The vertebrate GSC proteins are relatively small molecules of 240-250 amino acids, conserved along their entire length (77-85% identity outside the homeodomain and 98-100% inside the homeodomain). By contrast, Drosophila GSC is a larger protein whose homology with its vertebrate counterparts is limited to the homeodomain and to a small stretch (GEH for GSC-Engrailed Homology) of 7 amino acids located at the N-terminal part of the vertebrate proteins (residues 110-116 in Drosophila GSC). This sequence in the Engrailed homeoproteins appears to mediate part of their repression potential. Drosophila Goosecoid mimics the Xenopus Goosecoid when tested in Xenopus by rescuing the axis polarity of a UV-irradiated embryo (Goriely, 1996).

Goosecoid in invertebrates

Cngsc, a hydra homolog of the goosecoid gene, has been isolated. The homeodomain of Cngsc is identical to the vertebrate (65%-72%) and Drosophila (70%) orthologs. When injected into the ventral side of an early Xenopus embryo, Cngsc induces a partial secondary axis. During head formation, Cngsc expression appears prior to, and directly above, the zone where the tentacles will emerge, but is not observed nearby when the single apical tentacle is formed. This observation indicates that the expression of the gene is not necessary for the formation of a tentacle per se. Rather, it may be involved in defining the border between the hypostome and the tentacle zone. When the Cngsc+ tip of an early bud is grafted into the body column, it induces a secondary axis, while the adjacent Cngsc- region has much weaker inductive capacities. Thus, Cngsc is expressed in a tissue that acts as an organizer. Cngsc is also expressed in the sensory neurons of the tip of the hypostome and in the epithelial endodermal cells of the upper part of the body column. The plausible roles of Cngsc in organizer function, head formation and anterior neuron differentiation are similar to roles goosecoid plays in vertebrates and Drosophila. It suggests widespread evolutionary conservation of the function of the gene (Broun, 1999).

A single homolog of goosecoid, SpGsc, has been identified that regulates cell fates along both the animal-vegetal and oral-aboral axes of sea urchin embryos. SpGsc mRNA is expressed briefly in presumptive mesenchyme cells of the ~200-cell blastula and, beginning at about the same time, accumulates in the presumptive oral ectoderm through pluteus stage. Loss-of-function assays with morpholine-substituted antisense oligonucleotides show that SpGsc is required for endoderm and pigment cell differentiation and for gastrulation. These experiments and gain-of-function tests by mRNA injection show that SpGsc is a repressor that antagonizes aboral ectoderm fate specification and promotes oral ectoderm differentiation. SpGsc competes for binding to specific cis elements with SpOtx, a ubiquitous transcription activator that promotes aboral ectoderm differentiation. Moreover, SpGsc represses transcription in vivo from an artificial promoter driven by SpOtx. Since SpOtx appears long before SpGsc transcription is activated, it is proposed that SpGsc diverts ectoderm towards oral fate by repressing SpOtx target genes. Based on the SpGsc-SpOtx example and other available data, it is proposed that ectoderm is first specified as aboral by broadly expressed activators, including SpOtx, and that the oral region is subsequently respecified by the action of negative regulators, including SpGsc. Accumulation of SpGsc in oral ectoderm depends on cell-cell interactions initiated by nuclear ß-catenin function, which is known to be required for specification of vegetal tissues, because transcripts are undetectable in dissociated or in cadherin mRNA-injected embryos. This is the first identified molecular mechanism underlying the known dependence of oral-aboral ectoderm polarity on intercellular signaling (Angerer, 2001).

Transcription factors of the T-domain family regulate many developmental processes. A new member of the Tbx2 subfamily, coquillette, has been isolated from the sea urchin. Coquillette has a late zygotic expression whose localization is dynamic: at the blastula stage it is restricted to the aboral side of most of the presumptive ectoderm and endoderm territories and from gastrulation on, to the aboral-most primary mesenchyme cells. Perturbation of coquillette function delays gastrulation and strongly disorganizes the skeleton of the larva. Coquillette is sensitive to alteration of the oral-aboral (OA) axis and goosecoid, which controls oral and aboral fates in the ectoderm, is identified as a probable upstream regulator. Coquillette appears to be an integral part of the patterning system along the OA axis (Croce, 2003).

The first phase of coquillette expression occurs for a large part in the presumptive ectoderm. In this territory, several regulators of fate along the OA axis have been identified, including goosecoid. Goosecoid is a transcriptional repressor that promotes oral fate and represses aboral fate. A number of observations suggest that goosecoid may control coquillette. (1) Both goosecoid and coquillette expression begin at about the same time, at the swimming blastula stage. (2) Many genes that are restricted to the aboral ectoderm are initially activated throughout the ectoderm. Coquillette, however, is expressed only in the aboral ectoderm from the earliest time its expression can be detected, indicating that it is downstream of or simultaneous with oral-aboral specification. (3) The expression domains of goosecoid and coquillette are opposite one another. (4) Overexpression of goosecoid suppresses coquillette expression (Croce, 2003).

Goosecoid in fish

Gsc exhibits two independent phases of expression in zebrafish: early in cells anterior to the presumptive notochord (but not in cells of the notochord itself), and later in neural crest derivatives in the larval head. Zygotic Gsc transcripts are detected soon after the midblastula transition, and at the blastula stage form a gradient with a maximum at the dorsal side. ntl (a homolog of Drosophila T-related gene and optomotor blind) expression is initially activated at the dorsal side of the blastula. At this early stage, Gsc and ntl show overlapping domains of expression and are co-expressed in cells at the dorsal midline of the early gastrula. However, Gsc- and ntl-expressing cells become separated in the course of gastrulation, with Gsc being expressed in the axial hypoblast (prechordal plate) anterior to the ntl-expressing presumptive notochord cells. Studies with mutant embryos suggest that Gsc is independent of ntl function in vivo (Schulte-Merker, 1994).

Retinoid signaling plays an important role in embryonic pattern formation. Excess of retinoic acid during gastrulation results in axial defects in vertebrate embryos, suggesting that retinoids are involved in early anteroposterior patterning. To study retinoid signaling in zebrafish embryos, a novel method was developed to detect endogenous retinoids in situ in embryos, using a fusion protein of the ligand inducible transactivation domain from a retinoic acid receptor and a heterologous DNA binding domain. Using this method, retinoid signaling is shown to be localized in zebrafish embryos in the region of the embryonic shield, and towards the end of gastrulation in a posterior dorsal domain. To investigate the relationships between the spatial distribution of retinoid signaling and the regulation of retinoid target genes, the downregulation by retinoic acid of goodsecoid and otx1, two genes expressed in anterior regions of the embryo, was studied. These experiments show that expression of both genes is strongly downregulated in the anterior neurectoderm of zebrafish embryos treated with retinoic acid, whereas mesendodermal expression is only mildly affected. Interestingly, a significant downregulation of goosecoid expression by retinoic acid is observed only during midgastrulation but not in earlier stages. In agreement with these results, spatial expression of goosecoid and otx1 does not overlap with the region of retinoid signaling in the late gastrula. These data support the hypothesis that a localized retinoid signal is involved in axial patterning during early development, at least in part through the repression of anterior genes in posterior regions of the embryo. The data suggest that the action of retinoids is spatially as well as temporally regulated in the developing embryo (Joore, 1997).

An investigation was carried out of the role of cAMP-dependent protein kinase A (PKA) in the induction of the early mesodermal marker genes goosecoid and no tail by activin in zebrafish embryos. Upon treatment with activin, zebrafish blastula cells exhibit a rapid and transient increase in PKA activity. In these cells, activin rapidly induces the expression of the immediate early response genes goosecoid and no tail. Stimulation and inhibition of PKA by activin, respectively, enhances and reduces the induction of goosecoid and no tail mRNA expression. Similar effects of PKA stimulation and inhibition on the induction by activin of a 1.8 kb zebrafish goosecoid promoter construct are observed. The induction by activin of a fragment of the zebrafish goosecoid promoter that mediates an immediate early response to activin is blocked by inhibition of PKA. Activation of PKA alone has no effect in these experiments. Finally, inhibition of PKA in whole embryos by overexpression of a dominant negative regulatory subunit of PKA reduces the expression of no tail and goosecoid, whereas the expression of even-skipped1 remains unaltered. Overexpression of the catalytic subunit of PKA in embryos does not affect expression of goosecoid, no tail or even-skipped1. These data show that in dissociated blastulae, PKA is required, but not sufficient for activin signalling towards induction of goosecoid and no tail. In intact zebrafish embryos, PKA contributes to induction of goosecoid and no tail, although it is neither required nor sufficient (Joore, 1998).

Previous studies in both Xenopus and zebrafish have shown that goosecoid is one of the first genes to be transcribed at the onset of gastrulation. Goosecoid transcription still initiates when embryos are treated with protein synthesis inhibitors, indicating that it is mediated by preexisting factors and suggesting that goosecoid transcription is immediately downstream of the maternal mesoderm-inducing signal. However, goosecoid transcription continues long after this maternal signal has ceased to be active, indicating that there are mechanisms to maintain activin-induced transcription. This study has focused on understanding the factors required to maintain this transcription. An element has been identified within the zebrafish goosecoid promoter that is sufficient for activin inducibility in both Xenopus and zebrafish embryos. This element, the goosecoid activin element, interacts with two developmentally regulated proteins from Xenopus embryos. A maternal protein interacts through cleavage stages until the midblastula transition, and a second protein binds from the onset of gastrulation. The second protein is zygotically expressed, and its binding is required for activin inducibility in the assay system used. It is suggested that the zygotic protein that has been identified is a good candidate to be involved in the maintenance of goosecoid transcription. Furthermore, this zygotic protein is likely to contain a paired class homeodomain since a consensus binding site for such proteins is present within the goosecoid activin element and is essential for its function (McKendry, 1998).

Early embryonic development in many organisms relies upon maternal molecules deposited into the egg prior to fertilization. A maternal T-box gene in the zebrafish, eomesodermin (eomes), has been cloned and characterized. During oogenesis, the eomes transcript becomes localized to the cortex of the oocyte. After fertilization during early cleavage stages, eomes is expressed in a vegetal to animal gradient in the embryo, whereas Eomesodermin protein is distributed cytoplasmically throughout the blastoderm. Strikingly, following midblastula transition, nuclear-localized Eomesodermin is detected on the dorsal side of the embryo only. Overexpression of eomes results in Nodal-dependent and nieuwkoid/dharma independent ectopic expression of the organizer markers goosecoid (gsc), chordin and floating head (flh) and in the formation of secondary axes. The same phenotypes are observed when a VP16-activator construct is injected into early embryos, indicating that eomes acts as a transcriptional activator. In addition, a dominant-negative construct and antisense morpholino oligonucleotides leads to a reduction in gsc and flh expression. Together these data indicate that eomes plays a role in specifying the organizer (Bruce, 2003).

The organizer is essential for dorsal-ventral (DV) patterning in vertebrates. Goosecoid (Gsc), a transcriptional repressor found in the organizer, elicits partial secondary axes when expressed ventrally in Xenopus, similar to an organizer transplant. Although gsc is expressed in all vertebrate organizers examined, knockout studies in mouse suggested that it is not required for DV patterning. Moreover, experiments in Xenopus and zebrafish suggest a role in head formation, although a function in axial mesoderm formation is less clear. To clarify the role of Gsc in vertebrate development, gain- and loss-of-function approaches were used in zebrafish. Ventral injection of low doses of gsc produced incomplete secondary axes, which results from short-range repression of BMP signaling. Higher gsc doses resulted in complete secondary axes and long-range signaling, correlating with repression of BMP and Wnt signals. In striking contrast to Xenopus, the BMP inhibitor Chordin (Chd) is not required for Gsc function. Gsc produced complete secondary axes in chd null mutant embryos and gsc-morpholino knockdown in chd mutants enhanced the mutant phenotype, suggesting that Gsc has Chd-independent functions in DV patterning. Even more striking was that Gsc elicited complete secondary axes in the absence of three secreted BMP antagonists, Chd, Follistatin-like 1b and Noggin 1, suggesting that Gsc functions in parallel with secreted BMP inhibitors. These findings suggest that Gsc has dose dependent effects on axis induction and provide new insights into molecularly distinct short- and long-range signaling activities of the organizer (Dixon, 2009).

Xenopus Goosecoid

Goosecoid: Evolutionary homologs part 2/3 | part 3/3

Goosecoid: Biological Overview | Regulation | Developmental Biology | References

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