Identification of limb-specific Lmx1b auto-regulatory modules with Nail-patella syndrome pathogenicity

© The Author(s) 2021.LMX1B haploinsufficiency causes Nail-patella syndrome (NPS; MIM 161200), characterized by nail dysplasia, absent/hypoplastic patellae, chronic kidney disease, and glaucoma. Accordingly in mice, Lmx1b has been shown to play crucial roles in the development of the limb, kidney and eye. Although one functional allele of Lmx1b appears adequate for development, Lmx1b null mice display ventral-ventral distal limbs with abnormal kidney, eye and cerebellar development, more disruptive, but fully concordant with NPS. In Lmx1b functional knockouts (KOs), Lmx1b transcription in the limb is decreased nearly 6-fold, indicating autoregulation. Herein, we report on two conserved Lmx1b-associated cis-regulatory modules (LARM1 and LARM2) that are bound by Lmx1b, amplify Lmx1b expression with unique spatial modularity in the limb, and are necessary for Lmx1b-mediated limb dorsalization. These enhancers, being conserved across vertebrates (including coelacanth, but not other fish species), and required for normal locomotion, provide a unique opportunity to study the role of dorsalization in the fin to limb transition. We also report on two NPS patient families with normal LMX1B coding sequence, but with loss-of-function variations in the LARM1/2 region, stressing the role of regulatory modules in disease pathogenesis.This work was supported in part by grants from the Spanish Ministerio de Ciencia, Innovación y Universidades (M.A.R) (BFU2017-88265-P); the National Organization for Rare Disorders (K.C.O.), and the Loma Linda University Pathology Research Endowment Fund (K.C.O.)

Sp6 and Sp8 transcription factors control AER formation and dorsal-ventral patterning in limb development

The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. The induction of the AER is a complex process that relies on integrated interactions among the Fgf, Wnt, and Bmp signaling pathways that operate within the ectoderm and between the ectoderm and the mesoderm of the early limb bud. The transcription factors Sp6 and Sp8 are expressed in the limb ectoderm and AER during limb development. Sp6 mutant mice display a mild syndactyly phenotype while Sp8 mutants exhibit severe limb truncations. Both mutants show defects in AER maturation and in dorsal-ventral patterning. To gain further insights into the role Sp6 and Sp8 play in limb development, we have produced mice lacking both Sp6 and Sp8 activity in the limb ectoderm. Remarkably, the elimination or significant reduction in Sp6;Sp8 gene dosage leads to tetra-amelia; initial budding occurs, but neither Fgf8 nor En1 are activated. Mutants bearing a single functional allele of Sp8 (Sp6-/-;Sp8+/-) exhibit a split-hand/foot malformation phenotype with double dorsal digit tips probably due to an irregular and immature AER that is not maintained in the center of the bud and on the abnormal expansion of Wnt7a expression to the ventral ectoderm. Our data are compatible with Sp6 and Sp8 working together and in a dose-dependent manner as indispensable mediators of Wnt/βcatenin and Bmp signaling in the limb ectoderm. We suggest that the function of these factors links proximal-distal and dorsal-ventral patterning

Papel de los factores de transcripción Sp6 y Sp8 en el desarrollo de la extremidad

ABSTRACT: The Apical Ectodermal Ridge (AER) is a specialized thickened epithelium required for limb development that exerts its function through the production of Fibroblast growth factors. Its induction is a complex process that relies on interactions among Fgf, Wnt, and Bmp signaling pathways linked also to DV patterning. Sp6 and Sp8 members of the Specificity Protein family of transcription factors function downstream of Wnt/βCatenin signaling and upstream of Fgf8. Their individual genetic inactivation showed a mild phenotype for Sp6 and limb truncation due to the premature regression of the AER for Sp8. To investigate their possible functional redundancy, we generated Sp6;Sp8 null mutants and also Sp6-null;Sp8-conditional mutants using the Ap2-Cre and the Msx2-Cre lines. Our results show that double Sp6;Sp8 mutants are tetra-amelic. Initial budding occurs but Fgf8 is not detected in the limb ectoderm that entirely expresses the Wnt7a dorsal marker. The limb phenotype of the mutants bearing a single functional allele of Sp6 is indistinguishable from that of the double mutants whereas presence of a single functional allele of 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/βCatenin signaling in the limb ectoderm and that they are responsible of the linkage between the PD and DV.RESUMEN: La Cresta Ectodérmica Apical (AER) es un epitelio especializado necesario para el desarrollo de la extremidad que ejerce su función a través de la producción de Factores de Crecimiento Fibroblásticos. Su inducción es un proceso muy complejo que resulta de la interacción entre las vías de señalización de Fgf, Wnt y Bmp, y además está ligada al establecimiento DV. Sp6 y Sp8, dos miembros de la familia de factores de transcripción Specificity Protein, actúan “downstream” de Wnt/βCatenin y “upstream” de Fgf8. Los mutantes de Sp6 presentan un leve fenotipo mientras que la inactivación de Sp8 da lugar a truncamientos de la extremidad debidos a una regresión prematura de la AER. Para investigar su posible redundancia funcional hemos generado dobles mutantes Sp6;Sp8 así como mutantes condicionales para Sp8 en un “background” nulo para Sp6 con el uso de las líneas Msx2Cre y Ap2Cre. Nuestros resultados muestran que los dobles mutantes son tetra-amélicos, como también lo son los mutantes con una copia funcional de Sp6 en ausencia de Sp8. A pesar del inicio del esbozo de extremidad, Fgf8 nunca es detectado en el ectodermo que expresa en su totalidad el marcador de ectodermo dorsal Wnt7a. Sin embargo, los mutantes con una única copia funcional de Sp8 en ausencia de Sp6 presentan un fenotipo de mano y pie hendido. Concluimos que Sp6 y Sp8 actúan de manera redundante y son mediadores indispensables de la señalización por Wnt/βCatenin y son responsables de la relación entre el eje PD y el DV

Enhancer-gene specificity in development and disease

Enhancers control the establishment of spatiotemporal gene expression patterns throughout development. Over the past decade, the development of new technologies has improved our capacity to link enhancers with their target genes based on their colocalization within the same topological domains. However, the mechanisms that regulate how enhancers specifically activate some genes but not others within a given domain remain unclear. In this Review, we discuss recent insights into the factors controlling enhancer specificity, including the genetic composition of enhancers and promoters, the linear and 3D distance between enhancers and their target genes, and cell-type specific chromatin landscapes. We also discuss how elucidating the molecular principles of enhancer specificity might help us to better understand and predict the pathological consequences of human genetic, epigenetic and structural variants.Work in the Rada-Iglesias laboratory is funded by the Ministerio de Ciencia e Innovación and the Agencia Española de Investigación; by the European Regional Development Fund (PGC2018-095301-B-I00 and RED2018-102553-T); by the European Research Council (862022); and by the European Commission (H2020-MSCA-ITN-2019-860002)

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

Implementation of transgenic platform based on the application of CRISPR/Cas9 technology in mouse zygotes

Resumen del trabajo presentado en el 17th Spanish Society for Developmental Biology Meeting Virtual Meeting (2020), celebrado en modalidad virtual del 18 al 20 de noviembre de 2020.In recent years, the development of CRISPR technologies has provided an excellent tool for genomic editing. We have taken advantage of this technology to set up a CRISPR service at our Institute that based on the electroporation of mouse zygotes (day 0.5) generates desired mouse genomic modifications in a highly efficient, rapid and unexpensive manner. We have applied this methodology to the generation of several mouse models of human congenital hand malformations, especially focussing on the Split Hand Foot Malformation (SHFM), a rare and highly variable malformation characterized by the loss or deformity of the central digit rays. We have previously shown the involvement of Sp6 and Sp8 transcription factors in the generation of SHFM phenotypes in mouse and the interaction of these two factors with Dlx family members. Dlx genes are the genes involved in human SHFM types 1 (Dlx5/Dlx6) and 5 (Dlx1/Dlx2). With the aim of fully investigating the implication of the Dlx-Sp interactions in the pathogenesis of SHFM, we have generated a couple of mouse models: the Dlx5/Dlx6 double deletion and the Sp6- V5 tagged knock-in. Additionally, and based on studies in Drosophila indicating that Sp factors control appendage development through the Notch pathway, we have also generated a Jag2 KO mutant. We are currently using the generated animal models to advance our understanding of the mechanisms subjacent to the SHFM

Sp6 and Sp8 transcription factors are necessary mediators of Wnt/ ß Catenin function in the limb ectoderm

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.Peer Reviewe

Role of hox genes in regulating digit patterning

The distal part of the tetrapod limb, the autopod, is characterized by the presence of digits. The digits display a wide diversity of shapes and number reflecting selection pressure for functional adaptation. Despite extensive study, the different aspects of digit patterning, as well as the factors and mechanisms involved are not completely understood. Here, we review the evidence implicating Hox proteins in digit patterning and the interaction between Hox genes and the Sonic hedgehog/Gli3 pathway, the other major regulator of digit number and identity. Currently, it is well accepted that a self-organizing Turing-type mechanism underlies digit patterning, this being understood as the establishment of an iterative arrangement of digit/interdigit in the hand plate. We also discuss the involvement of 5’ Hox genes in regulating digit spacing in the digital plate and therefore the number of digits formed in this self-organizing system.The M.A.R. laboratory is supported by the Spanish Ministerio de Economía, Industria y Competitividad (BFU2014-57216-P and BFU2017-88265-P)

Loss of enhancer-mediated Lmx1b autoregulation disrupts limb dorsalization and causes nail patella sindrome

Trabajo presentado en el 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM, celebrado en Santander (España) del 10 al 13 de septiembre de 2018

A clinical and experimental overview of sirenomelia: insight into the mechanisms of congenital limb malformations

Sirenomelia, also known as sirenomelia sequence, is a severe malformation of the lower body characterized by fusion of the legs and a variable combination of visceral abnormalities. The causes of this malformation remain unknown, although the discovery that it can have a genetic basis in mice represents an important step towards the understanding of its pathogenesis. Sirenomelia occurs in mice lacking Cyp26a1, an enzyme that degrades retinoic acid (RA), and in mice that develop with reduced bone morphogenetic protein (Bmp) signaling in the caudal embryonic region. The phenotypes of these mutant mice suggest that sirenomelia in humans is associated with an excess of RA signaling and a deficit in Bmp signaling in the caudal body. Clinical studies of sirenomelia have given rise to two main pathogenic hypotheses. The first hypothesis, based on the aberrant abdominal and umbilical vascular pattern of affected individuals, postulates a primary vascular defect that leaves the caudal part of the embryo hypoperfused. The second hypothesis, based on the overall malformation of the caudal body, postulates a primary defect in the generation of the mesoderm. This review gathers experimental and clinical information on sirenomelia together with the necessary background to understand how deviations from normal development of the caudal part of the embryo might lead to this multisystemic malformation