Absence of digit tip regeneration in a mouse model lacking nails

Resumen del trabajo presentado en 17th Spanish Society for Developmental Biology Meeting Virtual Meeting, celebrado en modalidad virtual del 18 al 20 de noviembre de 2020.Epimorphic regeneration is a type of multi-tissue regeneration defined by the formation of a blastema. In contrast to amphibians, which can regenerate their entire limbs, mammals can only regenerate the distal tip of their digits, hence investigating the mechanisms involved is of maximum interest for regenerative medicine. Interestingly, in mice and humans this regeneration associates with the nail organ, particularly with the Wnt/ß-catenin active nail matrix. Nails are ectodermal appendages of the dorsal tip of the digits. Their development reflects the dorso-ventral polarity of the limb, established in the early limb bud ectoderm by the interaction of three central molecules. En1, expressed in the ventral ectoderm, restricts Wnt7a to the dorsal ectoderm. Wnt7a induces Lmx1b, the dorsal determinant, in the subjacent mesoderm. Lmx1b-null mice display bi-ventral distal limbs and die perinatally due to multisystemic defects. Recent studies have identified two Lmx1b limb-specific enhancers named LARM1 and LARM2. CRISPR/Cas9-mediated deletion of these two enhancers (LARM1/2-/-) yielded mice with a limb-restricted Lmx1b-null phenotype, but no other systemic defects. LARM1/2 null mutants show absence of nails in their digit tips, providing an opportunity to directly test the involvement of the nail in digit tip regeneration. As expected, LARM1/2 mutants fail to regenerate their digit tips, as histological and ¿CT analyses demonstrate. Importantly, disregarding the lack of regeneration, a blastema does form at the tip of the LARM1/2 stump. Our preliminary results point to a reduction of proliferation in the LARM1/2 blastema compared to wild-type, and to an absence of Wnt/ß-catenin active epidermis in mutants, as an explanation of the regenerative failure. Our results confirm that bi-ventral digits do not regenerate their digit tips and set the LARM1/2 mutant as a useful model to investigate the mechanisms of regenerative blastema, with the aim of enhancing regeneration

Time-sequenced transcriptomes of developing distal mouse limb buds: A comparative tissue layer analysis

[Background]: The development of the amniote limb has been an important model system to study patterning mechanisms and morphogenesis. For proper growth and patterning, it requires the interaction between the distal sub-apical mesenchyme and the apical ectodermal ridge (AER) that involve the separate implementation of coordinated and tissue-specific genetic programs.[Results]: Here, we produce and analyze the transcriptomes of both distal limb mesenchymal progenitors and the overlying ectodermal cells, following time-coursed dissections that cover from limb bud initiation to fully patterned limbs. The comparison of transcriptomes within each layer as well as between layers over time, allowed the identification of specific transcriptional signatures for each of the developmental stages. Special attention was given to the identification of genes whose transcription dynamics suggest a previously unnoticed role in the context of limb development and also to signaling pathways enriched between layers.[Conclusion]: We interpret the transcriptomic data in light of the known development pattern and we conclude that a major transcriptional transition occurs in distal limb buds between E9.5 and E10.5, coincident with the switch from an early phase continuation of the signature of trunk progenitors, related to the initial proximo distal specification, to a late intrinsic phase of development.Ministerio de Ciencia e Innovación, Grant/Award Number: BFU2017-88265-P; Swiss National Research Fund, Grant/Award Number: 310030B_138662; ERC grant RegulHox, Grant/Award Number: 588029Peer reviewe

Time‐sequenced transcriptomes of developing distal mouse limb buds: A comparative tissue layer analysis

Background The development of the amniote limb has been an important model system to study patterning mechanisms and morphogenesis. For proper growth and patterning, it requires the interaction between the distal sub‐apical mesenchyme and the apical ectodermal ridge (AER) that involve the separate implementation of coordinated and tissue‐specific genetic programs. Results Here, we produce and analyze the transcriptomes of both distal limb mesenchymal progenitors and the overlying ectodermal cells, following time‐coursed dissections that cover from limb bud initiation to fully patterned limbs. The comparison of transcriptomes within each layer as well as between layers over time, allowed the identification of specific transcriptional signatures for each of the developmental stages. Special attention was given to the identification of genes whose transcription dynamics suggest a previously unnoticed role in the context of limb development and also to signaling pathways enriched between layers. Conclusion We interpret the transcriptomic data in light of the known development pattern and we conclude that a major transcriptional transition occurs in distal limb buds between E9.5 and E10.5, coincident with the switch from an early phase continuation of the signature of trunk progenitors, related to the initial proximo distal specification, to a late intrinsic phase of development.</p

Time-sequenced transcriptomes of developing distal mouse limb buds: A comparative tissue layer analysis

Background: The development of the amniote limb has been an important model system to study patterning mechanisms and morphogenesis. For proper growth and patterning, it requires the interaction between the distal sub-apical mesenchyme and the apical ectodermal ridge (AER) that involve the separate implementation of coordinated and tissue-specific genetic programs. Results: Here, we produce and analyze the transcriptomes of both distal limb mesenchymal progenitors and the overlying ectodermal cells, following time-coursed dissections that cover from limb bud initiation to fully patterned limbs. The comparison of transcriptomes within each layer as well as between layers over time, allowed the identification of specific transcriptional signatures for each of the developmental stages. Special attention was given to the identification of genes whose transcription dynamics suggest a previously unnoticed role in the context of limb development and also to signaling pathways enriched between layers. Conclusion: We interpret the transcriptomic data in light of the known development pattern and we conclude that a major transcriptional transition occurs in distal limb buds between E9.5 and E10.5, coincident with the switch from an early phase continuation of the signature of trunk progenitors, related to the initial proximo distal specification, to a late intrinsic phase of development.Funding information: Ministerio de Ciencia e Innovación, Grant/Award Number: BFU2017-88265-P; Swiss National Research Fund, Grant/Award Number: 310030B_138662; ERC grant RegulHox, Grant/Award Number:588029AERHox genesLimb patterningLimb progenitorsRNA-seqSignalingTranscriptom

Two limb-specific enhancers control Lmx1b expression in a modular and autoregulatory manner

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

Deciphering Lmx1b regulation during development and evolution

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

Lmx1b regulation and function during limb development and evolution

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)

Absence of digit tip regeneration in Lmx1b-deficient nailless digits suggests a role for Lmx1b distinct from patterning

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

Dorso-ventral polarity is not required for digit tip regeneration

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

Failure of digit tip regeneration in the absence of Lmx1b suggests Lmx1b functions disparate from dorsoventral polarity

Summary: Mammalian digit tip regeneration is linked to the presence of nail tissue, but a nail-explicit model is missing. Here, we report that nail-less double-ventral digits of ΔLARM1/2 mutants that lack limb-specific Lmx1b enhancers fail to regenerate. To separate the nail’s effect from the lack of dorsoventral (DV) polarity, we also interrogate double-dorsal double-nail digits and show that they regenerate. Thus, DV polarity is not a prerequisite for regeneration, and the nail requirement is supported. Transcriptomic comparison between wild-type and non-regenerative ΔLARM1/2 mutant blastemas reveals differential upregulation of vascularization and connective tissue functional signatures in wild type versus upregulation of inflammation in the mutant. These results, together with the finding of Lmx1b expression in the postnatal dorsal dermis underneath the nail and uniformly in the regenerative blastema, open the possibility of additional Lmx1b roles in digit tip regeneration, in addition to the indirect effect of mediating the formation of the nail