACV) and the posterior crossveins. Two of the four mutants were strict recessive visibles (cv¹⁸, cv⁴³), whereas the other two (cv³⁴, cv⁵¹) showed semilethality (22% and 53%) not linked to the cv locus (Vilmos, 2005).

As flies heterozygous for cv¹⁸ and cv¹ have the same phenotype as cv¹ homozygotes, CG12410 is allelic to cv. Further evidence for allelism was obtained by rescuing both cv¹ and cv¹⁸ hemizygotes with CG12410 using UAS>EP1349 under the control of the ptc>Gal4 driver (Vilmos, 2005).

Although the most obvious phenotype is the absence of crossveins and a delta at the tips of the L3 and L4 veins as originally described (Bridges, 1920; Waddington, 1940), it was also found that the longitudinal veins in cv mutants show poorly defined edges and trajectories often broadening and meandering along their length in a manner similar to that seen in sog^- wing tissue, suggesting that cv has a role in refining the domains where veins and crossveins form (Vilmos, 2005).

The nature of the cv mutations was determined by PCR. As cv¹⁸, cv³⁴, cv⁴³ and cv⁵¹ alleles delete a region that extends from the P-element insertion site past the ATG start codon to the second intron of cv, these alleles are considered as physically verified nulls. The cv¹ mutation is due to a 412 retrotransposon inserted in the second intron of cv that introduces two poly(A) addition signals that should terminate the cv transcript prematurely (Vilmos, 2005).

The cv⁵² and cv¹² mutations show no phenotype; however, they delete all the DNA from the insertion to either the adjacent gene CG3160. Thus, regulatory sequences necessary for cv function do not extend past 475 bp upstream of the cv¹² breakpoint (Vilmos, 2005).

Endogenous cv messenger RNA was detected only in the developing pupal wing, with no evidence of earlier expression. Expression of cv first appears as diffuse staining in the regions of the vein primordia 24-28 h after pupariation (APF) and later refines to stripes of 2-3 cells localized at the vein-intervein boundaries and disappears by 40 h APF. By comparison, dpp and the sog-like cv-2 are expressed in the vein domain at these times, whereas sog and gbb are expressed in the intervein regions with concentrations at the boundaries that are coincident with cv (Vilmos, 2005).

Expression of UAS>cv along the anterior-posterior (A/P) border rescues both the ACV and the PCV in cv mutants, whereas tsg does not rescue either crossvein. Thus, anterior expression of cv can restore function in posterior cells. Similarly, cv expressed in the embryo does not rescue tsg mutants. Thus, Tsg and Cv are not functionally interchangeable proteins (Vilmos, 2005).

It has been well documented that the loss of BMP signalling in wings produces two phenotypes, one being reduction of wing size and the other loss of veins. The first phenotype involves an early abrogation of long-range BMP signalling, whereas the second results from a late local loss of signalling in veins (Vilmos, 2005).

It has also been shown that Tsg can inhibit BMP-like ligands by synergizing with Sog, or in other environments can promote BMP activity by displacing an inhibitory fragment of Sog generated by proteolytic cleavage. To compare the activities of Cv and Tsg, transgene combinations were expressed under the control of the wing driver A9>Gal4. Excess cv alone can induce small fragments of extra veins and a delta phenotype, consistent with a mild pro-BMP activity. In contrast, coexpression of cv and sog produces a phenotype resembling early loss of BMP signalling in the organizer that runs along the A/P boundary (i.e. reduction in size and loss of intervein regions). Interestingly, coexpression of cv and sog along the A/P border affects structures throughout the wing, whereas expression of these proteins in the posterior compartment affects only posterior structures. The asymmetric activity of cv+sog suggests a restricted mobility due either to local inhibition of a long-range signal such as Dpp or Gbb or to the existence of an asymmetric inhibitor of diffusion of Cv/Sog-containing complexes (Vilmos, 2005).

It has been postulated that Cv might synergize with Cv-2, a second Sog-like protein; however, when Cv-2 is coexpressed with either Cv or Tsg, no evidence is seen of the strong inhibition of BMP signalling observed when Cv or Tsg is coexpressed with Sog. On further underscoring a difference between Sog and Cv-2, both Cv and Sog mutants show similar effects on wing veins (loss of crossveins and expanded vein tips), whereas Cv-2 mutants show loss of vein tips as well as crossveins. Thus, Cv-2 is not interchangeable with Sog (Vilmos, 2005).

To evaluate ligand specificity, the ability was tested of Cv and Sog to suppress the wing disruptions caused by overexpressing Dpp and Gbb. Expression of cv, tsg or sog alone has no detectable effect on the overexpression of dpp. However, when expressed together with sog, cv and tsg behave fairly differently when challenged with excess Dpp. Although tsg with sog can rescue the effect of excess dpp, cv with sog does not suppress the dpp overexpression phenotype at all. Cv and Tsg also differ in their effect on Gbb overexpression. Although Cv or Sog alone has no effect on Gbb overexpression, Cv together with Sog re-establishes the intervein tissue and the longitudinal veins with a series of expanded crossveins between L2 and L3. In contrast, Tsg alone suppresses fairly effectively the excess Gbb effect, whereas Tsg together with Sog leads to an intermediate level of rescue. These observations indicate that Cv and Tsg have distinct activities with respect to the Dpp and Gbb ligands and also different requirements for Sog to inhibit BMP (Vilmos, 2005).

Shaping BMP Morphogen Gradients through Enzyme-Substrate Interactions

Bone morphogenetic proteins (BMPs) regulate dorsal/ventral (D/V) patterning across the animal kingdom; however, the biochemical properties of certain pathway components can vary according to species-specific developmental requirements. For example, Tolloid (Tld)-like metalloproteases cleave vertebrate BMP-binding proteins called Chordins constitutively, while the Drosophila Chordin ortholog, Short gastrulation (Sog), is only cleaved efficiently when bound to BMPs. This study identified Sog characteristics responsible for making its cleavage dependent on BMP binding. 'Chordin-like' variants that are processed independently of BMPs changed the steep BMP gradient found in Drosophila embryos to a shallower profile, analogous to that observed in some vertebrate embryos. This change ultimately affected cell fate allocation and tissue size and resulted in increased variability of patterning. Thus, the acquisition of BMP-dependent Sog processing during evolution appears to facilitate long-range ligand diffusion and formation of a robust morphogen gradient, enabling the bistable BMP signaling outputs required for early Drosophila patterning (Peluso, 2011).

To identify and characterize the Tld processing sites in Sog, the Sog cleavage fragments were purified and sequenced using tagged proteins generated in S2 insect cell cultures. The intermediate Sog cleavage fragments were captured using suboptimal amounts of enzyme and Dpp, the obligatory cosubstrate. The three major processing sites in Sog are in close proximity to the Cys-rich BMP binding modules. The positions of processing sites 1 and 3 correspond to the two major processing sites in Chordin. Sequencing of the N-termini revealed a conserved Asp residue at position P10, a hallmark of the astacin family of proteases that includes Tld, a conserved aliphatic residue (V) in position P3, and no significant homology with other Tld/BMP-1 substrates (Peluso, 2011).

Replacement of all four residues at processing site 1 (V183ALD) with Ala rendered Sog virtually uncleavable at this site in vitro. Full-length mutant Sog was still degraded over time, likely due to processing at the remaining unmodified sites, but the speed of its degradation was reduced. Additional Ala replacements at site 2 (V728PGD) further slowed down the Sog destruction. When only the conserved D (position P10) was replaced with E at processing site 1 were similar effects observed: undetectable cleavage at site 1 and a slower overall degradation of the mutant Sog. In this system, the precise cleavage kinetics at individual sites could not be observed, but the overall Sog destruction in various uncleavable Sog mutants was clearly slowed down, likely because of blocked/reduced cleavage at the modified site(s). Also any mutations at site 3 induced constitutive cleavage at this site, thus site 3 was kept intact for these studies (Peluso, 2011).

To test if these mutations (Sog-u) could render Sog uncleavable in vivo, Sog gain-of-function phenotypes were examined. Overexpression of Sog in the wing imaginal disc produces very mild phenotypes of venation defects. Sog together with Tsg produces a more potent BMP inhibitor; their combined overexpression inhibits BMP signaling and results in smaller wings with altered patterns of venation. Co-overexpression of Sog and Tsg with Tolloid-related (Tlr) is able to reverse the small wing phenotype and restore normal patterning in the case of wildtype Sog, but not in the case of Sog-u. In fact, overexpression of Sog-u by itself produced a significant loss of posterior crossvein, a structure that requires peak BMP signaling, suggesting that Sog-u is a better BMP inhibitor than the wild-type Sog. Moreover, the loss of posterior crossvein tissue was exacerbated when Tlr was coexpressed with Sog-u, indicating further reduction in the BMP activity. This is likely due to Tlr degrading endogenous Sog but not Sog-u. Thus, Sog-u appears resistant to cleavage and degradation in vivo and may act as a dominant negative by prolonged binding of the BMP ligands (Peluso, 2011).

Unlike in vertebrates, Drosophila Tld and Tlr process Sog only when bound to a BMP-type ligand. The binding of Sog to Tld requires several Tld protein interaction motifs besides the protease domain. Nevertheless, the requirement for the obligatory cosubstrate for Sog processing is thought to indicate a BMP-induced conformational modification that allows the Sog-BMP complex, but not Sog alone to fit into the catalytic pocket of the enzyme. In contrast, Chordin, which exhibits BMP-independent processing, should bind and fit into Tld's catalytic pocket without the need for a BMP-induced conformational change. Indeed, in spite of limited conservation between Sog and Chordin (40% similarity, 22% identity), it was found that Drosophila Tld can cleave the vertebrate Chordin in a BMP-independent manner (Peluso, 2011).

To focus on the enzyme-substrate interactions for Sog and Chordin, the Tld catalytic domain was modeled using the crystal structures available for related enzymes, the crayfish Astacin (the founder member of this zinc metalloprotease family), and the human Tld catalytic domain. As previously described, the catalytic pocket of Tld enzymes appears very tight in the proximity of the catalytic Zn, where scissile bonds align, and has a relatively wide cavity that accommodates residues P3 and P2 in the substrate. Bulky, hydrophobic residues in the substrate, such as in Chordin, might facilitate enzyme-substrate binding. Indeed, when the effect of changing processing site 1 was examined in Sog to either Chordin site 1 (Sog-1Ch1) or Chordin site 2 (Sog-1Ch2) indications were found of Tld cleavage at these sites in the absence of Dpp, although this cleavage was extremely weak. In addition, an aromatic residue in position P3 in the substrate could potentially stack against the aromatic ring, a key position near the active site, to further lower the substrate-enzyme binding energy and facilitate substrate binding (Peluso, 2011).

These predictions were tested and found that indeed changing several residues at the processing site could alter the cosubstrate requirements. For example, Sog processing at mutated site 1 (V183ALDV to FYGDP) occurred independently of the cosubstrate (Peluso, 2011).

This processing was enhanced when Dpp was added to the reaction partly because cleavage at unmodified sites 2 and 3 could not happen in the absence of the cosubstrate. Addition of Tsg similarly enhanced the processing of wild-type and mutant Sog. Nonetheless, when Dpp was in limiting amounts, the mutant Sog was processed more efficiently than the wild-type protein. Edman degradation confirmed that processing occurred at the expected G185-D covalent bond in the mutated site, and that mutagenesis did not create any promiscuous cleavage. Similar changes at processing site 2, separately or with site 1, further enhanced the speed of Sog degradation. The strongest effect was seen for a Sog variant in which both sites 1 and 2 were rendered BMP-independent for processing, designated Sog-i for 'independent of BMP for cleavage.' At the sequence level Sog-i is very different from Chordin, but it resembles Chordin in how it is processed by Tld: Sog-i exhibits significant BMP-independent processing by Tld, which is enhanced in the presence of BMP ligands. To emphasize these similarities Sog-i is referred as 'Chordinlike' Sog (Peluso, 2011).

Tests were performed to see if these changes impact Sog's ability to bind BMPs and/or inhibit their signaling. Purified Sog and Sog-i were found to be indistinguishable in their binding to Dpp homodimers and Dpp/Scw heterodimers in co-ip experiments. Also, in both cases, addition of Tsg equally increased Sog binding to the BMPs. The inhibitory activities of Sog and Sog-i on BMP signaling were compared in a cell-based assay; in the presence of Tsg, Sog inhibits Dpp-induced signaling in a concentration dependent manner. Equivalent amounts of Sog-i and Tsg produced a similar inhibitory response (Peluso, 2011).

While the BMP binding properties of Sog-i appeared to be largely unaffected, it was predicted that this 'Chordin-like' Sog would resemble Chordin when introduced into fly embryos, and be less efficient in promoting long-range BMP signaling. To model this process, a previously published spatiotemporal patterning model was modified by adding the BMP-independent processing of Sog. Briefly, the rate for Tld-mediated processing of Sog increases when the rate of BMP-dependent or BMP-independent cleavage increases. An increase in the rate of Tld processing will modify the Sog protein levels and the shapes of the Sog and Sog/Tsg distributions in the model. This results in a simultaneous reduction in the inhibition of Dpp signaling laterally and a reduction in the Dpp accumulation near the dorsal midline. To quantify the effect of BMP-independent cleavage of Sog-i on the net transport of BMP molecules toward the dorsal midline, the net diffusive flux of BMP ligand in the embryo was calculated by summing the contributions of free BMP and Sog-bound BMP. The flux provides the magnitude and direction of transport driven by the gradient of concentration. The Sog-i simulation clearly indicated a lower net transport toward the midline than the simulation with Sog-WT. Also investigated in this model was whether increased Sog-i expression could improve the transport toward the midline and whether the reduction in transport is solely the result of a reduction in Sog levels. Increasing the production of Sog-i increases the total amount of Sog-i in the system; however, transport of Dpp/Scw toward the midline is still reduced even with significantly increased levels of Sog-i greater than in Sog-WT embryos with normal patterning (Peluso, 2011).

To test the biological effect of these Sog variants on the BMP morphogen gradient profile, transgenic fly lines were constructed that allowed for normal spatial and temporal expression of Sog proteins at endogenous levels. The neural-ectoderm expression of tagged and nontagged Sog proteins, Sog-WT, Sog-WT-HA, and Sog-i-HA, in all of the transgenic lines obtained, overlapped the sog mRNA endogenous pattern. The relative Sog levels in these transgenic lines were quantified by immunofluorescence using anti-HA antibodies, anti-Sog antibodies, or both. It was found that indeed these transgenic lines have similar levels of Sog protein. In addition, all of the transgenic lines expressing Sog-WT (either HA-tagged or not-tagged) rescued the sog^YL26 mutants and trans-heterozygous combinations (sog-/-) to viable, and fertile adults (Peluso, 2011).

The profile of the BMP morphogen gradient was examined in stage 5 embryos by following the accumulation of activated/ phosphorylated Mad (P-Mad), the effector of the BMP signaling pathway. In the absence of Sog, the facilitated diffusion of BMP ligands does not occur and Dpp remains uniformly distributed over the dorsal domain. No gradient of BMP activity is generated, thus the P-Mad levels are low and constant over the entire dorsal domain of sog-/- mutant embryos. In contrast, stage 5 wild-type embryos have a sharp, step gradient of BMP signaling, in which P-Mad levels are high in the dorsal most cells and rapidly drop off to undetectable levels in more lateral regions. The P-Mad positive domain is wider and slightly reduced in intensity in heterozygous (sog+/-) embryos. Among the sog alleles tested, the sog^YL26/+ heterozygous embryos showed the widest P-Mad profile. The HA-tagged or untagged sog-WT transgenes were equally effective in restoring the sharp P-Mad profile in sog-/- embryos when in two copies, suggesting that the tag did not alter Sog activity. In contrast, addition of two sog-i copies to any sog-/- background produced a wide P-Mad positive domain with reduced signal intensities. In the latter embryos the boundaries of the P-Mad positive domains were more diffuse, with reduced slopes evident in the cross-section profile. Analogous studies of race expression, downstream of BMP signaling in the presumptive amnioserosa, indicated a similar effect. This suggests that Sog-i is indeed less efficient in supporting an adequate Dpp/Scw-Sog/Tsg flux toward the midline and consequently the formation of the steep BMP distribution profile (Peluso, 2011).

To quantify the differences in BMP signaling profiles between wild-type, sog+/-, and sog-/- embryos with 2x sog-i or 2x sog-WT transgenes, the P-Mad fluorescent staining of each embryo was decomposed into the product of an amplitude multiplied by the P-Mad distribution 'shape'. In brief, for each embryo, a region of interest was selected that encompasses a 4-cell-wide band centered at 33% embryo length. Each embryo was then processed through a Savitzky-Golay filter that removed noise while preserving the shape of the distribution and allowed for reliable calculation of the slope of the P-Mad profile (Peluso, 2011).

Shape was quantified by measuring the spatial-derivative of P-Mad in the cross-section. Starting on the left of a P-Mad cross-section plot, the derivative will be positive and change in magnitude at each position along the D/V axis directly proportional to the slope of P-Mad. As the slope decreases near the dorsal midline, the value of the derivative is approximately zero and then negative for the right side of the distribution where P-Mad is decreasing. The local average P-Mad slope for the population of 2x sog-WT embryos at each spatial location was virtually indistinguishable from the WT P-Mad slope. In contrast, the population average P-Mad slope for 2x sog-i embryos was noticeably shallower than WT and 2x sog-WT embryos with a lower magnitude of the spatial derivative near the midline and a higher magnitude in the lateral dorsal ectoderm. Moreover, differences in the overall intensity of P-Mad staining and/or in embryo-to-embryo variability do not account for this observation; even when the scaling for each population was chosen so the population means would have the same peak P-Mad levels, or when the absolute value of the local P-Mad slope was used for each individual in each population to calculate the population distributions of slopes, the differences remain clear: the replacement of sog-WT with sog-i leads to broader, shallower P-Mad profiles (Peluso, 2011).

The difference between the P-Mad profiles in WT and 2x sog-i was not equivalent to a decrease in the total amount of Sog protein in the system. Distributions of P-Mad slopes in both sog+/- and 2x sog-i differed from WT embryos, but the perturbations were not equivalent. For sog+/-, Sog protein levels were reduced approximately 50%, and the position of the peak slope shifted laterally away from the dorsal midline; however, the magnitude of the slope was still significantly greater than the magnitude of the slope for 2x sog-i embryos, though slightly less than WT. This means that the P-Mad profile in sog+/- is wider, but the steepness of the BMP activity gradient is similar to the steepness of the WT gradient. In contrast, the slope of the P-Mad profile in 2x sog-i embryos was significantly lower than WT embryos near where their peaks overlap and significantly greater than WT in the lateral dorsal ectoderm (Peluso, 2011).

Prior to gastrulation high levels of BMP signaling at the dorsal midline in early Drosophila embryos specify amnioserosa, an extraembryonic tissue required for gastrulation. The sharp and narrow BMP signaling domain in WT embryos induced formation of an amnioserosa field of approximately 200 cells in stage 13 embryos. The sog+/- heterozygous embryos have a wider BMP signaling domain that produced a larger amnioserosa field, about 50% bigger than that of the WT embryos. The spatial extent of the BMP signaling field above a certain threshold but below wild-type peak values appears to determine how many amnioserosa cells will be specified and consequently the size of the ensuing tissue (Peluso, 2011).

When sog-WT transgenes replaced the endogenous sog, the amnioserosa cell numbers were rescued to wild-type levels. However, addition of two sog-i copies to any sog-/- background tested produced statistically significant increases in the amnioserosa fields. The biological consequences of replacing Sog with Sog-i could not be explained simply by quantity differences between the sog-WT and sog-i transgenes. First, shallow BMP gradient profiles, broader target gene expression domains, along with increased cell allocation/ amnioserosa fields were observed using multiple independent sog-i transgenic lines with expression levels comparable with those of the sog-WT lines. Second, additional copies of sog-WT and sog-i transgenes did not significantly impact the BMP gradient profiles or amnioserosa fields. The P-Mad positive domains were more intense in either 4x groups, but in the sog-/-; 4x sog-i embryos the signaling domain remained wide, the boundaries diffuse and the slopes of the cross-section profiles reduced; also, the sog-/-; 4x sog-i embryos had significant embryo-to-embryo variability (Peluso, 2011).

To further search for alternative explanations for the Sog-i effects, Sog-WT and Sog-i versions of the 3D embryonic model were optimized against the 4x population data and it was asked whether the experimental observations could be captured by changes in Sog affinity to Dpp or changes in the processing rate of Sog by Tld independent of Dpp. It was found that the model with an enhanced processing rate achieved a greater fit: a modest increase in the processing of Sog by Tld without Dpp (increase BMP independent processing from about 8% to 19% of Tld processing rate in presence of Dpp), resulted in signaling profiles that matched the experimental data very well (Peluso, 2011).

In the 2x sog-WT simulations, the net reaction rate was negative in the lateral portions of the dorsal region and reached maximum near the dorsal midline. In the simulations for the 2x sog-i, the peak rate of Dpp release occurred laterally halfway between the neural-ectoderm and dorsal region. In contrast, no models obtained by decreasing the binding between Sog and Dpp (10x or more) could capture the experimentally observed loss of sharp boundaries. Thus, the shift in the net rate of cleavage, in conjunction with less effective net flux, produced less accumulation of Dpp near the dorsal midline in simulations of sog-i embryos (Peluso, 2011).

In conclusion, it was found that several residues at the Tld processing site make Sog dependent on a (BMP) cosubstrate for processing. Mutating these residues reduced the transport range of Sog-BMP complexes in vivo and altered the shape of the BMP signaling profiles and consequent cell fate allocation. Interestingly, BMP-dependent Chordin cleavage was also a requirement in mathematical modeling for scale invariance of Xenopus embryos. Here the cosubstrate requirement ensured transport of both ADMP and the BMP ligands and the reestablishment of a well-proportioned DV axis. How might shuttling of ligands persist in the absence of BMP-dependent cleavage of Chordin? An intriguing possibility is that Sizzled-mediated repression of Xolloid spatially restricts Chordin processing providing a nonuniform Chordin sink. In mathematical models of embryo patterning, lowering the processing rate of Tld results in signaling distributions that are sharper and result in a greater net transport of BMP ligands away from the Sog/Chordin source (Peluso, 2011).

An interesting and unexpected outcome of the comparison between sog-WT and sog-i embryos suggests that BMP-dependent Sog processing reduces embryo-to-embryo variability in P-Mad levels. Both sog-WT and sog-i embryos show sensitivity of P-Mad to gene dosage. However, when the coefficient of variation (standard deviation/width) within each genotype was calculated, it was found that embryos with one or two sog-WT copies showed less variability in signaling width than their 2x sog-i counterparts. The variability is greater at nearly all threshold positions, and the variability within this population increased dramatically at higher threshold levels. This suggests that BMP-dependent Sog destruction may reduce embryo-to-embryo variability between individuals in a population of the same genotype to provide robust patterning of the dorsal structures (Peluso, 2011).

Altogether, these results indicate that a 'Chordin-like' Sog is less able to reliably support patterning of the early Drosophila embryo. By modifying the Sog-Tld substrate-enzyme interaction with just a few residue changes, it appears that a new developmental function for Sog evolved that ensured reliable shuttling of BMPs and robust patterning. Further refinement of this shuttling mechanism, such as its speed or its directionality, expanded the repertoire of cell fate specification by BMP morphogen gradients and was likely exploited for diversified patterning during natural evolution (Peluso, 2011).

dHIP14-dependent palmitoylation promotes secretion of the BMP antagonist Sog

Analysis of diverse signaling systems has revealed that one important level of control is regulation of membrane trafficking of ligands and receptors. The activities of some ligands are also regulated by whether they are membrane bound or secreted. In Drosophila, several morphogenetic signals that play critical roles in development have been found to be subject to such regulation. For example, activity of the Hedgehog (Hh) is regulated by Raspberry, which palmitoylates Hh. Similarly, the palmitoylases Porcupine and Raspberry increase the activities of Wingless (Wg) and the EGF-ligand Spitz (Spi), respectively. In contrast to its vertebrate homologues, which have typical N-terminal signal sequences, the precursor form of Drosophila Hh contains an internal type-II secretory signal motif. The Short Gastrulation (Sog) protein is another secreted Drosophila protein that contains a type-II signal and differs from its vertebrate ortholog Chordin which contains a standard signal peptide. This study examined the regulation of Sog secretion and regulation by dHIP14, the ortholog of a mammalian palmitoylase first identified as Huntington Interacting Protein (HIP). It was shown that dHIP14 binds to Sog and Sog is palmitoylated. In S2 cells, dHIP14 promotes secretion of Sog as well as stabilizing a membrane associated form of Sog. The requirement was examineed for candidate cysteine residues in the N-terminal predicted cytoplasmic domain of Sog; Cys27, one of two adjacent cysteines (Cys27 and Cys28), is essential for the full activity of dHIP14 and its effect on Sog. Finally, dHIP14 was found to promote the activity of Sog in vivo. These studies highlight the growing importance of lipid modification in regulating signaling at the level of ligand production and localization (Kang, 2011).

Lipid modification of many ligands plays an important role in regulating their activity and range of action. In this study, evidence is provided that the type-II signal sequence of the BMP antagonist Sog is a target for palmitoylation by dHIP14. Reduction and gain-of-function studies in cell culture suggest this post-translational modification is important for secretion of Sog and for targeting intracellular Sog to a Triton-insoluble fraction, which may represent a membrane cargo compartment, possibly the Golgi, involved efflux of Sog. Mutational analysis suggests further that Cys27 in the cytoplasmic tail of the type-II signal sequence plays an important role in mediating dHIP14 activity. The extra secreted Sog from cells over-expressing dHIP14 is active as a BMP antagonist since supernatants from dHIP14 transfected cells are more effective than control supernatants in inhibiting BMP mediated pMAD phosphorylation. In vivo experiments also indicate that over-expression of dHIP14 mimics the effect of over-expression of Sog in the wing and, like Sog, can inhibit Gbb-dependent vein formation upstream of the BMP receptor during wing development. Localized ectopic expression of dHIP14 in the early embryo likewise phenocopies the effect of Sog by reducing pMAD activation along the dorsal midline. Cumulatively, these results suggest that palmitoylation increases secretion of an active form of Sog from cells, and may do so by elevating Sog flux through a membrane associated compartment (Kang, 2011).

The role that dHIP14 plays in increasing Sog secretion and its activity as a BMP antagonist at moderate-to-long range is similar to the positive regulatory role of palmitoylation on Wg or Hh secretion and activity, rather than to the negative effect this post-translational modification has on secretion of the EGF-R ligand, Spi. In the case of Spi, however, palmitoylation does increase the short range activity of Spi, presumably by concentrating on the cell surface, thereby increasing its likelihood of engaging EGF-R receptors on neighboring cells. Thus in all cases, palmitoylation plays a role in promoting ligand activity, albeit at differing ranges (Kang, 2011).

Another effect of dHIP14, which may also play a role in increasing secretion of Sog, is that it results in a stable Triton-insoluble form of Sog. This stable intracellular form of Sog may represent a membrane-bound intermediate within the secretory pathway or possibly also a pool of membrane tethered Sog at the cell surface. When palmitoylation is blocked pharmacologically or cells are treated with dHIP14 RNAi, the proportion of Triton-soluble Sog increases. This soluble fraction of Sog is unstable and its degradation is proteasome-dependent, although this effect may be indirect since soluble Sog would be expected to lie within a vesicular compartment where it would not come into direct contact with the cytoplasmically localized proteasome machinery. Since the ability of Sog to co-localize with dHIP14 in the Golgi compartment is compromised by the C27S mutation, one model to account for the data would be that palmitoylation is required to integrate Sog into the Golgi membrane thereby protecting it from degradation prior to reaching the cell surface, where it may be rapidly cleaved and liberated into the extracellular space. Possible role of palmitoylation on regulating the activity of distinct forms of Sog

Although this study focused on analyzing the effect of dHIP14 on full length Sog, and found that it played a role increasing its secretion, palmitoylases can also limit the range of action of other ligands such as Spi. In this latter case, palmitoylation functions instead to retain the ligand on the cell surface, thereby increasing its local concentration. It is interesting to consider whether such an alternative potential role of palmitoylation might differentially regulate the activity of shorter processed forms of Sog that have been observed to be produced during several stages of development including oogenesis, embryogenesis, and pupal wing development. Evidence obtained using antibodies raised to different portions of Sog and from genetic clonal analysis indicates that different forms of Sog diffuse to differing extents within the follicle cell epithelium during oogenesis, and potentially also in the wing epithelium where Sog diffuses from intervein cells into broad provein territories during the vein refinement process (Kang, 2011).

The most well characterized truncated forms of Sog, which contain only the first of four cysteine repeat (CR) domains plus some adjacent stem sequences, are referred as Supersog molecules since they have the ability to inhibit signaling mediated by Dpp:Dpp homodimers as well as by Dpp:Scw or Dpp:Gbb heterodimers. In contrast, full length Sog (with all four CR domains intact) only blocks signaling mediated by BMP heterodimers. Supersog fragments may be generated at least in part by alternative cleavage by the Tld protease, which can also cleave Sog immediately after the CR1 to inactivate it. In addition, a C-terminal fragment of Sog including the CR2-CR4 domains, which is complementary to Supersog has a mild BMP activating function. It has been anecdotally noted that the shorter Supersog fragments are not as readily secreted from cells in culture as full length Sog. There is also evidence that N-terminal fragments of Sog have reduced mobility in vivo in comparison to full length or C-terminal fragments during oogenesis, and clonal analysis of sog and integrin gene function during vein formation suggest that different forms of Sog have similar differing mobilities in the pupal wing. While further experimental analysis will be required to resolve this question, it is interesting to speculate that if Supersog-like molecules indeed have a greater tendency to remain tethered to the surface of lateral neuroectodermal cells in which sog is expressed, they might be able to more effectively inhibit the activity of Dpp:Dpp homodimers, which are thought to diffuse in from the adjacent dorsal epidermal ectoderm. This localized deployment of Supersog-like fragments would help explain how Sog acts as potently as it does to prevent BMP autoactivation within the neuroectoderm given that secreted full length Sog would only be able to block the activity of incoming Dpp:Scw heterodimers. The restriction of such Dpp:Dpp inhibiting forms of Sog to the neuroectoderm would also account for why Sog acts selectively to block peak BMP signaling mediated by Dpp:Scw heterodimers in the epidermal ectoderm (Kang, 2011).

If truncated forms of Sog are less diffusible than full length or C-terminal Sog fragments, then another question would be how palmitoylation might contribute to this difference. Perhaps there are two competing pathways for processing palmitoylated forms Sog at the cell surface, one which results in cleavage after the TM domain, thereby generating freely diffusible full length Sog, and another which results in Sog cleavage (by Tld or other proteases) some distance after CR1 to generate a tethered Supersog form and, potentially, a freely diffusible BMP promoting C-terminal fragment. Additional studies will be needed to address these various possibilities (Kang, 2011).

Multistep molecular mechanism for bone morphogenetic protein extracellular transport in the Drosophila embryo

In the Drosophila embryo, formation of a bone morphogenetic protein (BMP) morphogen gradient requires transport of a heterodimer of the BMPs Decapentaplegic (Dpp) and Screw (Scw) in a protein shuttling complex. Although the core components of the shuttling complex--Short Gastrulation (Sog) and Twisted Gastrulation (Tsg)--have been identified, key aspects of this shuttling system remain mechanistically unresolved. Recently, it was discovered that the extracellular matrix protein collagen IV is important for BMP gradient formation. This study formulates a molecular mechanism of BMP shuttling that is catalyzed by collagen IV. Dpp is shown to be the only BMP ligand in Drosophila that binds collagen IV. A collagen IV binding-deficient Dpp mutant signals at longer range in vivo, indicating that collagen IV functions to immobilize free Dpp in the embryo. In vivo evidence is provided that collagen IV functions as a scaffold to promote shuttling complex assembly in a multistep process. After binding of Dpp/Scw and Sog to collagen IV, protein interactions are remodeled, generating an intermediate complex in which Dpp/Scw-Sog is poised for release by Tsg through specific disruption of a collagen IV-Sog interaction. Because all components are evolutionarily conserved, it is proposed that regulation of BMP shuttling and immobilization through extracellular matrix interactions is widely used, both during development and in tissue homeostasis, to achieve a precise extracellular BMP distribution (Sawala, 2012).

There is ample experimental and theoretical support for the notion that BMP gradient formation in the early embryo involves the concentration of the most potent signaling species, the Dpp/Scw heterodimer, at the dorsal midline in a process involving Sog and Tsg. This study presents in vivo evidence for a role of collagen IV in two key aspects of this shuttling model, which have remained mechanistically unresolved. First, collagen IV functions to immobilize free Dpp, explaining why Sog and Tsg are needed for Dpp movement. Second, collagen IV acts as a scaffold for assembly of the Dpp/Scw-Sog-Tsg shuttling complex. The advantage to BMP gradient formation of assembling the shuttling complex on collagen IV has been suggested by analysis of organism-scale mathematical models. These models reveal that the in vitro binding affinity between BMPs and Sog is too low to account for the rate of shuttling complex formation required in vivo. However, by acting as a scaffold, collagen IV would increase complex formation by locally concentrating Dpp/Scw and Sog. Models with a 10–20% reduction in diffusion rates for Dpp/Scw and Sog and an increased apparent affinity of Dpp/Scw for Sog, show the best fit to in vivo data (Sawala, 2012).

The molecular model of shuttling complex assembly occurs in three steps. The first step involves independent binding of Dpp/Scw and Sog to collagen IV. The ability of Dpp-Δa to signal long range in sog⁻ embryos, where wild-type Dpp is trapped in its expression stripe, provides in vivo evidence that the Dpp-collagen IV interaction restricts movement of free Dpp ligands. The result also demonstrates that Sog and Tsg promote long-range movement of Dpp because they release Dpp from collagen IV, and not simply because they prevent Dpp–receptor interactions. Restriction of Dpp diffusion by collagen IV may stabilize the gradient by preventing ventral movement of Dpp/Scw after release from Sog/Tsg and promoting Dpp/Scw–receptor interactions at the dorsal midline. It will be interesting, ultimately, to directly visualize Dpp and Dpp-Δa directly in sog and tsg mutant embryos. Although current methods allow detection of high levels of receptor-bound Dpp, there are technical limitations associated with specifically detecting the pools of Dpp that would be informative here, i.e., Dpp/Scw heterodimer within the shuttling complex or Dpp-Δa/Scw diffusing between cells. The data show that Scw is unable to bind the NC1 domain of collagen IV. This lack of collagen IV-dependent immobilization can explain why Scw, unlike Dpp, is capable of long-range signaling in the absence of Sog (Sawala, 2012).

Step 2 of shuttling complex assembly involves remodeling of the protein interactions to generate a poised intermediate. Specifically, step 2 is driven by Scw-mediated disruption of the Sog CR4–collagen IV interaction, so that Dpp/Scw is transferred from collagen IV to the Sog CR3-CR4 domains. Scw displacement of the Sog CR4 domain from collagen IV provides molecular insight as to why Scw is needed for Dpp transport. In addition to the binding preference of Sog and Tsg for the Dpp/Scw heterodimer, only Scw has a high affinity for the Sog CR4 domain. Therefore, Dpp/Scw can be released from collagen IV into the shuttling complex, whereas the Dpp homodimer remains trapped on collagen IV (Sawala, 2012).

In the final step of the model, Tsg mobilizes the shuttling complex by disrupting the Sog CR1–collagen IV interaction. It has been noted that tsg mutants display a more severe reduction in BMP signaling than sog and sog tsg double mutants. This observation has been attributed to a potential Sog-independent pro-BMP activity of Tsg at the level of receptor binding. A second contributing factor is suggested by the model, where Sog and Tsg act at distinct steps to allow formation of the shuttling complex. In tsg mutants, Dpp/Scw is loaded onto Sog by collagen IV, but remains locked in this inhibitory poised complex, so that the only BMPs capable of signaling are Dpp and Scw homodimers, which are less potent than the Dpp/Scw heterodimer. By contrast, in sog or sog tsg mutants, Dpp/Scw is not shuttled dorsally but is still capable of signaling locally, adding to signaling by Dpp and Scw homodimers. The weaker level of Dpp/Scw signaling in tsg mutants also provides support for the proposed order of steps 2 and 3 in the assembly process, because this order gives rise to the inhibitory intermediate of Dpp/Scw-Sog. Previously it was shown that an N-terminal fragment of Sog, called Supersog, which contains the CR1 domain and a portion of the stem, can partially rescue the loss of peak Dpp/Scw signaling in tsg⁻ embryos. The model suggests that this property of Supersog comes from the ability of its CR1 domain to compete with full-length Sog for binding to collagen IV, thereby releasing Sog-Dpp/Scw, similar to the role of Tsg in shuttling complex assembly. It is noted that the CR1–collagen IV interaction appears weaker than that of CR4–collagen IV, which may facilitate release of Dpp/Scw by Tsg or Supersog-like fragments. After Tsg-mediated release from collagen IV, the mobile shuttling complex can diffuse randomly. Upon Tolloid cleavage of Sog, the liberated Dpp/Scw heterodimer rebinds collagen IV, which either promotes receptor binding or a further round of shuttling complex assembly, depending on the local concentration of Sog (Sawala, 2012).

In addition to collagen IV, the basic region in Dpp/BMP2/4 also binds to heparan sulfate proteoglycans (HSPGs), which can either restrict or enhance BMP long-range movement. Indeed, this study found that an HSPG-binding mutant, Dpp-ΔN, also binds only weakly to collagen IV, suggesting that the collagen IV- and HSPG-binding sites on Dpp overlap. It will be interesting to test how HSPGs and collagen IV interact to regulate BMP activity in tissues where they are coexpressed, such as the early vertebrate embryo. In the early Drosophila embryo, the absence of glycosaminoglycan chains, which largely mediate binding of HSPG to Dpp, make it possible to specifically focus on the Dpp–collagen IV interaction (Sawala, 2012).

A shuttling-based mechanism of BMP transport is also used in a number of other developmental contexts, including the early vertebrate embryo, specification of the vertebral field in mice, and establishment of the posterior cross-vein territory in the Drosophila wing disk. Restriction of BMP movement may also be important in other contexts, including several where collagen IV was already shown to regulate a short-range Dpp signal, such as the ovarian stem cell niche and the tip of malpighian tubules. The basic collagen IV binding motif is highly conserved among the Dpp/BMP2/4 subfamily and is also found in some other BMPs, including BMP3, consistent with reports that BMP3 and BMP4 can bind collagen IV. Overall, these findings support the idea that the collagen IV–BMP interaction is a conserved aspect of extracellular BMP regulation and suggest that the function of collagen IV in both long-range BMP shuttling and local restriction of BMP movement will impact on a number of other contexts in both flies and vertebrates (Sawala, 2012).

Post-transcriptional Regulation

Lack of abstrakt function does not compromise the ability of cells to undergo their differentiation program or cellular behaviour typical of their differentiated state. Thus, neurite outgrowth still occurs, and pole cells are able to migrate over large distances, but both types of cells appear to fail to recognize or react to the cues that direct their morphogenetic behavior in the spatially appropriate manner. One reason for this defect might be a loss of subcellular order or polarity. Various aspects of cell polarity were indeed affected in abs mutants. The first abnormality detected was in the localization of mRNAs in the blastoderm. In the wild-type blastoderm, the mRNAs of several genes are distributed unevenly in the cell. For example, the transcripts of short gastrulation (sog) and crumbs (crb) are tightly apposed to the apical cell surface. In abs^14B mutants, CRB mRNA is not seen apically but predominantly at the level of the nuclei, while SOG mRNA forms a gradient from the apical towards the basal side of the cell. In the case of crb, the mislocalization of RNA is also seen at later stages and in other tissues. For example, the tight apical localization in the hindgut epithelium is disturbed in abs mutants. Remarkably, the Crumbs protein is synthesized properly and targeted to the apical cell surface, showing that apical-basal cell polarity in the epithelial cells is correctly established and maintained in abs mutants (Irion, 1999).

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