The lifelong self‐renewal of the epidermis is driven by a progenitor cell population with high proliferative potential. To date, the upstream signals that determine this potential have remained largely elusive. Here, we find that insulin and insulin‐like growth factor receptors (IR and IGF‐1R) determine epidermal proliferative potential and cooperatively regulate interfollicular epidermal morphogenesis in a cell autonomous manner. Epidermal deletion of either IR or IGF‐1R or both in mice progressively decreased epidermal thickness without affecting differentiation or apoptosis. Proliferation was temporarily reduced at E17.5 in the absence of IGF‐1R but not IR. In contrast, clonogenic capacity was impaired in both IR‐ and IGF‐1R‐deficient primary keratinocytes, concomitant with an in vivo loss of keratin 15. Together with a reduction in label‐retaining cells in the interfollicular epidermis, this suggests that IR/IGF‐1R regulate progenitor cells. The expression of dominant active Rac rescued clonogenic potential of IR/IGF‐1R‐negative keratinocytes and reversed epidermal thinning in vivo. Our results identify the small GTPase Rac as a key target of epidermal IR/IGF‐1R signalling crucial for proliferative potential and interfollicular morphogenesis.
Two main processes are involved in driving ventral mesendoderm internalization in the Xenopus gastrula. First, vegetal rotation, an active movement of the vegetal cell mass, initiates gastrulation by rolling the peripheral blastocoel floor against the blastocoel roof. In this way, the leading edge of the internalized mesendoderm is established, that remains separated from the blastocoel roof by Brachet's cleft. Second, in a process of active involution, blastopore lip cells translocate on arc-like trails around the tip of Brachet's cleft. Hereby the lower, Xbra-negative part of the lip moves toward the interior, to contribute mainly to endoderm. In contrast, the upper, Xbra-expressing part moves toward the blastocoel roof-apposed surface of the involuted mesoderm, and eventually becomes inserted into this surface. Vegetal rotation and active mesoderm surface insertion persist over much of gastrulation ventrally. Both processes are also active dorsally. In fact, internalization processes generally spread from dorsal to ventral, though at different rates, which suggests that they are independently controlled. Ventrally and laterally, mesoderm occurs not only in the marginal zone, but also in the adjacent blastocoel roof. Such blastocoel roof mesoderm shares properties with the remaining, ectodermal roof, that are related to its function as substratum for mesendoderm migration. It repels involuted mesoderm, thus contributing to separation of cell layers, and it assembles a fibronectin matrix. These properties change as the blastocoel roof mesoderm moves into the blastopore lip during gastrulation.
In order to facilitate in situ detection of biomolecules in large sample series the processing of whole-mount specimens has been automated. A freely programmable liquid handling system is described by which embryos or similar biological materials are processed. Possible applications include in situ hybridization (ISH), immunocytochemistry (ICC) or reporter gene assays. Process times required for the preparation of whole-mount in situ hybridizations in Drosophila, Xenopus, Gallus and in hydroids were - in part - significantly reduced as compared with manual processing. Application of automated in situ detection (AISD) in random screening is demonstrated in hydroids. Potential further applications are discussed.
Cadherin switching is thought to contribute to melanoma progression. E-cadherin expression is downregulated, facilitating the release of contacts with keratinocytes, while N-cadherin expression is increased, potentially contributing to more migration. Proteolytic cleavage of the cadherin extracellular domain, a process called ectodomain shedding, is one way to decrease cadherin cell surface expression. In addition, the released ectodomain could actively contribute to a more invasive phenotype. To examine if melanoma progression correlates with increased cadherin ectodomain shedding, we tested the presence of N- and E-cadherin extracellular domains in different melanoma cell lines and the presence of E-cadherin in sera of patients. Shedding occurs and is regulated in several melanoma cell lines expressing these cadherins. No correlation could be found between cadherin shedding and invasive capacity of the cell lines. However, we did find a significant increase in serum E-cadherin levels of melanoma patients with advanced disease correlating with increased S100 tumor marker values, suggesting that increased cadherin shedding may contribute to melanoma progression.
Cadherin shedding affects migration and occurs in development and cancer progression. By examining the in vivo biological function of the extracellular cadherin domain (CEC1-5) independently of the shedding process itself, we identified a novel function for cadherins in convergent extension (CE) movements in Xenopus. CEC1-5 interfered with CE movements during gastrulation. Unexpectedly, CEC1-5 did not alter cell aggregation or adhesion to cadherin substrates. Instead, gastrulation defects were rescued by a membrane-anchored cadherin cytoplasmic domain, the polarity protein atypical PKC (aPKC) or constitutive active Rac, indicating that CEC1-5 modulates a cadherin-dependent signalling pathway. We found that the cadherin interacts with aPKC and, more importantly, that the extracellular domain alters this association as well as the phosphorylation status of aPKC. This suggests that CE movements require a dynamic regulation of cadherin-aPKC interaction. Our results show that cadherins play a dual role in CE movements: a previously identified adhesive activity and an adhesion-independent function that requires aPKC and Rac, thereby directly connecting cadherins with polarity. Our results also suggest that increased cadherin shedding, often observed in cancer progression, can regulate migration and invasion by modulating polarity protein activity.
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