NOGO-A induction and localization during chick brain development indicate a role disparate from neurite outgrowth inhibition

BACKGROUND: Nogo-A, a myelin-associated protein, inhibits neurite outgrowth and abates regeneration in the adult vertebrate central nervous system (CNS) and may play a role in maintaining neural pathways once established. However, the presence of Nogo-A during early CNS development is counterintuitive and hints at an additional role for Nogo-A beyond neurite inhibition. RESULTS: We isolated chicken NOGO-A and determined its sequence. A multiple alignment of the amino acid sequence across divergent species, identified five previously undescribed, Nogo-A specific conserved regions that may be relevant for development. NOGO gene transcripts (NOGO-A, NOGO-B and NOGO-C) were differentially expressed in the CNS during development and a second NOGO-A splice variant was identified. We further localized NOGO-A expression during key phases of CNS development by in situ hybridization. CNS-associated NOGO-A was induced coincident with neural plate formation and up-regulated by FGF in the transformation of non-neural ectoderm into neural precursors. NOGO-A expression was diffuse in the neuroectoderm during the early proliferative phase of development, and migration, but localized to large projection neurons of the optic tectum and tectal-associated nuclei during architectural differentiation, lamination and network establishment. CONCLUSION: These data suggest Nogo-A plays a functional role in the determination of neural identity and/or differentiation and also appears to play a later role in the networking of large projection neurons during neurite formation and synaptogenesis. These data indicate that Nogo-A is a multifunctional protein with additional roles during CNS development that are disparate from its later role of neurite outgrowth inhibition in the adult CNS

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

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

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