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 downregulated in affected placenta tissue, suggesting that Dlx3 is required for the maintenance of Esx1 expression, normal placental morphogenesis, and embryonic survival.

We report experiments with Xenopus laevis, using both intact embryos and ectodermal explants, showing that the transcription factor AP2␣ 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 AP2␣ 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. AP2␣ 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 AP2␣ requires some attenuation of endogenous BMP signaling; however, this process occurs at a lower threshold for AP2␣. Furthermore, AP2␣ expression domains are larger than for other NC factors. Loss-of-function experiments with antisense AP2␣ morpholino oligonucleotides result in severe reduction in the NC territory. These results support a central role for AP2␣ in NC induction. We propose a model in which AP2␣ expression, along with inactivation of NC inhibitory factors such as Dlx3, establish a feedback loop comprising AP2␣, Sox9, and Slug, leading to and maintaining NC specification.bone morphogenetic protein ͉ Wnt ͉ Sox9 ͉ Slug ͉ morpholino antisense oligonucleotides

The rat serum albumin gene has been isolated from a recombinant library containing the entire rat genome cloned in the X phage Charon 4A. Preliminary R-loop and restriction analysis has revealed that this gene is split into at least 14 fragments (exons) by 13 intervening sequences (introns), and that it occupies a minimum of 14.5 kilobases of genomic DNA.Recent advances in recombinant DNA technology have made it possible to obtain virtually any desired single-copy genomic sequence in cloned form, provided an appropriate probe is available. We have used these techniques to isolate the rat serum albumin gene. Serum albumin synthesis is one of the major characteristics of vertebrate liver. Observation of the activity and state of this gene during development and in adult tissues should be informative as to the process of terminal differentiation. Albumin synthesis is essentially constitutive, but does respond significantly to a variety of stimuli (1). It is also expressed to variable extents in different hepatoma cell lines (2). The availability of cloned albumin genomic DNA will greatly facilitate the study of this variable expression, particularly at the level of transcript processing.Determination of the sequence organization of the albumin gene is also of interest, especially with regard to the disposition of repetitive elements and intervening sequences. Although regulatory (3) and evolutionary (4) significance has been postulated, the functional role, if any, of these striking features of eukaryotic genomes remains unknown. The comparative studies that will be possible as other genes are extracted from the rat and related species can be expected to provide considerable insight into this fascinating problem. MATERIALS AND METHODSRat Genome Library. High molecular weight liver DNA was extracted from an adult male Sprague-Dawley rat (Simonsen Labs, Gilroy, CA) by the method of Blin and Stafford (5) and aliquots were digested with EcoRI (Boehringer Mannheim) under conditions adjusted to cleave either one-third or one-fifth of the EcoRI sites in an equivalent amount of bacteriophage X DNA. The fragments resulting from this partial digestion were sedimented through a 10-30% sucrose gradient; the material between 10 and 20 kilobases (kb) was recovered by ethanol precipitation. A sample of this rat DNA (2.5 ,g) was ligated with 8.5 ,ig of a preparation of Charon 4A "cloning fragments" (6, 7). This recombinant DNA was packaged in vitro by using extracts from defective X lysogens provided by N. Sternberg (6). The method used was that of Hohn and Murray (8). Approximately 2,000,000 independent clones were obtained. The library was amplified 100,000-fold by subconfluent plating on Escherichia coli strain DP5OSupF (9).cDNA Clones. cDNA was synthesized from purified albumin mRNA as described (10). This cDNA contained a small amount of full-length material and had a number average size of approximately 1000 nucleotides. It was rendered double-stranded by sequential treatment with E. coli DNA polymerase I and S1 nuc...

Expression of the Xenopus homolog of the mammalian transcription factor AP-2alpha (XAP-2) is activated throughout the animal hemisphere shortly after the midblastula transition, and becomes restricted to prospective epidermis by the end of gastrulation, under the control of BMP signal modulation. Elevated expression in the future neural crest region begins at this time. Ectopic expression of XAP-2 can restore transcription of epidermal genes in neuralized ectoderm, both in ectodermal explants and in the intact embryo. Likewise, loss of XAP-2 function, accomplished by injection of antisense oligonucleotides or by overexpression of antimorphic XAP-2 derivatives, leads to loss of epidermal and gain of neural gene expression. These treatments also result in gastrulation failure. Thus, AP-2 is a critical regulator of ectodermal determination that is required for normal epidermal development and morphogenesis in the frog embryo.

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.

DG81 is a cDNA clone derived from a subtracted library containing those RNA molecules that are present in gastrulae but absent from eggs of the frog Xenopus laevis. DG RNAs (where DG indicates differentially expressed in gastrula) represent the products of new transcription activated in the embryo at the midblastula transition or shortly thereafter. DG81 RNA is first detected in middle to late gastrulae, peaks in abundance in early tadpoles, and declines to background levels by the end of metamorphosis. Detailed developmental studies with a group of DG cDNA clones demonstrated that the rates of both their initial accumulation and their eventual disappearance differed, so that distinct types of behavior could be assigned to different DG genes. Some DG RNAs appear within <1 hr after the midblastula transition and persist for just 1 day of development, whereas others appear slightly later during gastrulation and persist through tadpole development. We selected DG clones representing different developmental behavior for further study of their biological and molecular properties. One of the cDNA clones studied, DG81, proved to be derived from a mRNA that encodes a type I epidermal keratin, as is outlined in the following sections of this paper. MATERIALS AND METHODSPreparation of RNA. Total RNA from gastrula stage embryos was prepared by a modification of the method of Chirgwin et al. (8). After homogenization in guanidinium thiocyanate the sample was extracted with 1 vol each of phenol, chloroform, and 10 mM Tris HCl, pH 7.5/1% NaDodSO4/1 mM EDTA and precipitated with 2 vol of 95% ethanol for 2 hr at -20°C. The RNA was redissolved in guanidinium thiocyanate solution before pelleting through a 5.7 M CsCl/1 mM EDTA gradient to be used for cDNA preparation or was precipitated two times with 2.5 M LiCl prior to RNA dot blot analysis. Poly(A)+ RNA was prepared by two cycles of oligo(dT) chromatography (9).RNA Blot Hybridization. RNA samples were bound to nylon membranes (Zetabind, AMF Cuno) as dots or as electroblots after separation on CH3HgOH/agarose gels (10) and hybridized with nick-translated probes under the conditions described by Church and Gilbert (11).cDNA Clones. DG81 is a clone selected from the DG library on the basis of its developmental behavior (see Results).Since DG81 is not full-length, homologous cDNA clones were selected from a library prepared by Susan Haynes from gastrula RNA by the method of Okayama and Berg (12). One such clone that is almost full-length (see Results) was named pC8128. We 5413The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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.

Abstract. The Distal-less-related homeodomain gene Dlx3 is expressed in terminally differentiated murine epidermal cells. Ectopic expression of this gene in the basal cell layer of transgenic skin results in a severely abnormal epidermal phenotype and leads to perinatal lethality. The basal cells of affected mice ceased to proliferate, and expressed the profilaggrin and loricrin genes which are normally transcribed only in the latest stages of epidermal differentiation. All suprabasal cell types were diminished and the stratum corneum was reduced to a single layer. These data indicate that Dlx3 misexpression results in transformation of basal cells into more differentiated keratinocytes, suggesting that this homeoprotein is an important regulator of epidermal differentiation.