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In vertebrates, there are six or more copies of genes related to the Drosophila pattern formation homeodomain gene Distal-less. Among this family, Dlx3 and Dlx5 share extensive sequence homology and have similar, but distinctive, expression patterns, suggesting that these two factors may have substantially redundant developmental functions. Here we show that at the earliest phases of embryogenesis in Xenopus, there are significant differences between Dlx3 and Dlx5 expression and that this correlates with different functions in the restriction of neural crest and neural plate boundaries, respectively., (Copyright 2001 Wiley-Liss, Inc.)

Three members of the vertebrate Distal-less gene family, Dlx3, 5 and 6, are transcribed in early gastrula embryos of Xenopus laevis. This expression is confined to ectoderm and is excluded from the presumptive neural plate region. Expression of all three genes is dependent upon BMP signaling, with significant differences in how the three genes respond to the BMP antagonist chordin. This correlates with the different expression domain boundaries in vivo for Dlx3 compared to Dlx5 and 6, suggesting that BMP signal attenuation could be the primary factor in determining these different patterns in the gastrula ectoderm.

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.

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.

Patterning of the embryonic ectoderm is dependent upon the action of negative (antineural) and positive (neurogenic) transcriptional regulators. Msx1 and Dlx3 are two antineural genes for which the anterior epidermal-neural boundaries of expression differ, probably due to differential sensitivity to BMP signaling in the ectoderm. In the extreme anterior neural plate, Dlx3 is strongly expressed while Msx1 is silent. While both of these factors prevent the activation of genes specific to the nascent central nervous system, Msx1 inhibits anterior markers, including Otx2 and cement gland-specific genes. Dlx3 has little, if any, effect on these anterior neural plate genes, instead providing a permissive environment for their expression while repressing more panneural markers, including prepattern genes belonging to the Zic family and BF-1. These properties define a molecular mechanism for translating the organizer-dependent morphogenic gradient of BMP activity into spatially restricted gene expression in the prospective anterior neural plate.

Dlx3 is a homeodomain transcription factor and a member of the vertebrate Distal-less family. Targeted deletion of the mouse Dlx3 gene results in embryonic death between day 9.5 and day 10 because of placental defects that alter the development of the labyrinthine layer. In situ hybridization reveals that the Dlx3 gene is initially expressed in ectoplacental cone cells and chorionic plate, and later in the labyrinthine trophoblast of the chorioallantoic placenta, where major defects are observed in the Dlx3 -/- embryos. The expression of structural genes, such as 4311 and PL-1, which were used as markers to follow the fate of different derivatives of the placenta, was not affected in the Dlx3-null embryos. However, by day 10.5 of development, expression of the paired-like homeodomain gene Esx1 was strongly down-regulated in affected placenta tissue, suggesting that Dlx3 is required for the maintenance of Esx1 expression, normal placental morphogenesis, and embryonic survival.

UVS.2 has been known as a cloned cDNA expressed selectively in the hatching gland cells of Xenopus laevis. To determine the molecular identity and function of UVS.2-encoded proteins, antibodies were raised against a bacterially-expressed fusion protein comprising glutathione-S-transferase (GST) and UVS.2. Anti-GST-UVS.2 antibodies inhibited the vitelline envelope digesting activity of the medium (hatching medium) in which dejellied prehatching embryos were cultured. On Western blotting, hatching medium contained 60 kDa and 40 kDa molecules reactive with these antibodies. Whole-mount immunostaining showed a specific localization of UVS.2 protein in the hatching gland cells which appeared first at stage 20, increased in number and intensity to stage 31 then decreased gradually thereafter. Immunoelectron microscopy revealed that UVS.2 protein is localized exclusively in the secretory granules in the hatching gland cells. A cDNA library from the dorsoanterior portion of stage 25 embryos was screened with UVS.2, and a 1.8 kb insert thus cloned contained additional 619bp and 204bp at the 5' and 3' ends of UVS.2, respectively. This clone, designated XHE, contained an open reading frame encoding 514 amino acids including both signal and propeptide sequences. The predicted mature enzyme comprising 425 amino acids consists of about 200 amino acid-long metalloprotease sequence of astacin family at the N-terminus, followed by two repeats of CUB domain each 110 amino acid-length. We conclude that UVS.2 represents an approximately 3/4 C-terminal portion of the hatching enzyme.

Pagliaccio (Pag) is a receptor tyrosine kinase of the Eph family that is expressed in Xenopus embryos in a diverse set of localized tissues. Pag is the Xenopus homolog of Hek-8 (human), Sek-1 (mouse), cek8 (chicken), and RTK-1 (zebrafish). We have investigated the function of this protein by injecting RNA encoding an epidermal growth factor receptor-Pag chimera into early Xenopus embryos. Activation of the chimeric receptor results in a kinase-dependent loss of cell-cell adhesion. This dissociation can be reversed by co-injection of RNA encoding C-cadherin, suggesting that one or more cadherins could be the functional targets for Pag activity.

Cadherins are calcium-binding transmembrane glycoproteins that are important mediators of cell-cell association. Here we describe a novel member of this gene family, zebrafish ventral neural cadherin (VN-cad). Multiple VN-cad transcripts are first detectable by Northern blots at 60% epiboly. In the developing neural tube, VN-cad RNA is first found in the neuroectoderm, directly above the notochord, and later was localized to the neural keel. At the 20-somite stage, VN-cad transcripts are confined to the ventral neural tube, otic vesicle, midbrain, and diencephelon. Transcription of VN-cad RNA continues in adult fish. The embryonic pattern of expression is not significantly disrupted in cyclops or no tail mutants, which lack the floor plate and notochord, respectively. Therefore, neither of these structures is absolutely required for VN-cad expression. The localized pattern of VN-cad expression suggests a possible role for this adhesion molecule in the initial formation and subsequent differentiation of the central nervous system.

Three cDNAs encoding receptor tyrosine kinases (RTKs) of the eph-subfamily have been identified based on their homology to Pagliaccio (Winning and Sargent, 1994, Mech. Dev. 46:219-229). These have been named TCK, Xelk (Xenopus homologue of elk), and PL7a (pag-like clone 7a). Each of these genes is expressed in a distinctive, tissue specific manner during early development. TCK is expressed in pre-somitic mesoderm, caudal somites, midbrain and cement gland. Xelk is expressed in the brain and spinal cord and in the first and fourth visceral arches. PL7a cDNA is expressed throughout the head and in the tip of the tail. All of the genes are represented in maternal mRNA, and are expressed in adult tissues. The Xelk cDNA encodes a protein which is 94% identical to rat elk and therefore is likely to represent the Xenopus homologue of this gene. TCK and PL7a are less related to previously identified eph-subfamily RTKs. The inability to unambiguously assign TCK and PL7a as Xenopus homologues of any previously identified eph RTK leads us to conclude that these cDNAs represent novel members of this family.

Xenopus Distal-less-like-2 (Xdll-2) is a gene encoding a homeodomain protein expressed predominantly in the epidermis of frog embryos. We report here that this epidermal expression is specified by a regulatory 5' flanking DNA region located within 933 bp of the start of transcription. This regulatory DNA also confers upon a globin reporter gene calcium-inducible expression in cultured murine keratinocytes and induction-dependent repression in frog ectodermal cells treated in vitro with activin A. These results reveal a new example of phylogenetically conserved, tissue-specific transcriptional regulation of a homeodomain gene.

This paper identifies a new, developmental role for transcription factor AP-2 in the activation of amphibian embryonic epidermal keratin gene expression. Keratin transcription factor KTF-1 is shown by several criteria to be identical or closely related to AP-2. KTF-1/AP-2 is shown to be tissue-specific from its first transcription in Xenopus embryos, and restricted to a small number of adult tissues, including skin. Epidermis-specific keratin transcription closely follows specification of the embryonic ectoderm in Xenopus, and is subject to regulation by growth factors and embryonic induction. We further show that in mouse basal keratinocytes, a KTF-1/AP-2-like factor is present and binds to a DNA sequence previously shown to be important in the regulation of the keratin K14 gene, which is actively expressed in these cells. Thus, the study of AP-2 and its role in the regulation of keratin gene transcription should enhance our understanding of both amphibian embryonic development and mammalian skin differentiation.

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.

Xenopus-posterior (Xpo) is a gene that is activated at or shortly after the midblastula transition (MBT). The RNA accumulates to a relatively low level, which remains constant until gastrulation, then rapidly and transiently increases in posterior ectoderm and mesoderm. A single copy of a putative finger motif, of the 'CCHC' type, is located near the carboxyl terminus. One or two copies of similar sequence motifs are found in the nucleocapsid protein of retroviruses where they are involved in protein-RNA interactions, and in cellular nucleic acid binding protein (CNBP), a protein that binds to the sterol regulatory element. Xpo expression is induced in ectodermal explants by treatment with basic fibroblast growth factor (bFGF) and with polypeptide growth factors found in medium conditioned by the Xenopus XTC cell line (XTC-CM). Taken together, these properties suggest a possible role for Xpo in the organization of the anteroposterior axis during development.

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.

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.

A modified cloning method designed to produce differential complementary DNA libraries permits the isolation of sequences that are present in the RNA population of any developmental stage or tissue, but are not present or are much less abundant in another stage or tissue. Selective complementary DNA cloning is especially useful when the differentially expressed RNA's are of low to moderate abundance in the cells in which they occur. A class of cytoplasmic polyadenylated RNA's differentially expressed in gastrula embryos of Xenopus laevis (DG RNA's) has been isolated. These DG RNA's occur very rarely or not at all in unfertilized eggs and blastulae, accumulate as the result of transcription before and during gastrulation, and, with some exceptions, decline in abundance as development proceeds. Many of these RNA molecules appear to be translated at the gastrula stage. Thus, DG RNA's may encode proteins that are important in the process of gastrulation.

This article considers some aspects of the storage of macromolecules in the oocyte of Xenopus laevis and the activation of previously unexpressed genes during early embryogenesis. The large quantity and complex nature of poly(A)+ RNA accumulated in the egg provides the cleavage embryo with a supply of mRNA sufficient to sustain protein synthesis for several hours of development. Onset of gene activity at the midblastula transition (MBT) leads to the synthesis and accumulation of molecules of various RNA classes, including tRNAs, rRNAs, mRNAs and mitochondrial RNAs. At gastrulation the poly(A)+ RNA population is still qualitatively similar to that of the egg but some sequences not present in egg RNA have accumulated by this time. Through the use of a subtractive cDNA cloning procedure we have prepared a library of sequences that represent genes activated for the first time between MBT and gastrula. A study of several of these cDNA clones suggests that genes in this class are restricted in their activity to embryonic and tadpole stages.

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.