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Funder: École Polytechnique Fédérale de Lausanne; Id: http://dx.doi.org/10.13039/501100001703, Funder: Medical Research Council; Id: http://dx.doi.org/10.13039/501100007155, Funder: Wellcome Trust; Id: http://dx.doi.org/10.13039/100010269, Funder: Wellcome; Id: http://dx.doi.org/10.13039/100010269, Funder: EPFL; Id: http://dx.doi.org/10.13039/501100001703, Why only certain species can regenerate their appendages (e.g. tails and limbs) remains one of the biggest mysteries of nature. Unlike anuran tadpoles and salamanders, humans and other mammals cannot regenerate their limbs, but can only regrow lost digit tips under specific circumstances. Numerous hypotheses have been postulated to explain regeneration‐incompetency in mammals. By studying model organisms that show varying regenerative abilities, we now have more opportunities to uncover what contributes to regeneration‐incompetency and functionally test which perturbations restore appendage regrowth. Particularly, Xenopus laevis tail and limb, and mouse digit tip model systems exhibit naturally occurring variations in regenerative capacities. Here, we discuss major hypotheses that are suggested to contribute to regeneration‐incompetency, and how species with varying regenerative abilities reflect on these hypotheses.

Senescence-associated β-galactosidase (SAβ-gal) is a convenient histological technique used to identify senescent cells. Its ease of use is helpful to initially screen and detect senescent cells in heterogeneous cell populations both in vitro and in vivo. However, SAβ-gal staining is not an unequivocal marker of the senescent state, and diagnosis of such usually requires additional markers demonstrating an absence of proliferation and expression of cell-cycle inhibitors. Nonetheless, SAβ-gal remains one of the most widely used biomarkers of senescent cells. Recently, by measuring SAβ-gal activity, the expression of the cyclin-dependent kinase inhibitor p21 (waf1/cip1) and demonstrating a lack of proliferation, we identified senescent cells in the developing embryo. This chapter describes the methods for identifying cellular senescence in the embryo, detailing protocols for the detection of SAβ-gal activity in both sections and at the whole mount level, and immunohistochemistry protocols for the detection of additional biomarkers of senescence. This work was funded by Grants SAF2010-18829 and SAF2013-49082-P to W.M.K. from the Spanish Ministry for Economy and Competitiveness, the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) from the Generalitat de Catalunya, and CRG core funding

Cellular senescence is an irreversible form of proliferative arrest, historically linked to tumour suppression and aging. Recent discoveries, however, have extended its known role to include complex biological processes such as tissue repair, tumour promotion and age-related disorders. These new insights are redefining our view of cellular senescence, that unlike a static endpoint, senescence represents a collective phenotype, composed of complex networks of effector programs. The biological outcome of these effector programs varies depending on the cellular context and nature of the stress. Here, we investigated the functional role of senescence in two polar contexts: first, within the epidermal stem cell population during the aging process, and second, during embryonic development. We identified that the primitive Keratin-15 positive (Krt-15) hair follicle stem cell population increases in number in an age-dependent manner, but exhibits decreased functional capacity and an inability to tolerate stress. While there was no evidence of Krt-15 stem cells entering directly into senescence, we identified an age-associated imbalance in epidermal Jak-Stat signalling surrounding the stem cells, reminiscent of extrinsic senescent cells that inhibit stem cell function. These findings suggest that epidermal stem cell decline contributes to the aging phenotype of tissue, and that this may be directed by extrinsic senescence. Conversely, we also describe cellular senescence as a normal developmental mechanism that occurs during mammalian embryonic development, specifically in the apical ectodermal ridge (AER) and the roof plate of the hindbrain neural tube. Interestingly, developmental senescence is strictly dependent on p21, wherein mice deficient in p21 present with defects in embryonic senescence, AER maintenance and abnormal limb patterning. Additionally, we found significant gene-expression overlap between developmental and oncogene-induced senescence in the adult, suggesting a commonality in function. Furthermore, we found that the underlying mesenchyme is a source for senescence instruction, while the fate of senescent cells is both apoptosis and immune-mediated clearance. These findings indicate that senescence also functions in non-pathological states and plays an instructive role during embryonic development, and suggests that senescence may have evolved initially as a developmental mechanism that was subsequently adapted for its role in adult life., La senescencia celular es una forma irreversible de paro proliferativo ligado históricamente a la supresión tumoral y al envejecimiento. Sin embargo, estudios recientes han demostrado que también juega un papel en complejos procesos biológicos como reparación tisular, promoción tumoral y desórdenes relacionados con la edad. Estos nuevos conocimientos están redefiniendo nuestra visión de la senescencia celular, no tanto como un proceso estático sino como un fenotipo colectivo compuesto por una compleja red de programas efectores. El resultado biológico de estos programas efectores varía dependiendo del contexto celular y la naturaleza del estrés. Aquí, hemos investigado el papel funcional de la senescencia en dos contextos antagónicos: primero, en la población de células madre del epidermis durante el proceso de envejecimiento, y segundo, durante el desarrollo embrionario. Hemos demostrado que la población de células madre del folículo piloso que expresan queratina 15 aumenta en número en función de la edad, pero presenta una disminución de la capacidad funcional y una incapacidad de tolerar el estrés. Si bien no hemos encontrado ninguna evidencia de que las células madre que expresan queratina 15 entren directamente a senescencia, hemos identificado un desequilibrio en la vía de señalización de Jak-Stat alrededor de las células madre, posiblemente procedente de células senescentes extrínsecas que inhiben la función de las células madre. Estos descubrimientos sugieren que el declive de las células madre epidérmicas contribuye al envejecimiento del tejido y que puede estar dirigido por la senescencia extrínseca. Por otra parte, también hemos descrito el proceso celular de la senescencia como un mecanismo normal en el desarrollo embrionario de los mamíferos, específicamente en la cresta ectodérmica apical (AER) y en la placa del techo del tubo neural. Cabe notar que la senescencia del desarrollo es estrictamente dependiente de p21. Así, los ratones deficientes de p21 presentan defectos en la senescencia embrionaria, el mantenimiento del AER y la formación de las extremidades. Además, hemos visto que hay un solapamiento importante en la expresión génica de la senescencia del desarrollo y la senescencia inducida por oncogenes en los adultos, sugiriendo una similitud en la función. Asimismo, hemos descubierto que el mesénquima subyacente actúa como instructor de la senescencia, y que el destino de las células senescentes es entrar en apoptosis y ser eliminadas por el sistema inmunitario. Estos hallazgos indican que la senescencia también tiene lugar en estados no patológicos y que juega un papel instructivo durante el desarrollo embrionario. Hacen pensar que la senescencia podría haber evolucionado inicialmente como un mecanismo del desarrollo que fue adaptándose para su papel en la vida adulta.

Background Congenital malformations involving the Müllerian ducts are observed in around 5% of infertile women. Complete aplasia of the uterus, cervix, and upper vagina, also termed Müllerian aplasia or Mayer–Rokitansky–Kuster–Hauser (MRKH) syndrome, occurs with an incidence of around 1 in 4500 female births, and occurs in both isolated and syndromic forms. Previous reports have suggested that a proportion of cases, especially syndromic cases, are caused by variation in copy number at different genomic loci. Methods In order to obtain an overview of the contribution of copy number variation to both isolated and syndromic forms of Müllerian aplasia, copy number assays were performed in a series of 63 cases, of which 25 were syndromic and 38 isolated. Results A high incidence (9/63, 14%) of recurrent copy number variants in this cohort is reported here. These comprised four cases of microdeletion at 16p11.2, an autism susceptibility locus not previously associated with Müllerian aplasia, four cases of microdeletion at 17q12, and one case of a distal 22q11.2 microdeletion. Microdeletions at 16p11.2 and 17q12 were found in 4/38 (10.5%) cases with isolated Müllerian aplasia, and at 16p11.2, 17q12 and 22q11.2 (distal) in 5/25 cases (20%) with syndromic Müllerian aplasia. Conclusion The finding of microdeletion at 16p11.2 in 2/38 (5%) of isolated and 2/25 (8%) of syndromic cases suggests a significant contribution of this copy number variant alone to the pathogenesis of Müllerian aplasia. Overall, the high incidence of recurrent copy number variants in all forms of Müllerian aplasia has implications for the understanding of the aetiopathogenesis of the condition, and for genetic counselling in families affected by it.