GATA factors play an essential role in endodermal specification in both protostomes and deuterostomes. In Drosophila, the GATA factor gene serpent (srp) is critical for differentiation of the endoderm. However, the expression of srp disappears around stage 11, which is much earlier than overt differentiation occurs in the midgut, an entirely endodermal organ. Another endoderm-specific Drosophila GATA factor gene, GATAe, has been identified. Expression of GATAe is first detected at stage 8 in the endoderm , and its expression continues in the endodermal midgut throughout the life cycle. srp is required for expression of GATAe, and misexpression of srp resulted in ectopic GATAe expression. Embryos that either lack GATAe or have been injected with double-stranded RNA (dsRNA) corresponding to GATAe fail to express marker genes that are characteristic of differentiated midgut. Conversely, overexpression of GATAe induces ectopic expression of endodermal markers even in the absence of srp activity. Transfection of the GATAe cDNA also induces endodermal markers in Drosophila S2 cells. These studies provide an outline of the genetic pathway that establishes the endoderm in Drosophila. This pathway is triggered by sequential signaling through the maternal torso gene, a terminal gap gene, huckebein (hkb), and finally, two GATA factor genes, srp and GATAe (Okumura, 2005).

The endoderm gives rise to major parts of the gut tube of multicellular organisms. Regulatory mechanisms that establish the endoderm have recently received considerable attention with respect to the development of protostomes and deuterostomes. Certain important components of this genetic regulatory pathway/network have now been identified. It remains unknown whether protostomes and deuterostomes share a common genetic mechanism of endoderm specification. However, GATA factor genes are expressed throughout endodermal development in both animal groups. GATA factors have one or two characteristic zinc-finger motifs corresponding to CXNCX₁₇CXNC, and act as transcription factors that bind to a consensus DNA sequence WGATAR of specific target genes (Okumura, 2005).

In vertebrates, GATA factor genes are classified into two groups, GATA-1/-2/-3 and GATA-4/-5/-6. While GATA-1, -2, and -3 are involved in hematopoiesis, GATA-4, -5, and -6 are essential for the development of endoderm-derived tissues, in that they activate endoderm-specific genes such as IFABP, gastric H⁺/K⁺-ATPase, HNF4, and albumin. The GATA factor genes are also essential for endodermal development in the protostomes Caenorhabditis elegans and Drosophila melanogaster. C. elegans has eleven GATA factor genes, and seven of these are known to be related to endodermal development, resulting in redundant regulatory pathways. end-1 is the earliest GATA factor gene that is expressed specifically in the endoderm lineage. end-1 mutant embryos fail to form the endoderm, whereas overexpression of end-1 can induce non-endodermal cells to switch to an endodermal fate. end-1 can also induce endoderm formation when it is expressed in Xenopus embryos, suggesting that the genetic mechanisms underlying endodermal development are at least partially shared between protostomes and deuterostomes (Okumura, 2005 and references therein).

In C. elegans, end-1 activates another GATA gene, elt-2, and end-1 expression ceases prior to overt differentiation of the endoderm. elt-2 continues to be expressed in the gut throughout life, and activates genes that are required for various gut functions (Fukushige, 1998). In the protostome D. melanogaster, serpent (srp) is a GATA gene that is essential for the specification of endoderm. srp is expressed in the anterior and posterior terminal regions of the blastoderm, which both give rise to the endoderm. In the srp mutant embryo, the prospective endodermal region differentiates into the ectodermal hindgut. In normal embryos, the prospective hindgut region abuts the prospective endoderm of the posterior terminal, and the hindgut is specified by the Brachyury ortholog, brachyenteron (byn). The initial area in which byn is expressed in the cellular blastoderm includes the prospective posterior endoderm. However, byn expression in the posterior half of this region is soon repressed by srp and the region develops into the endoderm. In the srp embryo, byn expression expands to the prospective endodermal regions. Activation of srp depends on a zygotic gap gene, huckebein (hkb), which is triggered by maternal Torso activity at the anterior and posterior terminal regions of the fertilized egg, followed by activation of Ras signaling. These events outline the genetic pathway that defines endodermal development in Drosophilato date, and represent one of the best examples of a genetic pathway that specifies organogenesis (Okumura, 2005).

Despite the significant progress that has been made in characterizing the pathway described above, another gene, as yet unknown, seems to be involved, since srp ceases to be expressed after stages 10–11 in the prospective endoderm, long before overt differentiation of midgut. Since several GATA factor genes are sequentially expressed during the development of the C. elegans endoderm, identification of a novel GATA gene in Drosophilais expected. Thus far, three GATA factor genes have been reported in Drosophila: pannier (pnr, also known as dGATAa), srp (also known as dGATAb), and grain (also known as dGATAc). By searching the Drosophilagenome sequence, several sequences were found containing novel GATA factor motifs, and two GATA factor genes, dGATAd and GATAe, were identified. While dGATAd is not expressed in the embryo, GATAe is specifically expressed in the endoderm after stage 8, and it continues to be expressed in the endodermal midgut of larvae and adult flies. In this study, the regulation and function of the GATAe was studied. GATAe, upon activation by srp, induces overt differentiation of the Drosophilaendoderm. This finding has enabled the delineation of almost the entire genetic pathway of Drosophilaendodermal development. This pathway is initiated by early maternal signals and results in the terminal differentiation of the midgut (Okumura, 2005).

srp is the first GATA factor gene to be expressed within the Drosophila endoderm, and it is essential for its endodermal specification. srp is expressed in the prospective endoderm in the cellular blastoderm stages, but its expression disappears by stages 10–11. This study shows that srp activates GATAe, and that GATAe is required for expression of specific genes in the differentiated midgut. GATAe induces the expression of late endodermal marker genes even in the absence of srp activity. Since GATAe expression in the endodermal midgut persists throughout the embryonic, larval, and adult stages of Drosophila, it seems likely that GATAe is also necessary for maintaining gene expression in the differentiated midgut. Inactivation of GATAe transcripts with dsRNA does not cause any marked morphological defects, but most of these embryos fail to hatch, suggesting that GATAe is essential for differentiated midgut and for viability of the larva. It should be noted that the endodermal midgut is subdivided into four chambers, and further arranged into 13 subdomains with distinct gene expression patterns. Homeotic genes expressed in the visceral muscle were shown to cause subdivision of the midgut into the four chambers, but the mechanisms that generate the various subdomains are still unknown (Okumura, 2005).

The sequential activation of srp and GATAe during endodermal development in Drosophila is analogous to the gene regulatory cascade that occurs during endodermal development in C. elegans. The earliest endodermal GATA factor expressed in C. elegans is end-1, which is expressed in the endoderm. end-1 then activates the subordinate GATA factor gene, elt-2, which activates late endodermal genes (Fukushige, 1998). These results suggest that the genetic mechanism underlying endodermal development is at least partially conserved between Drosophila and C. elegans. However, the molecular phylogenetic relationship of these endoderm-specific GATA factor genes has not yet been established (Okumura, 2005).

GATA factors are also essential for endodermal development in vertebrates. In the Xenopus embryo, GATA-4 and GATA-5 are expressed in the prospective endoderm, and both genes can induce formation of the endoderm. GATA-5 also plays an important role in endodermal development in zebrafish, where it functions as an upstream regulator of Sox17a, which is essential for endodermal specification. In Drosophila and C. elegans, the GATA factor genes GATAe and elt-2, respectively, continue to be expressed in the differentiated gut, as well as in the early stages of endodermal specification. In mammals, the GATA-4, and -6 proteins are expressed in the differentiated stomach and intestine, and bind to the gastric H⁺/K⁺-ATPase gene. Taken together, these findings suggest that vertebrate GATA factor genes also function in endodermal tissues after terminal differentiation (Okumura, 2005).

The Drosophila endoderm arises at the anterior and posterior terminal regions of the early blastoderm. Previous studies have revealed the gene regulatory pathway leading to endodermal development in some detail. A Torso-like protein secreted by the follicle cells covering both the anterior and posterior terminal regions of the egg triggers activation of the receptor tyrosine kinase, Torso, resulting in a graded Ras signal that peaks at both terminals. A zygotic gap gene, huckebein (hkb) is activated by high levels of the Ras signal, and hkb, in turn, activates srp. The present study revealed that srp activates another GATA factor gene, GATAe, and the latter leads to terminal differentiation of the midgut. GATAe may be necessary to maintain gene expression in the terminally differentiated midgut, since GATAe expression persists in the midgut throughout life. In addition to playing essential roles in the development of the endoderm, srp and GATAe also act to restrict the area of the adjacent hindgut. The ectodermal hindgut is specified by a Brachyury ortholog, brachyenteron (byn). The gene regulatory pathway leading to the activation of byn is closely linked with that of srp. Ras signaling in the posterior terminal region of the fertilized egg activates another gap gene, tailless (tll). tll is required for the activation of byn. The byn-positive domain in the early cellular blastoderm stages includes the prospective endoderm domain, but the expression in the prospective endoderm soon disappears in response to the repressive activity of srp. byn expression in the hindgut persists throughout life, as does GATAe expression in the midgut. These rather simple regulatory pathways lead to the activation of GATAe and byn, and consequently, to the terminal differentiation of the midgut and hindgut. It should be noted that these pathways not only delineate the process of endodermal development in Drosophila, but also highlight conserved genetic components underlying the endodermal development of multicellular animals (Okumura, 2005).

GENE STRUCTURE

While searching genomic databases in an attempt to identify novel Drosophila GATA transcription factor genes, two sequences were found containing novel GATA factor motifs. These cDNA sequences were cloned from Drosophila cDNA libraries and/or from EST clones. The two genes were named dGATAd and GATAe, respectively, in accordance with the nomenclature adopted for other DrosophilaGATA factor genes. Although embryonic dGATAd expression was not detected, GATAe was expressed specifically in the endoderm. GATAe is located at 89A13-B4, forming a cluster with two other known GATA factor genes, pannier (pnr, also known as dGATAa) and srp (also known as dGATAb). The exon-intron structure of GATAe was deduced by comparing the GATAe cDNA sequence with the genomic sequence (Okumura, 2005).

cDNA clone length - 3093 pb

Bases in 5' UTR - 326

Exons - 6

Bases in 3' UTR - 426

PROTEIN STRUCTURE

Amino Acids - 746

Structural Domains

GATAe encodes a predicted protein of 746 amino acids containing two GATA-type zinc finger motifs. The C-terminal finger, which is well conserved in various animals, is typical of the GATA family, corresponding to the C-X₂-C-X₁₇-C-X₂-C motif, with a flanking basic domain. In contrast, the N-terminal finger, C-X₂-C-X₁₂-C-X₂-C, is atypically short, although it also has a flanking basic domain. The N-terminal region of GATAe has several glutamine-rich domains, a feature also found in pnr and srp. Multiple alignment analysis of the amino acid sequence of the C-terminal finger and basic domain of GATAe with those of other known GATA factors showed that GATAe is closely related to certain endoderm-specific GATA factor genes, including Drosophila srp, C. elegans elt-2, and mouse GATA-4/5/6. However, distinct subgroupings could not be defined either between Drosophila and mouse or between Drosophila and C. elegans. Similar difficulties in classifying other known GATA genes into distinct subgroups have been previously reported (Okumura, 2005).

date revised: 25 March 2005

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