Gene name - serpent
Synonyms - dGATAb
Cytological map position - 89B1--B3
Function - transcription factor
Symbol - srp
Genetic map position - 3-58
Classification - zinc finger
Cellular location - nuclear
|Recent literature||Yue, Y., Yang, Y., Dai, L., Cao, G., Chen, R., Hong, W., Liu, B., Shi, Y., Meng, Y., Shi, F., Xiao, M. and Jin, Y. (2015). Long-range RNA pairings contribute to mutually exclusive splicing. RNA [Epub ahead of print]. PubMed ID: 26554032
Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down's syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. This study reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. These findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA-RNA interactions in gene regulatory networks.
There are three GATA homologs in Drosophila: Serpent (dGATAb), found in the midgut and ovary; Pannier (dGATA-a), found in the dorsal epidermis, and dGATAc, found in the procephalic region, the posterior spiracles, the gut, and the central nervous system.
Serpent has a pivotal role in promoting morphogenesis of the anterior and posterior midgut, although it does not set the borders of early tissue movements during gastrulation. Very early in development it acts like an effector gene, responsive to inductive actions of the terminal system, specifically huckebein. In this way serpent is induced in the gut through the action of Huckebein. serpent is also expressed in the fat body and in ovaries where it regulates yolk proteins (Abel, 1993).
The fat body is a loose sheet of cells located between the gut and somatic musculature. It has a segmental origin; its organization is dependent upon the activity of genes of the bithorax complex. serpent is the earliest known gene to be expressed in the fat body; serpent targets genes expressed later, like Alcohol dehydrogenase ( Adh). The fat body is mesodermal in origin, but is derived from cells deeper in the mesoderm than myogenic cells identified by their expression of nautilus (Abel, 1993).
serpent expression is seen in a patch of cells in the anterior portion of the mesodermal primordium. These cells invaginate with the portion of the ventral furrow that is anterior to the cephalic furrow. Slightly later these cells differentiate into prohemocytes and subsequently, become distributed throughout the body and differentiate as mature hemocytes. It is proposed that the mesodermal patch of srp constitutes the hemocyte primordium at blastoderm stage. These findings imply involvement of GATA in blood cell development and in endodermal differentiation. Such an involvement reflects an early phenomenon of metazoan evolution and may be common to most higher animals (Rehorn, 1997).
The somatic muscles, the heart, the fat body, the somatic part of the gonad and most of the visceral muscles are derived from a series of segmentally repeated primordia in the Drosophila mesoderm. The early development of the fat body is described and its relationship to the gonadal mesoderm, as well as the genetic control of the development of these tissues. The first sign of fat body development is the expression of serpent in segmentally repeated clusters within the trunk mesoderm in parasegments 4-9. Segmentation and dorsoventral patterning genes define three regions in each parasegment in which fat body precursors can develop. The primary and secondary dorsolateral fat body primordia are formed ventral to the visceral muscle primoridium in each parasegment. The ventral secondary cluster forms more ventrally in the posterior portion of each parasegment. Fat body progenitors in these regions are specified by different genetic pathways. Two dorsolateral regions require engrailed and hedgehog (within the even-skipped domain) for their development while the ventral secondary cluster is controlled by wingless. Ubiquitous mesodermal en expression leads to an expansion of the primary clusters into the sloppy-paired domain, resulting in a continuous band of serpent-expressing cells in parasegments 4-9. The observed effect of en on fat body development is seen not only on mesodermal overexpression but also when en is overexpressed in the ectoderm. Loss of wingless leads to an expansion of the dorsolateral fat body primordium. decapentaplegic and one or more unknown genes determine the dorsoventral extent of these regions. High levels of Dpp repress serpent, resulting in the formation of visceral musculature, an alternative cell fate (Reichmann, 1998).
In each of the parasegments from 10 to12, one of these primary dorsolateral regions generates somatic gonadal precursors instead of generating the fat body. The balance between fat body and somatic gonadal fate in these serially homologous cell clusters is controlled by at least five genes. A model is suggested in which tinman, engrailed and wingless are necessary to permit somatic gonadal develoment, while serpent counteracts the effects of these genes and promotes fat body development. In wg mutant embryos, all dorsolateral mesodermal cells, including those in parasegments 10-12, acquire fat body fate. This phenotype can be interpreted as the combined effects of two separate functions of wg: (1) wg is necessary to repress fat body development in the dorsolateral mesoderm underlying the wg domain in all parasegments; (2) wg is required in the primary cluster to permit somatic gonadal precursor instead of fat body development in parasegments 10-12. Loss of engrailed results in the absence of demonstrable somatic gonadal precursors, similar to the situation in tinman mutants. Ubiquitous mesodermal en expression leads to the formation of additional somatic gonadal precursor cells in parasegments 10-12. The homeotic gene abdominalA limits the region of serpent activity by interfering in a mutually repressive feed back loop between gonadal and fat body development. It is unlikely that abdA represses srp directly, since srp can be expressed in cells in which abdA is active. abdA might prevent srp from inhibition of a somatic gonadal precursor competence factor (Riechmann, 1998).
serpent is necessary for embryonic fat-cell differentiation in Drosophila and has been proposed to function in a cell-fate choice between fat cell and somatic gonadal precursors. Deregulated expression of serpent in the mesoderm induces the formation of ectopic fat cells and prevents the migration and coalescence of the somatic gonadal precursors. The ectopic fat cells do not arise from hyperproliferation of the primary fat-cell clusters but they do associate with the endogenous fat cells to form a fat body that is expanded in both the dorsal/ventral and anterior/posterior axes. Misexpression of serpent also affects the differentiation of muscle cells. Few body-wall muscle precursors are specified and there is a loss of most body-wall muscle fibers. The precursors of the visceral mesoderm are also absent and concomitantly the visceral muscle is absent. It is suggested that the ectopic fat cells might originate from cells that have the potential, but do not normally, differentiate into fat cells or from cells that have acquired a fat-cell fate (Hayes, 2001).
To test whether srp is sufficient to induce fat-cell development, the GAL4 targeted expression system was employed. twist-GAL4 and UAS-srp constructs were used to express srp throughout the mesoderm beginning at gastrulation. In stage 10-11 embryos, srp expression is readily detected in the primary fat-cell clusters in PS 4-9 and in the secondary fat-cell clusters in PS 10-12. In twist-GAL4;UAS-srp embryos, srp expression is expanded into a continuous band of cells throughout the mesoderm. Several molecular cell markers were used to monitor the effects of ectopic srp expression on the segregation of mesodermal derivatives. Two genes, alcohol dehydrogenase (Adh) and Drosophila collagen gene 1 (DCg1) were used to detect the fat body. Both Adh and DCg1 are embryonic terminal fat-cell differentiation markers. Adh expression is first detected during embryogenesis at stage 14 in the anterior wall of the midgut and the atrium of the posterior spiracle, and in the late precursor fat cells at stage 15. By stage 16, high levels of Adh transcripts are detected in the lateral fat body, and lower levels are found in the dorsal fat-cell projections. Within the developing fat cells, the temporal expression pattern of DCg1 is similar to Adh although DCg1 transcripts are easily detected at equivalent levels in both the dorsal fat-cell projections and the lateral fat body by stage 16. DCg1 is also expressed in the hemocytes. In twist-GAL4;UAS-srp embryos, the premature expression of srp throughout the mesoderm does not induce premature expression of Adh. As in wild-type embryos, Adh transcripts are first detected in the fat cells of experimental embryos at stage 15. The Adh-positive fat cells however, occupied most of the lateral mesoderm and formed an expanded lateral fat body (Hayes, 2001).
Because srp is a direct transcriptional activator of the Adh promoter in transient transfection assays, the increase in Adh-expressing cells might be the result of ectopic transactivation of Adh by srp rather than a reflection of the activation of the entire fat-cell genetic program. To test this possibility, the expression pattern of a second fat-cell marker, DCg1, was examined. In embryos carrying twist-GAL4;UAS-srp, the temporal pattern of DCg1 expression was also normal. DCg1 transcripts were first detected in hemocytes and then in fat cells at stage 15. The number of DCg1-expressing hemocytes was comparable to that of wild type. However, similar to the results obtained with Adh, there was an increase in the number of DCg1-expressing fat cells. The DCg1-expressing fat cells formed an expanded lateral fat body and dorsal fat-cell projections were expanded and fused with the lateral fat body in the posterior region of the embryo. An increase in lateral fat body cells was also detected in experimental embryos using a third fat-cell marker, the imaginal disc growth factor 3 gene, IDGF3. Similar results, albeit less extreme, were obtained using a second mesodermal GAL4 driver, 24B, that is first active in the presumptive mesoderm. These data demonstrate that ectopic expression of srp in the mesoderm leads to an increase in cells composing the lateral fat body and dorsal fat-cell projections (Hayes, 2001).
To determine whether mesoderm-specific components are necessary for srp to promote fat-cell development, srp was ectopically expressed in the ectoderm starting at stage 9 using the 68B-GAL4 driver. Ectopic expression of srp in the ectoderm is not sufficient to induce either Adh or DCg1 in the ectoderm, nor does it alter the normal development or morphology of the lateral fat body and dorsal fat-cell projections. These data suggest that srp requires mesodermal factors for induction of fat cells (Hayes, 2001).
At least two distinct mechanisms could account for the expansion of the fat body in the twist-GAL4;UAS-srp embryos: (1) the misexpression of srp could alter cell fates within the mesoderm by recruiting cells into the fat-cell developmental pathway; (2) premature and ectopic expression of srp in the mesoderm could cause hyperproliferation of endogenous precursor fat cells. To distinguish between these possibilities, the P-element enhancer-trap line, P[29D], was employed as a lacZ reporter gene for the primary fat-cell clusters in PS 4-9 and in the secondary fat-cell clusters in PS 10-12. Early expression of P[29D] is independent of srp. By marking precursor fat cell with the P[29D] lacZ reporter, a test could be made of whether expression of srp throughout the mesoderm causes hyperproliferation of endogenous fat cells. It was found that lacZ expression is not significantly altered in twist-GAL4;UAS-srp embryos. Specifically, the number and organization of the fat cells marked by P[29D] appear normal. Thus, the increase in fat cells is likely not to be due to hyperproliferation of endogenous fat-cell lineage although the possibility that the ectopic fat cells originate from the secondary fat-cell clusters not marked by P[29D] could not be eliminated (Hayes, 2001).
The forced expression of srp in the mesoderm results in the production of ectopic fat cells. Because it is not likely that the endogenous fat-cell lineage undergoes hyperproliferation, it is suggested that srp might be capable of inducing fat-cell development in cells that normally would not contribute to the fat body. Such a capability has been proposed for srp in the developmental choice model between somatic gonadal precursors (SGPs) and precursor fat cells. If srp activity can direct a fat-cell fate upon a common precursor that otherwise would be specified as SGPs, then it follows that ectopic expression of srp should repress the specification of SGPs in PS 10-12 and these cells should be replaced by fat cells (Hayes, 2001).
To test for srps role in the repression of SGP specification, twist-GAL4;UAS-srp embryos were examined using 412 as a cell marker for the SGPs and the gonadal mesoderm. In wild-type stage-12 embryos, 412 is expressed in PS 10-12 in the SGPs and in cell clusters in PS 2-9 and 14. During germband retraction, expression of 412 declines but persists in the SGPs (PS 10-12). It is unclear which cell lineage(s) the PS 2-9 and PS 14 clusters of cells represent. However, by over-staining for 412 transcripts, late 412 expression can be detected in the dorsal-most cells of the lateral fat body. At stage 13, the SGPs migrate and coalesce to form the gonadal mesoderm cells that will eventually ensheath the germ cells. At stage 16, 412 transcripts are still detected in the gonadal mesoderm (Hayes, 2001).
In stage-12 twist-GAL4;UAS-srp embryos, the expression of 412 is similar to that observed in wild-type embryos. The SGPs are specified and initial association of pole cells with gonadal precursors appears normal. The first alteration in the development of the gonadal mesoderm is detected at stage 13 when the SGPs fail to migrate and coalesce. The number of 412-expressing cells gradually declines in the experimental embryos and the few remaining 412-expressing cells are dispersed in the posterior region of the embryo. Misexpression of srp in the mesoderm does not affect the formation of the SGPs, however it does disrupt the ability of the SGPs to migrate and coalesce into a gonad (Hayes, 2001).
To confirm the effects of srp on SGP and gonad formation, twist-GAL4;UAS-srp embryos were examined for expression of a second gonadal mesoderm cell maker, clift (cli). cli is expressed throughout the mesoderm but by early stage 11 is lost in most mesodermal cells. During late stage 11, cli expression is detected in SGPs and in lateral muscle precursors, as well as in the ectoderm. Based on mutational analysis of a transcript-producing cli mutant, cliIIe, cli is necessary for maintenance of SGPs and their migration and coalescence into a gonad (Hayes, 2001).
In twist-GAL4;UAS-srp embryos, misexpression of srp does not affect early cli expression in the mesoderm. However, in older embryos, cli transcripts are not detected in either the SGPs or in the precursors of the lateral muscles although cli expression is still detected in the ectoderm. Because cli is necessary for the migration and coalescence of the SGPs, the inability of the SGPs to form a mature gonad is most likely due to the loss of cli expression in these cells. It is suggested that in the experimental embryos, srp does not prevent the specification of the SGPs but can block the differentiation of the SGPs by repressing cli expression in these cells (Hayes, 2001).
To test whether other lineages are affected by misexpression of srp, twist-GAL4;UAS-srp embryos were examined for heart and visceral muscle precursors. To mark heart precursors, the homeobox gene tinman (tin) was employed. tin is involved in the specification of the dorsal mesoderm and in the formation of heart muscle precursors. In embryos carrying twist-GAL4;UAS-srp, heart precursors develop normally to form the heart. Thus, ectopic expression of srp does not affect heart development (Hayes, 2001).
The bagpipe (bap) gene was used to mark the visceral mesoderm, which gives rise to visceral muscle precursors. The visceral mesoderm is made up of 10 metameric clusters of cells located in the dorsal mesoderm. bap is expressed in these cells and is necessary for the formation of the visceral muscle. twist-GAL4;UAS-srp embryos lack the bap-expressing cells and exhibit a bap-like phenotype, in which the midgut fails to undergo its normal constrictions. The loss of bap-expressing cells in the experimental embryos might reflect a cell-fate change that allows the replacement of visceral muscle precursors by fat-cell precursors (Hayes, 2001).
Finally, the effect of srp misexpression on somatic or body-wall muscle was examined. The absence of cli-expressing lateral (body wall) muscle precursors suggests that these muscles might be absent in the experimental embryos. The body-wall muscle is derived from the lateral region of the slp domain that exhibits the highest levels of twist expression. nautilus (nau) and S59 were used as cell markers for a subset of the founder or precursor cells for body-wall muscle and the tropomyosin I (Tm1) gene was used as a marker for body-wall muscle fibers. nau is first active at stage 10 and is expressed in a dynamic pattern. nau expression is detected in cells flanking the ventral midline and later in lateral and dorsolateral cell clusters. S59 expression is initially detected in a single, large mesodermal cell in a segmentally repeating pattern. At late stage 11, these cells give rise to two founder muscle cells and in each segment a second cluster of four S59-expressing cells appears that also contributes to precursor muscles. In embryos carrying twist-GAL4;UAS-srp, the early nau and S59 pattern of expression is disrupted and various nau- and S59-expressing cells are absent. To determine whether there is a general effect on body-wall muscle, twist-GAL4; UAS-srp embryos were stained for the body-wall muscle marker Tm1. Consistent with the loss of nau- and S59-expressing cells, a dramatic loss of muscle fibers was found. The few remaining muscle fibers were mono- and bi-nucleate and their number and location varied from embryo to embryo. These remaining muscle fibers might reflect an incomplete loss of the founder muscle cells and/or myoblasts. It is possible that the remaining fibers are due to loss of bap expression, which leads to visceral mesoderm precursors assuming a somatic muscle identity. It is concluded that the ectopic expression of srp in the mesoderm can disrupt body-wall muscle differentiation (Hayes, 2001).
Bases in 5' UTR - 281
Exons - 6
Bases in 3' UTR - 581
Serpent has a single Cys-4 zinc finger domain.
date revised: 25 April 2001
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