In human cutaneous malignant melanoma, a predominance of activated mutations in the N-ras gene has been documented. To obtain a mouse model most closely mimicking the human disease, a transgenic mouse line was generated by targeting expression of dominant-active human N-ras (N

Here we describe the generation of the Nes-Cre1 transgenic mouse line in which Cre recombinase expression is controlled by the rat nestin promoter and intron 2 enhancer. This line has previously been used for conditional loss-of-function studies of various genes in the central nervous system and first branchial arch ectoderm. Here we report the detailed temporal and spatial recombination pattern of Nes-Cre1 using three different reporters of Cre-mediated recombination, ROSA26R (R26R), Z/AP, and Z/EG. Cre/loxP recombination was detected in embryos as early as the head-fold stage. By embryonic day (E)15.5 recombination occurred in virtually all cells of the nervous system and unexpectedly also in somite-derived tissues and kidneys. Tissues with little or no recombination included heart, liver, thymus, and lung. This study suggests that Nes-Cre1-mediated recombination occurs in progenitor cell types present in the neuroectoderm, the developing mesonephros, and the somites.

In hydra, the endodermal epithelial cells carry out the digestive function together with the gland cells that produce zymogens and express the evolutionarily conserved gene Kazal1. To assess the hydra Kazal1 function, we silenced gene expression through double-stranded RNA feeding. A progressive Kazal1 silencing affected homeostatic conditions as evidenced by the low budding rate and the induced animal death. Concomitantly, a dramatic disorganization followed by a massive death of gland cells was observed, whereas the cytoplasm of digestive cells became highly vacuolated. The presence of mitochondria and late endosomes within those vacuoles assigned them as autophagosomes. The enhanced Kazal1 expression in regenerating tips was strongly diminished in Kazal1(-) hydra, and the amputation stress led to an immediate disorganization of the gland cells, vacuolization of the digestive cells and death after prolonged silencing. This first cellular phenotype resulting from a gene knock-down in cnidarians suggests that the Kazal1 serine-protease-inhibitor activity is required to prevent excessive autophagy in intact hydra and to exert a cytoprotective function to survive the amputation stress. Interestingly, these functions parallel the pancreatic autophagy phenotype observed upon mutation within the Kazal domain of the SPINK1 and SPINK3 genes in human and mice, respectively

Hydra regenerate throughout their life. We previously described early modulations in cAMP-response element-binding protein (CREB) DNA-binding activity during regeneration. We now show that the Ser-67 residue located in the P-box is a target for posttranslational regulation. The antihydra CREB antiserum detected CREB-positive nuclei distributed in endoderm and ectoderm, whereas the phosphoSer133-CREB antibody detected phospho-CREB-positive nuclei exclusively in endodermal cells. During early regeneration, we observed a dramatic increase in the number of phospho-CREB-positive nuclei in head-regenerating tips, exceeding 80% of the endodermal cells. We identified among CREBbinding kinases the p80 kinase, which showed an enhanced activity and a hyperphosphorylated status during head but not foot regeneration. According to biochemical and immunological evidence, this p80 kinase belongs to the Ribosomal protein S6 kinase family. Exposure to the U0126 mitogen-activated protein kinase kinase inhibitor inhibited head but not foot regeneration, abolished CREB phosphorylation and activation of the early gene HyBra1 in head-regenerating tips. These data support a role for the mitogen-activated protein kinase͞ribosomal protein S6 kinase͞ CREB pathway in hydra head organizer activity. S pecies that display regenerative capacities can be found in most animal phyla, from invertebrates to vertebrates, likely representing an ancestral feature lost several times during evolution. The central issue concerning regeneration is the unfolding of developmental programs in adult tissues, ending ultimately in a de novo morphogenesis. That issue is poorly understood, but some stages might rely on conserved ancestral mechanisms. Hydra, which belong to the Cnidaria phylum, a sister group to bilaterians, bud and regenerate throughout their life. Hence their developmental programs stay permanently accessible. Hydra are made up of two cell layers, ectoderm and endoderm, separated by the mesoglea. After midgastric section, regeneration of the missing part is achieved within 3 days, resulting from differentiation of cells from the gastric region in the absence of cell proliferation during the first day.Although early modulations in gene expression have been identified during regeneration (see ref. 1), little is known about the signaling mechanisms that control regeneration (2, 3). Diacylglycerol treatment induces formation of multiple heads (4), whereas inhibition of PKC and mitogen-activated protein kinase (MAPK) pathways selectively block head regeneration and initiation of budding (5-7). In hydra, conserved regulatory elements have been tested in gel retardation assays, and a significant modulation during both apical and basal regeneration was detected with the cAMP response element (CRE). The hydra CRE-binding protein (CREB) gene was cloned, showing a high level of similarity with vertebrate cognate genes (8, 9). In vertebrates, CREB mediates the response to a large array of extracellular signals to the nucleus through posttranslational modifi...

The secreted signaling molecule fibroblast growth factor 8 (Fgf8) is an essential component of certain embryonic signaling centers including the mid-hindbrain (isthmic) organizer, the first branchial arch (BA1), and the apical ectodermal ridge (AER). In these signaling centers Fgf8 transcripts are expressed in a dynamic and transient fashion, but the mechanism by which this highly specific expression pattern is established remains largely unknown. We used DNA sequence comparisons coupled to transgenic approaches to obtain insight into the structure and function of regulatory elements in the Fgf8 locus. First, a bacterial artificial chromosome (BAC) containing the mouse Fgf8 gene partially rescues the embryonic lethality of Fgf8-deficient mice and controls Fgf8-specific gene expression of a coinjected lacZ reporter transgene. Second, sequence comparison of vertebrate Fgf8 loci revealed evolutionarily highly conserved noncoding sequences that were unexpectedly located mainly 3' of the Fgf8 coding region. Third, in transgenic mice some of these elements were sufficient to target expression to the AER, tail bud, and brain, including the isthmic organizer, indicating that they may represent Fgf8 cis-acting elements. Collectively, these data identify novel regulatory elements of the Fgf8 gene sufficient to drive expression to regions of known Fgf8 activity.

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Supplementary Table 1 from Metastasizing Melanoma Formation Caused by Expression of Activated N-Ras^Q61K on an INK4a-Deficient Background