Identification of limb-specificLmx1bauto-regulatory modules with Nail-Patella Syndrome pathogenicity

Abstract

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 murine 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. Interestingly, 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 in the limb and are necessary for Lmx1b-mediated limb dorsalization. Remarkably, we also report on two NPS patient families with normal LMX1B coding sequence, but loss-of-function variations in the LARM1/2 region, stressing the role of regulatory modules in disease pathogenesis.

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

“…We have recently characterized two limb-specific Lmx1b enhancers, termed LARM1 and LARM2 , that mediate Lmx1b limb dorsalization (Haro et al 2021). Mice homozygous for the removal of these two enhancers ( ΔLARM1/2 ) replicate the Lmx1b-null limb phenotype consisting of double-ventral distal limbs with sole pads in both surfaces and no nails (Haro et al 2021; Fig 1A-A’).…”

“…We have recently characterized two limb-specific Lmx1b enhancers, termed LARM1 and LARM2 , that mediate Lmx1b limb dorsalization (Haro et al 2021). Mice homozygous for the removal of these two enhancers ( ΔLARM1/2 ) replicate the Lmx1b-null limb phenotype consisting of double-ventral distal limbs with sole pads in both surfaces and no nails (Haro et al 2021; Fig 1A-A’). In contrast to Lmx1b-null mutants that die perinatally, ΔLARM1/2 homozygous mutants survive, as they show no other Lmx1b-related systemic defects, allowing the analysis of fully differentiated double-ventral limbs.…”

“…Lmx1b -null mutants display double ventral digits with no nail but die perinatally because of other systemic Lmx1b-associated defects (Chen et al 1998). However, in the absence of two limb-specific Lmx1b enhancers ( ΔLARM1/2 mutants), Lmx1b-null phenotype is recapitulated only in the limbs of mutants that are otherwise viable and similar to wild-type (Haro et al 2021). The double-ventral digits of ΔLARM1/2 mutants offer an extraordinary opportunity to test of the nail involvement in digit tip regeneration.…”

“…These were reviewed and approved by the Bioethics Committee of the University of Cantabria. We have used the previously published Δ LARM1/2 and the Del(27) mutant lines generated by CRISPR/Cas9 (Haro et al 2021; Allou et al 2021). A minimum of 2 independent specimens were analyzed for each experiment and time point analyzed.…”

“…Lmx1b plays essential roles in a wide range of developmental processes including the development of the kidney, the anterior segment of the eye, and central nervous system (specifically dopaminergic and serotonergic neurons) (Sweeney et al 2003). Recently, we characterized two limb-specific Lmx1b enhancers, termed LARM1 and LARM2 ( L mx1b a ssociated r egulatory m odule 1 and 2) that are key regulators of Lmx1b expression in the limb (Haro et al 2017; 2021). Accordingly, deletion of both LARM enhancers results in mice ( ΔLARM1/2 ) that replicate the Lmx1b-null phenotype exclusively in the limb and are, therefore, viable.…”