High throughput sequencing of a mouse keratinocyte library was used to identify an expressed sequence tag with homology to the epidermal growth factor (EGF) family of growth factors. We have named the protein encoded by this expressed sequence tag Epigen, for epithelial mitogen. Epigen encodes a protein of 152 amino acids that contains features characteristic of the EGF superfamily. Two hydrophobic regions, corresponding to a putative signal sequence and transmembrane domain, flank a core of amino acids encompassing six cysteine residues and two putative N-linked glycosylation sites. Epigen shows 24 -37% identity to members of the EGF superfamily including EGF, transforming growth factor ␣, and Epiregulin. Northern blotting of several adult mouse tissues indicated that Epigen was present in testis, heart, and liver. Recombinant Epigen was synthesized in Escherichia coli and refolded, and its biological activity was compared with that of EGF and transforming growth factor ␣ in several assays. In epithelial cells, Epigen stimulated the phosphorylation of c-erbB-1 and mitogen-activated protein kinases and also activated a reporter gene containing enhancer sequences present in the c-fos promoter. Epigen also stimulated the proliferation of HaCaT cells, and this proliferation was blocked by an antibody to the extracellular domain of the receptor tyrosine kinase c-erbB-1. Thus, Epigen is the newest member of the EGF superfamily and, with its ability to promote the growth of epithelial cells, may constitute a novel molecular target for wound-healing therapy.

Temporal colinearity describes a correspondence between the spatial ordering of Hox genes within their clusters (in the direction 3′ to 5′) and the time of their first expression (earlier to later) during embryonic development (Izpisúa‐Belmonte et al. [1991] EMBO J. 10:2279–2289). It suggests that activation of each Hox gene might be controlled in some way by its position within the cluster. So far, in situ hybridization experiments on vertebrate embryos have provided clear evidence of temporal colinearity only for “posterior” Hox genes (5′ located, AbdB related). We now describe a search in the chick embryo for evidence of temporal colinearity in the expression of some anterior Hox genes (Hoxb‐1, b‐3, b‐4, b‐6, and a‐9). Clear evidence for temporal colinearity was seen in neural tube tissue adjacent to the first few somites. Here, there were delays in the expression of Hoxb‐3 following b‐1, Hoxb‐4 following b‐3, and Hoxb‐6 following b‐4. Temporal colinearity was also detected in anterior primitive streak tissue. Hox gene expression reached both the neural tube and the anterior streak following forward spreading from posteriormost parts of the primitive streak. Overall, therefore, temporal colinearity was seen as sequential waves of Hox gene expression that proceeded forward (3′ genes before 5′ genes) along the developing chick embryo. Within posterior primitive streak tissue, there was only limited evidence for temporal colinearity. We discuss these results in terms of possible models for the establishment of Hox gene expression patterns. © 1996 Wiley‐Liss, Inc.

By use of wholemount in situ hybridization, we show how expression of the chicken homeobox gene Hoxd-4 commences in the posterior part of the primitive streak and then spreads forward, covering most of the primitive streak by the 2 somite stage, covering the entire primitive streak by the 5 somite stage, reaching the somite lkomite 2 level of the neural tube by the 9 somite stage, and reaching the rhombomere G/rhombomere 7 junction of the hindbrain by the 15 somite stage. Forward spreading does not depend upon cell migration, as was evidenced by vital dye (DiI) cell marking experiments. Furthermore, forward spreading does not apparently require tissue continuity since it could not be blocked by impermeable (glass) barriers surgically implanted to divide embryonic tissues. As forward spreading of chick Hoxd-4 proceeds, the domain of expression separates, at late primitive streak stages, into "anterior" and "posterior zones," with an intervening "intermediate zone" of weak or non-expression. Clear anterior and posterior zones were also found for Hoxa-3 and a-4 expression in late primitive streak stage mouse embryos. We present evidence that the anterior zone corresponds with the "definitive" domain of Hox gene expression, as has earlier been extensively characterized in midgestation embryos. The posterior zone is transitory, probably persisting only for the duration of the primitive streak, and it is a region of intense Hox expression in primitive streak tissue, Hensen's node, and adjacent regions of neurectoderm and mesoderm. We suggest that the posterior zone marks the source of a morphogen which is the primary activator of Hox gene expression, and we discuss possible models for the mechanism of forward spreading in expression. 0 1994 Wiley-Liss, Inc.

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