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Resumen del trabajo presentado en Experimental Biology 2018, celebrado en San Diego (Estados Unidos) del 21 al 25 de abril de 2018., The developing vertebrate limb has long proved as an excellent system for studying the mechanisms involved in pattern formation and morphogenesis and more recently in transcriptional regulation and morphological evolution. To elucidate the stage-specific expression profiles of the components of the developing limb, we have generated the temporal transcriptome of the limb progenitors and of the overlying ectoderm separately. Our study has uncovered a collinear activation of Hoxc genes in the limb ectoderm that we have validated by in situ hybridization. However, while members of the HoxA and HoxD clusters show complex and dynamic patterns of expression during limb development that correlate with the morphology of the different limb segments, no specific function for the HoxC or HoxB clusters has been identified (1-3). To investigate the function of Hoxc genes in the limb ectoderm, we have reexamined the HoxC cluster null mice. Remarkably, and despite exhibiting normal terminal phalanges, these mice didn¿t form claws (anonychia). Morphological and immunohistochemical analysis identified a failure in the differentiation of the main components of the nail/claw organ. To unravel the transcriptional regulation of Hoxc genes in the limb ectoderm, we used the ATACseq technique. Using this approach, we identified two putative regulatory regions which activity was tested in mouse transgenic enhancer assays. It is currently considered that Hox genes have played a key role in the evolution of morphological traits, probably associated with changes in their regulatory landscapes (4). Given that the form and size of the distal limb integumentary organ (nail, claw or hoof) correlates with that of the distal phalanx and that the development of hooves was a major innovation in the evolution of a cursorial lifestyle, we are also exploring the possible implication of Hoxc genes in the nail/claw/hoof transition.

Resumen del trabajo presentado al XL Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Barcelona del 23 al 26 de octubre de 2017., Hox genes are a family of important developmental regulators critical for the establishment of the basic body plan of bilaterian animals. In vertebrates, Hox genes are organized in four clusters (A, B, C and D) two of which, HoxA and HoxD cluster, are critical for the outgrowth and patterning of the tetrapod limb (Kmita et al. 2005; Woltering et al. 2014). In contrast, no specific function for the HoxC or HoxB clusters has been identified during limb development, as no obvious limb defect is observed in the complete absence of either of these clusters (Suemori & Noguchi, 2000). Recently, with the generation of the temporal expression profiles of the limb progenitors and of the ectoderm, separately, we have exposed a temporal collinear activation of the HoxC cluster in the limb ectoderm. Furthermore, by in situ hybridization we found a progressive restriction of 5¿Hoxc genes to the nail primary fi eld, correlating with the nail defect exhibited by Hoxc13 mutants (Godwin & Cappechi, 1998). A re-examination of the phenotype of mice lacking the entire HoxC cluster (Suemori & Noguchi, 2000) revealed remarkable nail development defects suggesting the involvement of Hoxc genes in nail/claw morphogenesis. As a first step to identify the cis-regulatory mechanisms underlying Hoxc gene transcription, we performed ATAC-seq of the limb ectoderm and identified two conserved regions 5' of the HoxC cluster that we are currently evaluating for activity in mouse transgenesis. Moreover, given the role of Hox genes in the evolution of form, we are expanding our analysis to other species with a particular development of nail organ (hoof) in order to investigate the role of Hoxc genes in the nail-claw-hoof transition and its putative contribution to the diversity of distal limb structures.

In this commentary we focus on the function of FGFs during limb development and morphogenesis. Our goal is to understand, interpret and, when possible, reconcile the interesting findings and conflicting results that remain unexplained. For example, the cell death pattern observed after surgical removal of the AER versus genetic removal of the AER-Fgfs is strikingly different and the field is at an impasse with regard to an explanation. We also discuss the idea that AER function may involve signaling components in addition to the AER-FGFs and that signaling from the non-AER ectoderm may also have a significant contribution. We hope that a re-evaluation of current studies and a discussion of outstanding questions will motivate new experiments, especially considering the availability of new technologies, that will fuel further progress toward understanding the intricate ectoderm-to-mesoderm crosstalk during limb development., Grant sponsor: Spanish Ministry of Science; Grant number: BFU2014-57216-P; Grant sponsor: University of Southern California.

Resumen del póster presentado al 11th Meeting Spanish Society for Developmental Biology, celebrado en Girona del 19 al 21 de octubre de 2016., Limbs develop from limb buds composed of a core of limb progenitor cells covered by the surface ectoderm. Cell trac- king experiments in chick showed that the limb progenitors located at the distal tip of the limb bud progressively res- trict their developmental potential in the proximo-distal axis. The mechanisms and models by which the limb progeni- tors acquire progressively more distal fates are subject to intense investigation and debate. To shed light into this issue we have generated expression profiles for each of the progressive differentiation step by RNA-seq. In parallel we have also generated the temporal transcriptome of the distal ectoderm including the Apical ectodermal ridge (the AER). We seek to unravel expression pattern specific of each of the progressive stages of the limb progenitors and of the AER trying to identify essential descriptors of their biological state (morphogenetic potential). Of 46,480 Ensembl-anno- tated genes, 3,416 were differentially expressed between ectoderm and mesoderm. Intriguingly, we observed that the number of genes changing between stages is similar between ectoderm and mesoderm, with the bulk of changes occurring from E9.5 to E10.5. Expression pattern validation was confirmed by in situ hybridization and trustful pre- viously published data. Gene Ontology analysis revealed, in general terms, a downregulation of transcription factors and an upregulation of extracellular molecules with age. We have identified a specific expression of HoxC genes in the limb ectoderm, which is currently on evaluation.

PMCID: PMC4482672, [Background]: Precise temporal and spatial expression of the clustered Hox genes is essential for patterning the developing embryo. Temporal activation of Hox genes was shown to be cluster-autonomous. However, gene clustering appears dispensable for spatial colinear expression. Generally, a set of Hox genes expressed in a group of cells instructs these cells about their fate such that the differential expression of Hox genes results in morphological diversity. The spatial colinearity is considered to rely both on local and long-range cis regulation. [Results]: Here, we report on the global deregulation of HoxA and HoxD expression patterns upon inactivation of a subset of HOXA and HOXD proteins. [Conclusions]: Our data suggest the existence of a >self-regulation> mechanism, a process by which HOX proteins establish and/or maintain the spatial domains of the Hox gene family and we propose that the functionally dominant HOX proteins could contribute to generating the spatial parameters of Hox expression in a given tissue, i.e., HOX controlling the establishment of the ultimate HOX code., Grant sponsor: the Spanish Government; Grant number: BFU2011-24972; Grant sponsor: the Canadian Institutes for Health Research; Grant number: MOP-82880; Grant number: 126110. This work was supported by the Spanish Government to M.R. and by the Canadian Institutes for Health Research as well as the Canada Research Chair program to M.K. R.S was supported by a Formación Profesorado Universitario fellowship from the Spanish Ministry of Science and Innovation and currently supported by the Angelo Pizzagalli postdoctoral fellowship.

Resumen del trabajo presentado a los Congresos: 17th International Congress of Developmental Biology, 72nd Annual Meeting of the Society for Developmental Biology, VII Latin American Society of Developmental Biology Meeting y XI Congreso de la Sociedad Mexicana de Biologia del Desarrollo, celebrados en Cancún (México) del 16 al 20 de junio de 2013., The apical ectodermal ridge (AER) is a specialized epithelium located at the distal dorso-ventral (DV) rim of the developing limb that is crucial for limb bud development. The induction of the AER is a complex process that relies on intricate interactions among the FGF, WNT, and BMP signaling pathways operating within the ectoderm and between the ectoderm and mesoderm of the early limb bud. Furthermore, induction of the AER is linked to the establishment of DV patterning. Sp6 and Sp8 are two members of the Specificity Protein family of transcription factors that are expressed in the limb bud ectoderm and function downstream of WNT/ β Catenin signaling and upstream of Fgf8. Their individual genetic inactivations result in a mild syndactyly phenotype for Sp6 and limb truncation, due to the premature regression of the AER, for Sp8. To investigate a possible functional redundancy between Sp6 and Sp8, we generated double Sp6;Sp8 null mutants. We also generated Sp6-null; Sp8-conditional mutants using a Sp8 floxed allele with the Ap2-Cre and with the Msx2-Cre deleter lines. Our results show that double Sp6;Sp8 mutants are tetra-amelic. Initial budding occurs, but Fgf8 and Bmp4 are not activated in the limb ectoderm and the dorsal marker Wnt7a persists throughout the limb bud ectoderm. The phenotype of mutants bearing a single functional copy of Sp6 (Sp6 +/-;Sp8-/-) is indistinguishable from that of the double mutants, whereas the presence of a single functional allele of Sp8 (Sp6-/-;Sp8+/-) results in a Split Hand Foot Malformation phenotype. We conclude that Sp6 and Sp8 work in a redundant manner as indispensable mediators of WNT/ β Catenin signaling in the limb ectoderm and that their function links the Proximo-distal and DV axes.

Resumen del trabajo presentado a los Congresos: 17th International Congress of Developmental Biology, 72nd Annual Meeting of the Society for Developmental Biology, VII Latin American Society of Developmental Biology Meeting y XI Congreso de la Sociedad Mexicana de Biologia del Desarrollo, celebrados en Cancún (México) del 16 al 20 de junio de 2013.-- et al., One of the most intriguing questions in developmental biology is how organ growth and patterning are coordinated during embryogenesis. Limb development relies on an exquisite coordination between growth and patterning but the underlying mechanisms remain elusive. Previous studies showed that A-P and P-D limb bud growth and patterning relies on a positive feedback loop between Sonic Hedgehog (Shh), the BMP antagonist Gremlin1 (Grem1), both expressed in mesenchymal cells, and Fibroblast growth factors (Fgfs) produced in the Apical Ectodermal Ridge (AER). In addition, the collinear expression of HoxA and HoxD genes has a key role in A-P and P-D patterning by establishing positional identity along these axes. Here, we show that HoxA and HoxD genes are required at early stages for Grem1 activation and proper Fgf10 and Fgf8 expression and therefore are mandatory for the establishment of the Shh-Grem1-Fgf feedback loop. Our results provide evidence that, in addition to this early function, HoxA and HoxD genes remain indispensable for proper limb growth at later stages. Together, our results reveal a dual role of Hox genes in controlling limb growth and establishing the limb architecture and we propose that the intricate interactions between Hox function and growth pathways act as the molecular network coordinating limb bud growth and patterning.

10 páginas, 8 figuras., Eph receptors and their ligands, the ephrins, have been implicated in early patterning and axon guidance in vertebrate embryos. Members of these families play pivotal roles in the formation of topographic maps in the central nervous system, the formation of brain commissures, and in the guidance of neural crest cells and motor axons through the anterior half of the somites. Here, we report a highly dynamic expression pattern of the chick EphA7 gene in the developing limb. Expression is detected in discrete domains of the dorsal mesenchyme from 3 days of incubation. The expressing cells are adjacent to the routes where axons grow to innervate the limb at several key points: the region of plexus formation, the bifurcation between dorsal and ventral fascicles, and the pathway followed by axons innervating the dorsal muscle mass. These results suggested a role for EphA7 in cell-cell contact-mediated signalling in dorsal limb patterning and/or axon guidance. We carried out experimental manipulations in the chick embryo wing bud to alter the dorsoventral patterning of the limb. The analyses of EphA7 expression and innervation in the operated wings indicate that a signal emanating from the dorsal ectoderm regulates EphA7 in such a way that, in its absence, the wing bud lacks EphA7 expression and shows innervation defects at the regions where the gene was downregulated. EphA7 downregulation in the dorsal mesenchyme after dorsal ectoderm removal is more rapid than that of Lmx-1, the gene known to mediate dorsalisation in response to the ectodermal signal. These results add a new gene to the dorsalisation signalling pathway in the limb. Moreover, they implicate the Eph receptor family in the patterning and innervation of the developing limb, extending its role in axon pathfinding to the distal periphery., This work was supported by grants from the Spanish Ministry of Education and Culture (DGICYT-PM95-0024) and the European Union Biotech Programme (BIO4-CT96-0659) to M. A. N., and DGICYT-PM95-0088 to M. A. R.; M. A. and M. E. P. were the recipients of predoctoral fellowships (PFPI) from the Spanish Ministry of Education and Culture and M. T. H. of an ICI fellowship.