Biology subjects

Ros, M. A.

Publications and source records attributed to Ros, M. A..

9 recordsLinked to original sources

bioRxiv2025

Targeted p63 isoform switch corrects dominant mutations in AEC syndrome without disrupting epidermal homeostasis

The transcription factor p63 is a master regulator of stratified epithelial development, and its disruption causes severe congenital defects affecting the skin, limbs, and craniofacial structures in both humans and mice. Among p63-related disorders, Ankyloblepharon-Ectodermal Defects-Cleft Lip/Palate (AEC) syndrome is caused by dominant mutations primarily affecting the Sterile Alpha Motif (SAM) domain and the Transactivation Inhibitory Domain (TID) of the TP63 gene, which are unique to the p63 isoform. These mutations promote protein aggregation and transcriptional dysregulation, ultimately leading to debilitating skin erosions, suggesting that isoform-specific strategies could be therapeutically relevant. To explore a therapeutic strategy based on isoform switching, we generated a conditional mouse model with deletion of exon 13, resulting in replacement of p63 by the shorter p63{beta} isoform, which is expressed in the skin at lower levels. Although we found that p63 is required for limb and palate development, p63{beta} proved sufficient to support epidermal formation, postnatal skin homeostasis, and wound healing. At the molecular level, the switch from p63 to p63{beta} preserved chromatin binding and global transcriptional programs in keratinocytes. We next used genome editing to delete exon 13 in human primary keratinocytes, inducing a switch from p63 to p63{beta}. This isoform switch maintained normal proliferation and global gene expression. Importantly, p63{beta} expression in AEC patient-derived keratinocytes rescued protein aggregation, restored mechanical integrity, and normalized epidermal gene expression. Together, these findings demonstrate that p63{beta} can functionally compensate for p63 in the skin and establish and indicate that isoform switching could offer a new treatment option for AEC syndrome.

molecular biology↗

bioRxiv2025

Dorsoventral limb patterning in paired appendages emerged via regulatory repurposing of an ancestral posterior fin module

Limbs exhibit adaptive differentiation along their dorsoventral (DV) axis, determined by the dorsal expression of the LIM homeobox gene Lmx1b. The paired appendages (i.e. the pectoral and pelvic fins from which limbs evolved) arose in an early jawless ancestor via co-option of a midline-fin genetic program including modules for anteroposterior (AP) and proximodistal (PD) patterning. Unlike the AP and PD axes, median fins lack an unambiguous DV axis, leaving the origin of this DV pattern in paired appendages unresolved. Here, we describe Lmx1b expression in the posterior midline fins of cichlids, sturgeons and catsharks, revealing an ancestral role for this gene predating the origin of paired appendages. In median fins, Lmx1b activation depends on shh from the ZPA, whereas in paired fins it relies on ectodermal wnt signalling, indicating the evolution of novel regulatory inputs for dorsal patterning. We observe ephA4b, a putative Lmx1b target, is co-expressed with Lmx1b in dorsal pectoral and posterior midline fins and downregulated alongside Lmx1b, suggesting a role in both fin types related to axon guidance. We propose that novel regulation drove the repurposing of Lmx1b from posterior to dorsal fin determinant, with co-option of conserved downstream targets. Altogether, our findings demonstrate that the DV axis of paired appendages represents an evolutionary innovation arising from the integration of ancestral midline fin and flank determinants with novel regulatory inputs.

evolutionary biology↗

bioRxiv2023

Fgf signalling triggers an intrinsic mesodermal timer that determines the duration of limb patterning

Complex signalling between the apical ectodermal ridge (AER - a thickening of the distal epithelium) and the mesoderm controls limb patterning along the proximo-distal axis (humerus to digits). However, the essential requirement for AER-Fgf signalling during in vivo development makes it difficult to understand the exact roles that it fulfils. To overcome this barrier, we developed an amenable ex vivo chick wing tissue explant system that faithfully replicates in vivo parameters. Using inhibition experiments and RNA-sequencing, we identify a transient role for Fgfs in triggering the distal patterning phase. Fgfs are then dispensable for the maintenance of an intrinsic mesodermal transcriptome, which controls proliferation/differentiation timing and the duration of patterning. We also uncover additional roles for Fgf signalling in maintaining AER-related gene expression and in suppressing myogenesis. We describe a simple logic for limb patterning duration, which is potentially applicable to other systems, including the main body axis.

developmental biology↗

bioRxiv2020

Identification of limb-specific Lmx1b auto-regulatory modules with Nail-Patella Syndrome pathogenicity.

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.

developmental biology↗

bioRxiv2020

SPECIFIC ECTODERMAL ENHANCERS CONTROL THE EXPRESSION OF HoxC GENES IN DEVELOPING MAMMALIAN INTEGUMENTS

Vertebrate Hox genes are key players in the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that the HoxC gene cluster was globally co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws. Accordingly, deletion of the HoxC cluster led to mice lacking nails (anonychia) and also hairs (alopecia), a condition stronger than the previously reported loss of function of Hoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two ectodermal, mammalian-specific enhancers located upstream of the HoxC gene cluster, which act synergistically to regulate Hoxc gene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation of Hoxc genes in the ectoderm, suggesting that these two enhancers may have evolved along with mammals to provide the level of HOXC proteins necessary for the full development of hairs and nails. Significance StatementIn this study, we report a unique and necessary function for the HoxC gene cluster in the development of some ectodermal organs, as illustrated both by the hair and nail phenotype displayed by mice lacking the Hoxc13 function and by the congenital anonychia (absence of nails) in full HoxC cluster mutants. We show that Hoxc genes are activated in a colinear manner in the embryonic limb ectoderm and are subsequently transcribed in developing nails and hairs. We identify two mammalian-specific enhancers located upstream of the HoxC cluster with and exclusive ectodermal specificity. Individual or combined enhancer deletions suggest that they act in combination to raise the transcription level of several Hoxc genes during hairs and nails development.

developmental biology↗

bioRxiv2020

Sp8 regulatory function in the limb bud ectoderm

Sp8 and Sp6 are two closely related Sp genes expressed in the limb ectoderm, where they regulate proximo-distal and dorso-ventral patterning. Genetic studies in mice have shown that they act in a dose-dependent manner, with Sp8 exerting a substantially stronger effect than Sp6. Here, we integrate ChIPmentation-seq and RNA-seq analyses to elucidate their genome-wide regulatory networks and mechanisms of action. Our results show that Sp8 has a dual mode of action either directly binding GC-rich motifs or indirectly binding AT-rich motifs through Dlx. In contrast, Sp6 primarily acts through AT-rich motifs, via interaction with cofactors such as Dlx5. Both factors regulate key components of the Wnt, FGF, and BMP signaling pathways, central to limb patterning, and display both cooperative and independent roles. We further show that Sp8 and Sp6 can form homo- and heterodimers, as well as interact with Dlx5, revealing a complex regulatory network. Our findings provide molecular insight into limb morphogenesis and have potential implications for understanding congenital limb malformations.

developmental biology↗

bioRxiv2019

A positional information gradient of sonic hedgehog is required for flight feather formation in avian wings

Flight is a triumph of evolution that enabled the radiation and success of birds. A crucial step was the development of forelimb flight feathers that may have evolved for courtship or territorial displays in ancestral theropod dinosaurs. Classical tissue recombination experiments performed in the chick embryo provide evidence that signals operating during early limb development specify the position and identity of feathers. Here we show that a positional information gradient of Sonic hedgehog (Shh) signalling in the embryonic chick wing bud specifies the pattern of adult flight feathers in a defined spatial and temporal sequence that reflects their different identities. We reveal that the Shh signalling gradient is interpreted into specific patterns of flight feather-associated gene expression. Our data suggests that flight feather evolution involved the co-option of the pre-existing digit patterning mechanism and therefore uncovers an embryonic process that played a fundamental step in the evolution of avian flight.

developmental biology↗

bioRxiv2018

Mutual transcriptional repression between Gli3 and Hox13 genes determines the anterior-posterior asymmetry of the autopod

In the present study we have investigated the molecular causes of the absence of digit 1 in the Hoxa13 mutant and why the absence of Hoxa13 protein, whose expression spans the entire autopod, specifically impacts the anterior-most digit. We show that in the absence of Hoxa13, the expression of Hoxd13 does not extend into the anterior mesoderm consequently leaving the presumptive territory of digit1 devoid of distal Hox expression and providing an explanation for the agenesis of digit 1. We provide compelling evidence that the lack of Hoxd13 transcription in the anterior mesoderm is due to increased Gli3R activity, in turn resulting from the loss of transcriptional repression exerted by Hoxa13 on Gli3. Our results are compatible with a mutual transcriptional repression between Gli3 and Hox13 genes that determines the anterior-posterior asymmetry of the autopod.

developmental biology↗

bioRxiv2018

An intrinsic cell cycle timer terminates limb bud outgrowth

The longstanding view of how proliferative outgrowth terminates following the patterning phase of limb development involves the breakdown of reciprocal extrinsic signalling between the mesenchyme and the overlying epithelium (e-m signalling). However, by grafting mesenchyme cells from late stage chick wing buds to an early epithelial environment we show that this mechanism is not required. RNA sequencing reveals that mesenchyme cells terminate growth by an intrinsic cell cycle timer in the presence of e-m signalling. In this process, e-m signalling is required permissively to allow the intrinsic cell cycle timer to run its course. We provide evidence that a temporal switch from BMP antagonism to BMP signalling controls the intrinsic cell cycle timer during limb outgrowth. Our findings have general implications for other patterning systems in which extrinsic signals and intrinsic timers are integrated.

developmental biology↗