GATAe: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - GATAe
Synonyms - dGATAe
Cytological map position - 89A12
Function - transcription factor
Keywords - endoderm development
Symbol - GATAe
FlyBase ID: FBgn0038391
Genetic map position - 3R
Classification - Zn-finger, GATA type
Cellular location - nuclear
|Recent literature||Okumura, T., Takeda, K., Kuchiki, M., Akaishi, M., Taniguchi, K. and Takashi, A. Y. (2015). GATAe regulates intestinal stem cell maintenance and differentiation in Drosophila adult midgut. Dev Biol [Epub ahead of print]. PubMed ID: 26719127
Adult intestinal tissues, exposed to the external environment, play important roles including barrier and nutrient-absorption functions. These functions are ensured by adequately controlled rapid-cell metabolism. GATA transcription factors play essential roles in the development and maintenance of adult intestinal tissues both in vertebrates and invertebrates. This study investigated the roles of GATAe, the Drosophila intestinal GATA factor, in adult midgut homeostasis with its first-generated knock-out mutant as well as cell type-specific RNAi and overexpression experiments. These results indicate that GATAe is essential for proliferation and maintenance of intestinal stem cells (ISCs). Also, GATAe is involved in the differentiation of enterocyte (EC) and enteroendocrine (ee) cells in both Notch (N)-dependent and -independent manner. The results also indicate that GATAe has pivotal roles in maintaining normal epithelial homeostasis of the Drosophila adult midgut through interaction of N signaling. Since recent reports showed that mammalian GATA-6 regulates normal and cancer stem cells in the adult intestinal tract, these data also provide information on the evolutionally conserved roles of GATA factors in stem-cell regulation.
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).
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).
Drosophila responds to infection by producing a broad range of antimicrobial agents in the fat body and more restricted responses in tissues such as the gut, trachea, and malpighian tubules. The regulation of antimicrobial genes in larval fat depends on linked Rel/NF-kappaB and GATA binding sites. Serpent functions as the major GATA transcription factor in the larval fat body. However, the transcriptional regulation of other tissue-specific responses is less well understood. This study presents evidence that dGATAe regulates antimicrobial gene expression in the midgut. Regulatory regions for antimicrobial genes Diptericin and Metchnikowin require GATA sites for activation in the midgut, where Grain (dGATAc), dGATAd, and dGATAe are expressed in overlapping domains. Ectopic expression of dGATAe in the larval fat body, where it is normally absent, causes dramatic up-regulation of numerous innate immunity and gut genes, as judged by microarray analysis and in situ hybridization. Ectopic dGATAe also causes a host of symptoms reminiscent of hyperactive Toll (Toll10b) mutants, but without apparent activation of Toll signaling. Based on this evidence it is proposed that dGATAe mediates a Toll-independent immune response in the midgut, providing a window into the first and perhaps most ancient line of animal defense (Senger, 2006).
Previous studies have established the importance of Serpent in mediating the systemic immune response in the larval fat body. Serpent is also essential for the differentiation of the fat body during embryogenesis; srp mutants are lethal and lack fat cell differentiation. Serpent is not merely a transient determinant of fat body development. It is proposed that dGATAe plays an analogous role in the anterior midgut: it functions as a tissue determinant in the embryo but mediates immunity in larvae (Senger, 2006).
dGATAe is expressed throughout the developing midgut during embryogenesis. As seen for srp in the fat body, dGATAe expression persists in the definitive midgut of feeding larvae. Thus, both srp and dGATAe might have dual roles in development and physiology. Early expression is required for tissue differentiation, and late expression is required for the immune response. srp mediates immunity in the fat body, whereas dGATAe mediates expression of specific immunity genes in the midgut (Senger, 2006).
Evidence that dGATAe functions in the early development of the midgut stems from microarray assays. Misexpression of dGATAe in the fat body leads to ectopic induction of a number of genes required for digestion, including trypsin-like serine proteases, a sugar transporter, and genes involved in lipid metabolism. All of these genes display restricted expression in the midgut of developing embryos, in regions where dGATAe is also expressed. It is proposed that at least some of these genes are immediate and direct targets of dGATAe in the developing gut, and consequently, they are efficiently activated by dGATAe in the fat body (Senger, 2006).
dGATAe might also activate genes required for immunity gene expression in the anterior midgut of feeding larvae. By analogy to Serpent, dGATAe might activate different components of signaling pathways required for immunity. When larvae ingest pathogenic bacteria such as E. carotovora, these pathways are induced to trigger expression of Drs, Dpt, Mtk, and other immunity genes in the anterior midgut. When misexpressed in the fat body, dGATAe only moderately affects the levels of Dpt or Mtk, although the regulatory regions of these genes showed GATA-dependent activity in the midgut. This low activity is attributed to the presence of repressors in the fat body that cannot be overcome by ectopic dGATAe (Senger, 2006).
It is possible that dGATAe-mediated immunity in the anterior midgut does not depend on the Toll signaling pathway, because none of the signaling components of this pathway are up-regulated in the fat body on misexpresion of dGATAe. This misexpression is nonetheless sufficient to induce Drs and dro5 in the absence of infection or injury. It is proposed that dGATAe plays two roles in the differentiated midgut. One role is activating 'housekeeping' genes that are required for digestion. The second role of dGATAe in the midgut is triggering unknown signaling pathways that lead to the activation of immunity genes such as Drs, Dpt, and Mtk. Drs may be especially sensitive to dGATAe in the fat body because it is “poised” for induction (Senger, 2006).
In summary, it has been argued that dGATAe is critical for anterior midgut formation and function in a manner analogous to Serpent in the fat body. It is possible that the dGATAe immunity pathway is an evolutionarily ancient form of innate immunity. Under typical living conditions, the gut is the first line of defense, because ingestion is the most likely basis for contact with pathogens. The immunity signaling pathway(s) governing dGATAe activity is not yet known. However, ectopic expression of dGATAe in the fat body leads to the activation of a number of signaling components, including RhoL, Takl2, and Tetraspanins. The latter are integral membrane proteins that have been implicated in the immune responses of higher organisms, including antigen presentation. Future studies will assess the role, if any, of these genes in the gut-specific immune response (Senger, 2006).
Search PubMed for articles about Drosophila GATAe
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
Senger, K., Harris, K. and Levine, M. (2006). GATA factors participate in tissue-specific immune responses in Drosophila larvae. Proc. Natl. Acad. Sci. 103(43): 15957-62. PubMed Citation: 17032752
date revised: 20 August 2012
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