Thomas D. Sargent

Induction of neural crest in Xenopus by transcription factor AP2α

We report experiments with Xenopus laevis, using both intact embryos and ectodermal explants, showing that the transcription factor AP2α is positively regulated by bone morphogenetic protein (BMP) and Wnt signaling, and that this activation is an essential step in the induction of neural crest (NC). Ectopic expression of AP2α is sufficient to activate high-level expression of NC-specific genes such as Slug and Sox9, which can occur as isolated domains within the neural plate as well as by expansion of endogenous NC territories. AP2α also has the property of inducing NC in isolated ectoderm in which Wnt signaling is provided but BMP signaling is minimized by overexpression of chordin. Like other NC regulatory factors, activation of AP2α requires some attenuation of endogenous BMP signaling; however, this process occurs at a lower threshold for AP2α. Furthermore, AP2α expression domains are larger than for other NC factors. Loss-of-function experiments with antisense AP2α morpholino oligonucleotides result in severe reduction in the NC territory. These results support a central role for AP2α in NC induction. We propose a model in which AP2α expression, along with inactivation of NC inhibitory factors such as Dlx3, establish a feedback loop comprising AP2α, Sox9, and Slug, leading to and maintaining NC specification.

Cell interactions and the control of gene activity during early development of Xenopus laevis

During embryonic development, regulation of the zygotic genome may be mediated by inductive interactions and by cell-autonomous inheritance of informational material from the egg; we have studied certain aspects of such regulatory events in Xenopus laevis. Embryos cultured in Ca2+/Mg2+-free medium can be dissociated and dispersed, eliminating cell-cell contact and thus precluding inductive interactions. Such manipulations revealed that activation of the muscle-specific alpha-actin genes is absolutely dependent upon cell contacts. Conversely, the endoderm-specific DG42 gene and the ectoderm-specific DG81 cytokeratin gene are activated in embryo cells dispersed throughout blastula stages and therefore appear to be controlled by inherited factors. Nevertheless, abnormal cell arrangements may prevent expression of the cytokeratin gene, suggesting that animal pole cells can be diverted from their normal ectodermal fate by inductive contact with vegetally derived cells. The interactions required for alpha-actin induction and inhibition of cytokeratin expression are independent of strong adhesion between embryonic cells mediated by high concentrations of divalent cations.

Development of neural inducing capacity in dissociated Xenopus embryos

During gastrulation in amphibians, dorsal ectoderm is converted by induction from an epidermal pathway to central nervous system (CNS). We show in this report that the ability of the embryo to manufacture this neural inducing signal is not dependent upon intercellular communication between early cleavage and early gastrula stages. This result is consistent with the interpretation that the cell lineage that induces CNS formation arises itself by a cell-autonomous mechanism, perhaps specified by materials inherited from the fertilized egg.

Accumulation and decay of DG42 gene products follow a gradient pattern during Xenopus embryogenesis

The DG42 gene is expressed during a short window during embryogenesis of Xenopus laevis. The mRNA for this gene can be first detected just after midblastula, peaks at late gastrula, and decays by the end of neurulation. The sequence of the DG42 cDNA and genomic DNA predicts a 70,000-Da protein that is not related to any other known protein. Antibodies prepared against portions of the DG42 open reading frame that had been expressed in bacteria detected a 70,000-Da protein in the embryo with a temporal course of appearance and decay that follows that of the RNA by several hours. Localization of the mRNA in dissected embryos and immunohistochemical detection of the protein showed that DG42 expression moves as a wave or gradient through the embryo. The RNA is first detected in the animal region of the blastula, and by early gastrula is found everywhere except in the outer layer of the dorsal blastopore lip. By midgastrula DG42 protein is present in the inner ectodermal layer and the endoderm; it disappears from dorsal ectoderm as the neural plate is induced and later decays in a dorsoventral direction. The last remnants of DG42 protein are seen in ventral regions of the gut at the tailbud stage.

Regulation and function of Dlx3 in vertebrate development

Dlx3 is a homeodomain transcription factor in vertebrates, related to Distal‐less in Drosophila, that is expressed in differentiating epidermal cells, in neural crest, hair follicles, dental epithelium and mesenchyme, the otic and olfactory placodes, limb bud, placenta, and in the cement gland, which is located in the extreme anterior neural plate in Xenopus embryos. This factor behaves as a transcriptional activator, and positively regulates gene expression in the skin, and negatively regulates central nervous system markers in Xenopus epidermis and anterior neural plate. A mutation in the DLX3 gene is associated with a hereditary syndrome in humans, and loss of Dlx3 function is a developmental lethal in gene‐targeted mice, where it is essential for proper modeling of the labyrinthine layer of the placenta. In this review, we discuss the evolution, expression, regulation, and function of Dlx3 in mouse, amphibians, and zebrafish. Published 2000 Wiley‐Liss, Inc.

Developmental regulation of transcription factor AP-2 during Xenopus laevis embryogenesis

We have isolated a cDNA clone encoding the Xenopus homologue of the transcription factor AP-2 (XAP-2). The predicted amino acid sequence derived from the Xenopus cDNA shows very strong conservation with the amino acid sequence of human AP-2, suggesting that this protein is evolutionarily conserved, at least among vertebrates. This is further substantiated by the demonstration that an in vitro translation product of XAP-2 cDNA bound specifically to an AP-2 binding site from the human MT-IIA gene. Northern blot analysis of Xenopus embryo RNA revealed the existence of three major XAP-2 mRNA species that were only detectable after the midblastula transition (when embryonic transcription is activated), with peak accumulation of the transcripts occurring during gastrulation. Therefore, in contrast to other Xenopus transcription factors, XAP-2 is not maternally derived but arises exclusively from zygotic transcription. Unlike the situation in cultured human teratocarcinoma (NT2) cells, retinoic acid treatment did not induce XAP-2 mRNA in Xenopus embryos, even though the treatment had a pronounced morphogenetic effect on the embryos. Our results suggest that XAP-2 may play a distinctive role during Xenopus embryogenesis.

Specification of the otic placode depends on Sox9 function in Xenopus

The vertebrate inner ear develops from a thickening of the embryonic ectoderm, adjacent to the hindbrain, known as the otic placode. All components of the inner ear derive from the embryonic otic placode. Sox proteins form a large class of transcriptional regulators implicated in the control of a variety of developmental processes. One member of this family, Sox9, is expressed in the developing inner ear, but little is known about the early function of Sox9 in this tissue. We report the functional analysis of Sox9 during development of Xenopus inner ear. Sox9 otic expression is initiated shortly after gastrulation in the sensory layer of the ectoderm, in a bilateral patch of cells immediately adjacent to the cranial neural crest. In the otic placode, Sox9 colocalizes with Pax8 one of the earliest gene expressed in response to otic placode inducing signals. Depletion of Sox9 protein in whole embryos using morpholino antisense oligonucleotides causes a dramatic loss of the early otic placode markers Pax8 and Tbx2. Later in embryogenesis, Sox9 morpholino-injected embryos lack a morphologically recognizable otic vesicle and fail to express late otic markers (Tbx2, Bmp4, Otx2 and Wnt3a) that normally exhibit regionalized expression pattern throughout the otocyst. Using a hormone inducible inhibitory mutant of Sox9, we demonstrate that Sox9 function is required for otic placode specification but not for its subsequent patterning. We propose that Sox9 is one of the key regulators of inner ear specification in Xenopus.

Differential expression of a Distal-less homeobox gene Xdll-2 in ectodermal cell lineages.

Neural induction in Xenopus requires the activation of new sets of genes that are necessary for cellular and regional specification of the neural tube. It has been reported earlier that members of the Distal-less homeobox gene family are specifically activated in distinct regions of the central nervous system (CNS) of Xenopus embryos (Dirksen et al., 1993; Papalopulu and Kintner, 1993). In this paper we describe in detail a Xenopus homeobox containing gene Xdll-2, which belongs to the Distal-less gene family. In contrast to other previously described Xenopus family members, Xdll-2 is expressed in the embryonic ectoderm and is specifically repressed in the CNS. This repression can be mimicked in isolated animal caps by treatment with activin. Expression of Xdll-2 persists in the epidermis and some neural crest cells. Because of its spatial and temporal expression pattern this gene is a good candidate to have a regulatory function in the initial formation of the epidermis. Its high level of expression in adult skin indicates that its function is continuously required in this tissue.

Molecular approach to dorsoanterior development in Xenopus laevis

RNA from ventralized Xenopus laevis embryos has been used to generate a subtracted library consisting of genes whose expression is dependent upon dorsoanterior development. Clones were selected on the basis of two criteria: (1) at least 10-fold reduction in ventralized versus control embryo RNA, and (2) absence of hybridization to RNA from adult muscle. One of these clones, designated UVS.2, was characterized further. UVS.2, was found by in situ hybridization to be expressed exclusively in the anterior neural fold of neurula stage embryos. By the tailbud stage, the UVS.2 protein and transcript were localized in specialized cephalic ectoderm, in a region probably corresponding to the hatching gland. UVS.2 expression could be induced in ectodermal explants by contact with dorsal mesodermal tissue. Furthermore, in lithium-treated embryos, which have exaggerated dorsoanterior structures, UVS.2 expression was increased four- to fivefold. A molecular marker such as UVS.2 will be useful in defining and studying the induction and organization of cephalic tissues.

Cloning and expression of a novel zinc finger gene, Fez, transcribed in the forebrain of Xenopus and mouse embryos

We have identified and cloned a novel zinc finger gene, Fez (forebrain embryonic zinc-finger), as a potential downstream determinant of anterior neural plate formation in Xenopus. Fez was isolated as one of several neural-specific genes that was induced by the neuralizing factor, noggin (Smith and Harland, 1992. Cell 70, 829-840), in uncommitted ectoderm. Fez has an open reading frame comprising 466 amino acids, and contains six C(2)H(2) type zinc finger domains, which are highly conserved among Drosophila, zebrafish, mouse, and human. In Xenopus, the expression of Fez begins at stage 12 in the rostral end of the neural plate, and by stage 45, it is localized to several telencephalic regions, including the olfactory bulbs, nervus terminalis, and ventricular zone. The mouse homologue of Fez is similarly expressed in the mouse forebrain by embryonic day 11.