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Trabajo presentado en el 19th International Congress of Developmental Biology, celebrado en Guia (Portugal) del 16 al 20 de octubre de 2022., Regeneration in mammals is limited to the digit tips. The amputation of the distal tip of the terminal phalanx triggers the formation of an undifferentiated pool of progenitor cells called a blastema that will regenerate the multiple tissues required to restore the missing part. Digit tip regeneration is linked to the presence of the dorsal nail organ, as only amputations that conserve the nail matrix regenerate. However, the regenerative potential of digits without nails has not yet been tested. To evaluate nailless digits, we used dLARM1/2 mutants that develop digits with no nails due to the limb-specific loss of the dorsal limb determinant Lmx1b. We report that dLARM1/2 mutant digits are unable to regenerate, although they form a blastema. To test whether the lack of regeneration was due to the absence of dorso-ventral (DV) polarity, a concept suggested in amphibia, we evaluated Del(27) mutants that also lack DV polarity with nails on both dorsal and ventral aspects of distal phalanges due to ventral overexpression of Lmx1b. Unlike dLARM1/2 mutants, Del(27) mutant digits regenerated indicating that DV polarity is dispensable for regeneration. Interestingly, Lmx1b expression (which continues in the nail dermis postnatally) is detected uniformly in the blastema of WT, but not dLARM1/2 mutant mice. In addition, comparison of dLARM1/2 vs WT blastemal transcriptomes revealed that the mutants had defective pro regenerative processes such as vasculogenesis and extracellular matrix remodeling, while inflammatory pathways were exacerbated. Gene expressions typical of the nail dermis were also lost in dLARM1/2 digit tips, suggesting regulation by Lmx1b. Collectively, our data suggest a direct role for Lmx1b in digit tip regeneration maintaining the nail dermis and promoting a competent blastema.

Trabajo presentado en el 19th International Congress of Developmental Biology, celebrado en Guia (Portugal) del 16 al 20 de octubre de 2022., The dorso-ventral (DV) limb asymmetry is crucial for its function; however, little is known about how it is accomplished during morphogenesis. Lmx1b is the key regulator of DV patterning during limb development as it is both necessary and sufficient to specify dorsal fates, but its regulation remains poorly known. We recently demonstrated a complex autoregulatory mode of transcriptional Lmx1b regulation mediated by two limb-specific enhancers, LARM1 and LARM2. The deletion of both enhancers (dLARM1/2) reproduces the Lmx1b-null phenotype in the limb, while the removal of each enhancer produced a double ventral phenotype restricted to the posterior (dLARM1) or anterior (dLARM2) half of the limb. To investigate the regulatory logic of the Lmx1b locus in detail, we dissected the LARM1 element into its enhancer and silencer sequences and explored the functional contribution of the two Lmx1b-associated lncRNAs. The results confirm the presence of a functional repressor contained in LARM1 and underscore the positional specificity of the LARM sequences. The data suggests that Lmx1b transcription uses different regulatory elements in different positions probably reflecting the integration of the local transcriptional environment. Indeed, published ChIP-seq data showed the Gli3 and Hox13 are significantly enriched in the LARM sequences exposing their interaction with major limb regulators. Finally, considering that the double-ventral limbs of dLARM1/2 mutants fail to support locomotion, even to lift the body weight, we hypothesized that the elaboration of the DV asymmetry by modification of Lmx1b regulation, or its functional targets, had to accompany the fin to limb transition. We have identified orthologs of the LARMs in fishes and all of them show activity in the pectoral fin in zebrafish reporter essays, revealing the deep roots of Lmx1b regulation., Spanish Ministry of Science and Innovation Grant PID2020-114525GB-I00 to Marian Ros. Sofía Zdral is supported by a PhD fellowship of the University of Cantabria and Alejandro Castilla Ibeas by a PhD fellowship of the Ministry of Science and Innovation (FSE/AEI/PRE2018-083421).

Trabajo presentado en la 16th International Conference on Limb Development, regeneration, and evolution, celebrada en Cambridge (Estados Unidos) del 08 al 11 de agosto de 2022., The dorso-ventral (DV) organization of limb morphology is crucial for its function but little is known about how it is implemented during morphogenesis. Lmx1b is the key regulator of DV patterning as it is both necessary and sufficient to specify dorsal fates, but its regulation remains poorly known. Recently, we reported that Lmx1b is subject to a complex and autoregulatory mode of transcriptional regulation mediated by two limb-specific enhancers termed LARM1 and LARM2. Interestingly, these enhancers display anterior-posterior (AP) spatial modularity with LARM1 controlling Lmx1b expression in the anterior and LARM2 in the posterior half of the limb. To investigate the regulatory logic of the Lmx1b locus in higher detail, we have dissected the LARM1 enhancer and explored the functional contribution of two Lmx1b associated lncRNAs. The results confirm the presence of a functional repressor and underscore the AP positional specificity of the LARM sequences. Conjointly, the data suggests that Lmx1b transcription uses different regulatory elements in different positions probably reflecting the integration of the local transcriptional environment. Finally, since the double-ventral limbs of dLARM1/2 mutants fail to support locomotion, we hypothesized that the elaboration of the DV axis, possible driven by changes in the regulatory genome, had to accompany the fin transition towards appendages capable of lifting the body weight. In this direction, we have identified orthologs of the LARM sequences from chondrichthyans. All these sequences show activity in the pectoral fin in zebrafish reporter assays, revealing the deep roots of Lmx1b regulation.

Trabajo presentado en el 16th International Conference on Limb Development, Regeneration, and Evolution, celebrado en Cambridge (Estados Unidos) del 08 al 11 de agosto de 2022., Mammalian limb regeneration is restricted to the distal third of terminal phalanges. Amputation of distal digit tips results in blastema formation and restoration of the missing part. This regenerative response has been linked to the presence of the nail, but digits without nails have not been evaluated. Here we interrogate the regenerative ability of the double-ventral digits of dLARM1/2 mutants, which lack dorsal nails due to the limb-specific absence of Lmx1b. We report that these nail-less digits fail to regenerate, supporting the nail requirement. Because the absence of dorso-ventral (DV) polarity could also impair regeneration, as reported in amphibians, we tested the double-dorsal double-nail digits of Del(27) mutants, which lack DV polarity due to the limb-restricted loss of En1. Since Del(27) digits did regenerate, we concluded that DV polarity is not required for digit tip regeneration. We documented continued dorsal expression of Lmx1b postnatally in the wild-type (WT) nail dermis. Moreover, there is uniform Lmx1b expression in WT regeneration-competent blastemas. Transcriptomic comparison between WT and dLARM1/2 mutant blastemas revealed differential up-regulation of gene signatures functionally associated with vascularization and connective tissue remodeling in WT mice. In contrast, the dLARM1/2 non-regenerative blastemas that lack Lmx1b expression show differential up-regulation of genes associated with inflammation and inflammatory processes. Conjointly, our data implicate a direct role for Lmx1b in digit tip regeneration by maintaining the nail dermis and promoting a competent blastema.

Trabajo presentado en The Company of Biologists Workshop Molecular mechanisms of developmental and regenerative biology (EMBO), celebrado en modalidad virtual del 26 al 29 de abril de 2022., The regeneration of the digit tip is an exceptional model of multi-tissue regeneration in mammals, with remarkable potential to advance regenerative medicine. Digit tip regeneration positively correlates with the presence of the nail organ, most likely because of the environment of the nail stem cells, but the underlying mechanisms are still unclear. The fact that the nail is a dorsal structure links regeneration with dorso-ventral (DV) polarity. Here, to evaluate the nail function more directly, and to disentangle it from DV positional information, we tested the regenerative ability of double-ventral digits and double-dorsal digits resulting from the limb-specific loss of Lmx1b and of En1 expression, respectively. Our results indicate that DV polarity is not required for digit tip regeneration but that an excessive inflammatory response prevents regeneration in the absence of Lmx1b. The possible causes of the exacerbated inflammation in the absence of Lmx1b are discussed.

Trabajo presentado en el EMBO Workshop: Enhanceropathies: understanding enhancer function to understand human disease, celebrado en Santander (España) del 06 al 09 de octubre de 2021., Lmx1b is the key regulator of dorso-ventral (DV) pattern in the limb, as it is both necessary and sufficient to specify dorsal fates. Accordingly, targeted disruption of Lmx1b results in double ventral limbs. In humans, mutations in the LMX1B gene are responsible for the autosomal dominant Nail-Patella Syndrome (OMIM #161200), characterized by incomplete limb dorsalization. Here, we report on two enhancers located upstream of the gene, LARM1 and LARM2, that regulate Lmx1b specifically in the limb. These sequences contain several canonical Lmx1b binding sites that mediate a positive loop of Lmx1b autoregulation. Deletion of these enhancers together results in mice with double ventral limbs, like these of Lmx1b-nul mice. Remarkably, deletion of each individual enhancer produced a double ventral phenotype clearly restricted to the posterior half of the limb in LARM1 mutants and to the anterior in LARM2 mutants, indicating differential activity of these enhancers along the anterior-posterior axis. These results reflect an unexpected complex, modular and autoregulatory mode of Lmx1b transcriptional regulation during limb development. We also report on several NPS patient families with normal LMX1B coding sequence but variations in the LARM region that exhibit limb restricted phenotypes, stressing the role of regulatory modules in disease pathogenesis.

Resumen del trabajo presentado en el 17th Spanish Society for Developmental Biology Meeting Virtual Meeting, celebrado en modalidad virtual del 18 al 20 de noviembre de 2020., Vertebrate Hox genes are critical developmental regulators in the establishment of the basic body plan of bilaterian animals and, subsequently, in the organization of secondary axial structures such asthe appendages. At postnatal stages and in adulthood, Hox genes also play important roles in homeostasis and disease. Here we concentrate in the study of the function of the HoxC cluster in the ectoderm, using the mouse limb bud as a model. We report that Hoxc genes are activated in a colinear manner in the embryonic limb ectoderm and are subsequently transcribed in developing nails and hairs. Accordingly, mice lacking the complete HoxC cluster display anonychia (absence of the nail organ), a condition stronger than the misshaped nails previously reported in the loss of function of Hoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identify two mammalian-specific enhancers located upstream of the HoxC cluster that show ectodermal restricted activity in mouse transient transgenic reporter assays. The individual and combined deletions of these two enhancers indicate that they act together to control the transcription level of Hoxc gene necessary for correct hair and nail development.