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.)

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

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

Two Lmx1b-associated cis-regulatory modules (LARM1/2) mediate Lmx1b auto-amplification during limb dorsalization and their disruption can cause a limb-specific form of Nail-Patella syndrome

Trabajo presentado en Limb Development and Regeneration: New Tools for a Classic Model System EMBO Workshop, celebrado en Barcelona (España) del 2 al 5 de julio de 2019.During limb development, the Lmx1b homeodomain transcription factor is restricted to dorsal mesenchyme and is required for distal dorsalization. Lmx1b also contributes to kidney, eye and cerebellar development. Haploinsufficiency of the human LMX1B genes causes Nail-Patella Syndrome (NPS) characterized by nail dysplasia, absent or hypoplastic patellae, eye abnormalities, and progressive kidney disease. In the functional Lmx1b knockout mouse, transcription of the mutant Lmx1b mRNA in the limb is reduced, indicating a role for Lmx1b in an auto-amplifying feedback loop. Previous Lmx1b-targeted ChIP-seq data identified two Lmx1b-associated regulatory modules (LARM1/2) bound by Lmx1b. Herein, we demonstrate that LARM1/2 activity overlaps Lmx1b expression in the dorsal limb mesoderm and is dependent on conserved Lmx1b binding sites for activity. We also demonstrate interaction between the LARM enhancers and the Lmx1b promoter. In addition, we performed a targeted CRISPR-Cas excision of the LARM1/2 region in mice; loss of LARM1/2 reduced the expression of Lmx1b in normal mice and generated a phenotype with nearly symmetrical ventral-ventral distal limbs. LARM1/2 loss was limb-specific and did not affect any other Lmx1b-related organs. We extended our investigation to humans with an NPS phenotype that lacked a variation within the LMX1B coding sequence. In two families with a limb-restricted phenotype, loss-of-function variations in the LARM region were identified. These data indicate that the limb-specific expression of Lmx1b is amplified through an auto-regulatory loop involving LARM1/2. Furthermore, disruption of LARM1/2 function can cause isolated limb malformations in mice and human NPS

LHX2 Mediates the FGF-to-SHH Regulatory Loop during Limb Development

During limb development, fibroblast growth factors (Fgfs) govern proximal–distal outgrowth and patterning. FGFs also synchronize developmental patterning between the proximal–distal and anterior–posterior axes by maintaining Sonic hedgehog (Shh) expression in cells of the zone of polarizing activity (ZPA) in the distal posterior mesoderm. Shh, in turn, maintains Fgfs in the apical ectodermal ridge (AER) that caps the distal tip of the limb bud. Crosstalk between Fgf and Shh signaling is critical for patterned limb development, but the mechanisms underlying this feedback loop are not well-characterized. Implantation of Fgf beads in the proximal posterior limb bud can maintain SHH expression in the former ZPA domain (evident 3 h after application), while prolonged exposure (24 h) can induce SHH outside of this domain. Although temporally and spatially disparate, comparative analysis of transcriptome data from these different populations accentuated genes involved in SHH regulation. Comparative analysis identified 25 candidates common to both treatments, with eight linked to SHH expression or function. Furthermore, we demonstrated that LHX2, a LIM Homeodomain transcription factor, is an intermediate in the FGF-mediated regulation of SHH. Our data suggest that LHX2 acts as a competency factor maintaining distal posterior SHH expression subjacent to the AER