serpent is required for endodermal development in Drosophila. srp is first expressed at the cellular blastoderm stage in yolk cells, and in prospective regions of the endoderm, amnioserosa, and hemocyte primordium. Endodermal expression of srp disappears by stages 10-11. Embryos lacking srp activity fail to develop endoderm; instead, the prospective endodermal region develops into the ectodermal hindgut. Since srp is expressed in the endoderm earlier than GATAe is expressed, activation of GATAe expression by srp was examined. Expression of GATAe in the prospective endoderm region is abolished in the srp mutant (srp2/srp2), whereas GATAe expression in the Malpighian tubules is not affected. Conversely, ubiquitously misexpressed srp causes strong ectopic expression of GATAe in the foregut, and in the hindgut. Note that the foregut and hindgut arise immediately anterior and posterior to the endoderm, respectively. This ectopic expression pattern is transient. During embryogenesis, GATAe is also induced ectopically in the salivary gland and segmentally in the ventral nerve cord. These results strongly suggest that srp activates GATAe in the endoderm. fork head (fkh) is expressed throughout the prospective gut, and the gut primordia degenerate during germband retraction in fkh mutants. However, GATAe expression is not affected in the fkh mutant (fkhXT6/fkhXT6) (Okumura, 2005).
GATAe expression in the Malpighian tubules is not affected in the srp mutant, indicating that this expression depends on some other gene. Krüppel (Kr) is known to be required for the development of Malpighian tubules, so whether the GATAe expression in this organ depends on Kr was investigated. GATAe expression is completely abolished in the prospective region of the Malpighian tubules of Kr mutant embryos, indicating that Kr is required for GATAe expression in the Malpighian tubules (Okumura, 2005).
Whether the GATAe plays a role in expression of an early marker gene in the endoderm was examined. The Race gene is an early marker for endoderm and amnioserosa. Race expression begins in the invaginating endoderm slightly before GATAe expression begins, and Race expression persists throughout embryogenesis. Race expression in the endoderm is not affected in either Df(3R)sbd45 embryos or in embryos treated with GATAe dsRNA. These results show that GATAe is not essential for the expression of Race during normal development. Nevertheless, misexpression of GATAe in the present study induces ectopic expression of Race in a portion of the hindgut. Since Race is a target of srp, it is likely that misexpressed GATAe activates the srp target gene because of the possible structural similarities between the protein products of GATAe and srp (Okumura, 2005).
In the posterior terminal region of the blastoderm, prospective regions of the posterior endoderm and hindgut abut each other. byn is responsible for determination of the prospective hindgut. byn is expressed throughout the prospective posterior endoderm during early stage 5, but soon disappears in this region. It is the srp gene that represses byn in the prospective endoderm. Thus, the boundary between the endoderm and hindgut is established by the repressive activity of srp on byn. Whether GATAe also represses byn was examined, since GATAe continues to be expressed after endodermal srp expression ceases. Ectopic expression of byn in the prospective endodermal domain is observed in embryos that lack GATAe, although the area of ectopic expression is much smaller than that observed in the srp mutant, in which ectopic expression of byn is observed throughout the entire prospective endoderm. Moreover, when misexpressed in the prospective hindgut domain, both GATAe and srp strongly repress byn expression. Thus, GATAe is required to maintain the endodermal identity that is initially established by srp (Okumura, 2005).
In situ hybridization on whole-mount embryos was used to determine the expression pattern of GATAe during embryogenesis. GATAe mRNA is first detected in the posterior endoderm at stages 7-8, and in the anterior endoderm at stage 8. Malpighian tubule primordia also express GATAe from stage 10 onwards. Expression of GATAe in the endoderm, in the midgut (which is exclusively derived from the endoderm) and within Malpighian tubules continues throughout embryonic development. GATAe expression is not detected in any embryonic tissues other than endoderm and Malpighian tubules (Okumura, 2005).
RT-PCR was used to examine whether GATAe expression in the endodermal midgut continues in post-embryonic stages. The midguts of third instar larvae were dissected into anterior, middle, and posterior segments, whereas adult fly midguts were dissected into anterior and posterior segments. Each of these midgut segments exhibit GATAe mRNA expression, indicating that GATAe is expressed in the midgut throughout life (Okumura, 2005).
To test whether GATAe is required for the later stages of endodermal development, leading to terminal differentiation of the midgut, the role of GATAe in the expression of midgut-specific integrin βν, and the midgut-specific gap junction gene, inx7, was examined. integrin βν and inx7 are both expressed throughout the endoderm from embryonic stage 11 onwards. RT-PCR analyses indicate that integrin βν and inx7 are expressed in the midgut of third instar larvae and adult flies. Thus, these genes can serve as markers of the differentiated midgut (Okumura, 2005).
The Df(3R)sbd45 embryo lacks GATAe and pnr loci, but, retains the srp locus. In contrast to the srp mutant, in which the prospective endodermal region develops into ectodermal tissues, including a portion of the hindgut, the Df(3R)sbd45 embryo forms apparently normal midgut primordium surrounding the yolk, with constrictions that are characteristic of the late stages of normal midgut. However, the midgut primordium fails to express integrin βν and inx7. Another midgut-specific gene, midgut expression 1 (mex1) is also not expressed in this embryo. Since the Df(3R)sbd45 strain also lacks the pnr locus, the hypothesis was tested that this phenotype is caused by the lack of GATAe, but not by the lack of pnr. Both integrin βν and inx7 were normally expressed in both pnrVX6/Df(3R)sbd45 heterozygotes and pnrVX6 (null) homozygotes. Unlike the misexpression of GATAe, misexpressed pnr does not induce integrin βν or inx7 expression in the hindgut. When GATAe is ubiquitously misexpressed in the Df(3R)sbd45 homozygotes, expression of integrin βν is restored, although inx7 expression is not restored under these conditions. To further confirm that the loss of integrin βν and inx7 expression is due to the lack of GATAe activity, GATAe transcripts were inactivated with RNA interference (RNAi) using dsRNA. The gross morphology of the midgut primordium of dsRNA-injected embryos appears to be normal, forming normal constrictions in late stages. However, embryos injected with dsRNA corresponding to GATAe fail to express either of the integrin βν and inx7. The loss of midgut markers in response to dsRNA treatment is not a secondary effect of the loss of srp activity, since Srp protein is detected at normal levels in embryos injected with dsRNA. When GATAe is ubiquitously misexpressed in the srp mutant embryos, expression is restored. In addition, misexpression of GATAe in the hindgut of wild-type embryos strongly induces ectopic expression of integrin βν and inx7. Misexpressed pnr does not induce these marker genes. Furthermore, both integrin βν and inx7 are also induced in S2 cells transfected with GATAe cDNA. These results clearly demonstrate that GATAe induces gene expression in the differentiated midgut (Okumura, 2005).
Fukushige, T., Hawkins, M. G. and McGhee, J. D. (1998). The GATA-factor elt-2 is essential for formation of the Caenorhabditis elegans intestine. Dev. Biol. 198(2): 286-302. 9659934
Okumura, T., Matsumoto, A., Tanimura, T. and Murakami, R. (2005). An endoderm-specific GATA factor gene, dGATAe, is required for the terminal differentiation of the Drosophilaendoderm. Dev. Biol. 278(2): 576-86. 15680371
date revised: 25 March 2005
Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.