Retinoic acid regulates avian lung branching through a molecular network

Retinoic acid (RA) is of major importance during vertebrate embryonic development and its levels need to be strictly regulated otherwise congenital malformations will develop. Through the action of specific nuclear receptors, named RAR/RXR, RA regulates the expression of genes that eventually influence proliferation and tissue patterning. RA has been described as crucial for different stages of mammalian lung morphogenesis, and as part of a complex molecular network that contributes to precise organogenesis; nonetheless, nothing is known about its role in avian lung development. The current report characterizes, for the first time, the expression pattern of RA signaling members (stra6, raldh2, raldh3, cyp26a1, rar alpha, and rar beta) and potential RA downstream targets (sox2, sox9, meis1, meis2, tgf beta 2, and id2) by in situ hybridization. In the attempt of unveiling the role of RA in chick lung branching, in vitro lung explants were performed. Supplementation studies revealed that RA stimulates lung branching in a dose-dependent manner. Moreover, the expression levels of cyp26a1, sox2, sox9, rar beta, meis2, hoxb5, tgf beta 2, id2, fgf10, fgfr2, and shh were evaluated after RA treatment to disclose a putative molecular network underlying RA effect. In situ hybridization analysis showed that RA is able to alter cyp26a1, sox9, tgf beta 2, and id2 spatial distribution; to increase rar beta, meis2, and hoxb5 expression levels; and has a very modest effect on sox2, fgf10, fgfr2, and shh expression levels. Overall, these findings support a role for RA in the proximal-distal patterning and branching morphogenesis of the avian lung and reveal intricate molecular interactions that ultimately orchestrate branching morphogenesis.The authors would like to thank Ana Lima for slide sectioning and Rita Lopes for contributing to the initiation of this project. This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the Project POCI-01-0145-FEDER-007038; and by the Project NORTE-01-0145- FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

Mutations in fam20b and xylt1 Reveal That Cartilage Matrix Controls Timing of Endochondral Ossification by Inhibiting Chondrocyte Maturation

Differentiating cells interact with their extracellular environment over time. Chondrocytes embed themselves in a proteoglycan (PG)-rich matrix, then undergo a developmental transition, termed “maturation,” when they express ihh to induce bone in the overlying tissue, the perichondrium. Here, we ask whether PGs regulate interactions between chondrocytes and perichondrium, using zebrafish mutants to reveal that cartilage PGs inhibit chondrocyte maturation, which ultimately dictates the timing of perichondral bone development. In a mutagenesis screen, we isolated a class of mutants with decreased cartilage matrix and increased perichondral bone. Positional cloning identified lesions in two genes, fam20b and xylosyltransferase1 (xylt1), both of which encode PG synthesis enzymes. Mutants failed to produce wild-type levels of chondroitin sulfate PGs, which are normally abundant in cartilage matrix, and initiated perichondral bone formation earlier than their wild-type siblings. Primary chondrocyte defects might induce the bone phenotype secondarily, because mutant chondrocytes precociously initiated maturation, showing increased and early expression of such markers as runx2b, collagen type 10a1, and ihh co-orthologs, and ihha mutation suppressed early perichondral bone in PG mutants. Ultrastructural analyses demonstrated aberrant matrix organization and also early cellular features of chondrocyte hypertrophy in mutants. Refining previous in vitro reports, which demonstrated that fam20b and xylt1 were involved in PG synthesis, our in vivo analyses reveal that these genes function in cartilage matrix production and ultimately regulate the timing of skeletal development

NOVOcan: a molecular link among selected glial cells

The nervous system is generated from cells lining the ventricular system. Our understanding of the fate potentials and lineage relationships of these cells is being re-evaluated, both because of recent demonstrations that radial glia can generate neurons and because of the identification of fate-determining genes. A variety of intrinsic and extrinsic molecules, including proteoglycans, regulate embryonic and postnatal brain development. Using probes modeled after species conserved domains of heparan sulfate proteoglycans, we cloned a novel gene called novocan, raised monoclonal antibodies against a segment of the predicted amino acid sequence of the expressed protein (NOVOcan) and used the antibodies to establish the cell and tissue localization of NOVOcan in postnatal rat brains by immunohistochemistry. NOVOcan was expressed in cells lining the ventricles, including a variety of radial glia during early postnatal development. Later, as radial glia disappeared and ependymal cells appeared, NOVOcan was detected in ependymal cells and in tanycytes, a specialized form of ependymal cell resembling radial glia. NOVOcan was absent in two known progeny of radial glia, mature astrocytes and neurons. Whereas NOVOcan was also absent in mature oligodendrocytes (OLGs), it was present in OLG precursors in developing white matter. These studies set the stage for determining the roles of NOVOcan in brain cell lineage patterns as well as in other aspects of development. © 2003 Elsevier B.V. All rights reserved

Retinoic acid as a modulator of proximal-distal patterning and branching morphogenesis of the avian lung

Retinoic acid modulates numerous cellular events, namely, proliferation, differentiation, apoptosis, and patterning, hence influencing both embryo development and adult homeostasis. In vitro explant culture is a valuable technique for studying the impact of growth factors and signaling molecules, such as retinoic acid, in organ development since tissue architecture is maintained. This technique allows controlled supplementation of culture medium and straightforward analysis of its effect on morphogenesis. This chapter describes the detailed protocol for culturing embryonic chick lung explants and testing the impact of retinoic acid in branching and patterning, based on morphometric and molecular analysis.FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the Project UID/Multi/50026/2019; and by the Project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER