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Resumen del trabajo presentado en el 4th Meeting of the portuguese society for developmental biology, celebrado en Oporto (Portugal) del 07 al 10 de noviembre de 2018., SP6 and SP8 are two members of the SP family of transcription factors that are conjointly required in the limb bud ectoderm for correct proximo-distal and dorso-ventral patterning (Talamillo et al., 2010; Haro et al., 2014). To identify the SP8 regulatory network in the limb ectoderm, we performed ChIPmentation and RNA-seq experiments in the E10.5 limb bud ectoderm. The genome-wide binding pattern of SP8 yielded 1,451 conserved and significantly enriched regions while the comparison between the transcriptional signature of WT and Sp8 null mutant embryos found 892 differentially expressed genes (DEG). The combination of the ChIPmentation and DEG datasets identified 183 direct SP8 target genes, 30% repressed and 70% activated by SP8. Further analyses revealed that SP8 acted on these direct targets mainly from distally located binding sites. We also found that AT-rich motifs, described as the preferential motif of SP7 to indirectly bind the DNA through DLX5, were predominant when SP8 functions as an activator. Accordingly, Co¿Immunoprecipitation (CoIP) experiments showed binding between SP8 and DLX5. Interestingly, SP1 like CG-rich motifs were more frequent when SP8 acts as a repressor. Finally, CoIP and Bimolecular Fluorescence Complementation assays also showed that SP8 and SP6 form homo and heterodimers providing some insights into their functional redundancy.

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 XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016., Limbs develop from limb buds composed of a core of limb progenitor cells covered by the surface ectoderm. A variety of experiments in chick and mouse embryos indicate that the fate of the distal limb progenitors is progressively restricted: early progenitors can form the whole proximo-distal elements of the limb while late progenitors can only form the digits. Here we have used RNA-seq to reveal the transcriptional features associated with this developmental transition. The analysis was performed in mouse forelimbs of 4 different stages (E9.5, E10.5, E11.5 and E12.5) and restricted to the distal 150μm with ectodermal and mesodermal components analyzed separately. Two biological replicates were used for each condition. Of 46,480 Ensembl-annotated genes, 3,416 were differentially expressed between ectoderm and mesoderm. In each tissue, the time serie analysis revealed that major changes occur between E9.5 and E10.5. The accuracy of the expression patterns was confirmed by in situ hybridization and by the truthful coincidence with published expression patterns. According to their temporal expression, six different dynamics of gene expression were identified. In general, the average expression of transcription factors decreased with age, whereas the average expression of extracellular molecules increased with age. The differentially expressed genes were further subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and showed diverse functional categories and signaling pathways. We are exploring their relevance in orchestrating age-specific developmental capacities of each tissue.

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.