Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell-death by anoikis.

To investigate the role of the nuclear receptor NR5A1 in testis after sex determination, we have analyzed mice lacking NR5A1 in Sertoli cells (SC) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impairs the expression of genes characteristic of the SC identity (e.g., Sox9, Amh), causes SC death from E14.5 through a Trp53-independent mechanism related to anoikis, and induces disorganization of the testis cords. Together, these effects cause germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SC change their molecular identity: some acquire a "pre-granulosa-like" identity, while other revert to a "supporting progenitor-like" cell identity, most of them being "intersex" because they express both testicular and ovarian genes. Fetal Leydig cells (LC) do not display significant changes, indicating that SC are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LC were absent from the postnatal testes. In addition, adult mutant males display persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which can be explained by the loss of AMH and testosterone synthesis due to SC failure.

Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell death by anoikis.

To investigate the role of the nuclear receptor NR5A1 in the testis after sex determination, we analyzed mice lacking NR5A1 in Sertoli cells (SCs) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impaired the expression of genes characteristic of SC identity (e.g. Sox9 and Amh), caused SC death from E14.5 onwards through a Trp53-independent mechanism related to anoikis, and induced disorganization of the testis cords. Together, these effects caused germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SCs changed their molecular identity: some acquired a 'pre-granulosa-like' cell identity, whereas other reverted to a 'supporting progenitor-like' cell identity, most of them being 'intersex' because they expressed both testicular and ovarian genes. Fetal Leydig cells (LCs) did not display significant changes, indicating that SCs are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LCs were absent from postnatal testes. In addition, adult mutant males displayed persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which could be explained by the loss of AMH and testosterone synthesis due to SC failure.

Local retinoic acid signaling directs emergence of the extraocular muscle functional unit.

Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments.

Retinoic acid signaling pathways.

Retinoic acid (RA), a metabolite of retinol (vitamin A), functions as a ligand for nuclear RA receptors (RARs) that regulate development of chordate animals. RA-RARs can activate or repress transcription of key developmental genes. Genetic studies in mouse and zebrafish embryos that are deficient in RA-generating enzymes or RARs have been instrumental in identifying RA functions, revealing that RA signaling regulates development of many organs and tissues, including the body axis, spinal cord, forelimbs, heart, eye and reproductive tract. An understanding of the normal functions of RA signaling during development will guide efforts for use of RA as a therapeutic agent to improve human health. Here, we provide an overview of RA signaling and highlight its key functions during development.

Two functionally redundant sources of retinoic acid secure spermatogonia differentiation in the seminiferous epithelium.

In mammals, all-trans retinoic acid (ATRA) is instrumental to spermatogenesis. It is synthesized by two retinaldehyde dehydrogenases (RALDH) present in both Sertoli cells (SCs) and germ cells (GCs). In order to determine the relative contributions of each source of ATRA, we have generated mice lacking all RALDH activities in the seminiferous epithelium (SE). We show that both the SC- and GC-derived sources of ATRA cooperate to initiate and propagate spermatogenetic waves at puberty. In adults, they exert redundant functions and, against all expectations, the GC-derived source does not perform any specific roles despite contributing to two-thirds of the total amount of ATRA present in the testis. The production from SCs is sufficient to maintain the periodic expression of genes in SCs, as well and the cycle and wave of the SE, which account for the steady production of spermatozoa. The production from SCs is also specifically required for spermiation. Importantly, our study shows that spermatogonia differentiation depends upon the ATRA synthesized by RALDH inside the SE, whereas initiation of meiosis and expression of STRA8 by spermatocytes can occur without ATRA.

Constitutive WNT/CTNNB1 activation triggers spermatogonial stem cell proliferation and germ cell depletion.

The differentiation of germ cells into oogonia or spermatogonia is the first step that eventually gives rise to fully mature gametes. In the female fetal gonad, the RSPO1/WNT/CTNNB1 signalling pathway is involved in primordial germ cell proliferation and differentiation into female germ cells, which are able to enter meiosis. In the postnatal testis, the WNT/CTNNB1 pathway also mediates proliferation of spermatogonial stem cells and progenitor cells. Here we show that forced activation of the WNT/CTNNB1 pathway in fetal gonocytes using transgenic mice leads to deregulated spermatogonial proliferation, and exhaustion of the spermatocytes by apoptosis, resulting in a hypoplastic testis. These findings demonstrate that a finely tuned timing in WNT/CTNNB1 signalling activity is required for spermatogenesis.

Retinoic acid signaling is dispensable for somatic development and function in the mammalian ovary.

Retinoic acid (RA) is a potent inducer of cell differentiation and plays an essential role in sex-specific germ cell development in the mammalian gonad. RA is essential for male gametogenesis and hence fertility. However, RA can also disrupt sexual cell fate in somatic cells of the testis, promoting transdifferentiation of male Sertoli cells to female granulosa-like cells when the male sexual regulator Dmrt1 is absent. The feminizing ability of RA in the Dmrt1 mutant somatic testis suggests that RA might normally play a role in somatic cell differentiation or cell fate maintenance in the ovary. To test for this possibility we disrupted RA signaling in somatic cells of the early fetal ovary using three genetic strategies and one pharmaceutical approach. We found that deleting all three RA receptors (RARs) in the XX somatic gonad at the time of sex determination did not significantly affect ovarian differentiation, follicle development, or female fertility. Transcriptome analysis of adult triple mutant ovaries revealed remarkably little effect on gene expression in the absence of somatic RAR function. Likewise, deletion of three RA synthesis enzymes (Aldh1a1-3) at the time of sex determination did not masculinize the ovary. A dominant-negative RAR transgene altered granulosa cell proliferation, likely due to interference with a non-RA signaling pathway, but did not prevent granulosa cell specification and oogenesis or abolish fertility. Finally, culture of fetal XX gonads with an RAR antagonist blocked germ cell meiotic initiation but did not disrupt sex-biased gene expression. We conclude that RA signaling, although crucial in the ovary for meiotic initiation, is not required for granulosa cell specification, differentiation, or reproductive function.

Retinoic Acid Receptors Control Spermatogonia Cell-Fate and Induce Expression of the SALL4A Transcription Factor.

All-trans retinoic acid (ATRA) is instrumental to male germ cell differentiation, but its mechanism of action remains elusive. To address this question, we have analyzed the phenotypes of mice lacking, in spermatogonia, all rexinoid receptors (RXRA, RXRB and RXRG) or all ATRA receptors (RARA, RARB and RARG). We demonstrate that the combined ablation of RXRA and RXRB in spermatogonia recapitulates the set of defects observed both upon ablation of RAR in spermatogonia. We also show that ATRA activates RAR and RXR bound to a conserved regulatory region to increase expression of the SALL4A transcription factor in spermatogonia. Our results reveal that this major pluripotency gene is a target of ATRA signaling and that RAR/RXR heterodimers are the functional units driving its expression in spermatogonia. They add to the mechanisms through which ATRA promote expression of the KIT tyrosine kinase receptor to trigger a critical step in spermatogonia differentiation. Importantly, they indicate also that meiosis eventually occurs in the absence of a RAR/RXR pathway within germ cells and suggest that instructing this process is either ATRA-independent or requires an ATRA signal originating from Sertoli cells.

Retinoic acid improves nephrotoxic serum-induced glomerulonephritis through activation of podocyte retinoic acid receptor α.

Proliferation of glomerular epithelial cells, including podocytes, is a key histologic feature of crescentic glomerulonephritis. We previously found that retinoic acid (RA) inhibits proliferation and induces differentiation of podocytes by activating RA receptor-α (RARα) in a murine model of HIV-associated nephropathy. Here, we examined whether RA would similarly protect podocytes against nephrotoxic serum-induced crescentic glomerulonephritis and whether this effect was mediated by podocyte RARα. RA treatment markedly improved renal function and reduced the number of crescentic lesions in nephritic wild-type mice, while this protection was largely lost in mice with podocyte-specific ablation of Rara (Pod-Rara knockout). At a cellular level, RA significantly restored the expression of podocyte differentiation markers in nephritic wild-type mice, but not in nephritic Pod-Rara knockout mice. Furthermore, RA suppressed the expression of cell injury, proliferation, and parietal epithelial cell markers in nephritic wild-type mice, all of which were significantly dampened in nephritic Pod-Rara knockout mice. Interestingly, RA treatment led to the coexpression of podocyte and parietal epithelial cell markers in a small subset of glomerular cells in nephritic mice, suggesting that RA may induce transdifferentiation of parietal epithelial cells toward a podocyte phenotype. In vitro, RA directly inhibited the proliferation of parietal epithelial cells and enhanced the expression of podocyte markers. In vivo lineage tracing of labeled parietal epithelial cells confirmed that RA increased the number of parietal epithelial cells expressing podocyte markers in nephritic glomeruli. Thus, RA attenuates crescentic glomerulonephritis primarily through RARα-mediated protection of podocytes and in part through the inhibition of parietal epithelial cell proliferation and induction of their transdifferentiation into podocytes.

Endogenous retinoic acid signaling is required for maintenance and regeneration of cornea.

Retinoic acid (RA) is a biologically active metabolite of vitamin A (retinol) that serves as an important signaling molecule in orchestrating diverse developmental processes including multiple roles during ocular development. Loss-of-function studies using gene knockouts of RA-synthesizing enzymes encoded by Aldh1a1, Aldh1a2, and Aldh1a3 (also known as Raldh1, Raldh2, and Raldh3) have provided valuable insight into how RA controls eye morphogenesis including corneal development. However, it is unclear whether endogenous RA is required for maintenance and regeneration of adult cornea. Here, we investigated the role of Aldh1a genes in the adult cornea using a novel conditional Aldh1a1,2,3-flox/flox;Rosa26-CreERT2 loss-of-function mouse model to determine the biological function of RA. Our findings indicate that loss of RA synthesis results in corneal thinning characterized by reduced thickness of the stromal layer, impaired corneal epithelial cell proliferation, and increased apoptosis. Corneal thinning in Aldh1a-deficient mice was significantly rescued by RA administration, indicating an important role of endogenous RA signaling in adult corneal homeostasis and regeneration. Thus, Aldh1a1,2,3-flox/flox;Rosa26-CreERT2 mice provide a useful model for investigating the mechanistic role of RA signaling in adult corneal maintenance and could provide new insights into therapeutic approaches for controlling corneal repair to prevent vision loss.

Role of retinoic acid receptor (RAR) signaling in post-natal male germ cell differentiation.

All-trans retinoic acid (atRA), the active metabolite of vitamin A, plays critical functions in spermatogenesis, a complex, highly organized and regulated process comprising three phases. During the proliferative phase, undifferentiated spermatogonia divide to maintain a stem cell population and expand a progenitor cell population, of which a fraction enters the differentiation pathway yielding primary spermatocytes. During the meiotic phase, primary spermatocytes undergo recombination, segregation and reduction by half of chromosomes to produce haploid round spermatids. During the morphogenetic, post-meiotic phase, spermatids differentiate and elongate to ultimately form spermatozoa. Studies performed during the past 20 years have significantly improved our knowledge on the location of the proteins transducing the atRA signal, on the target genes of atRA and on its mechanism of action. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Testicular differentiation occurs in absence of R-spondin1 and Sox9 in mouse sex reversals.

In mammals, male sex determination is governed by SRY-dependent activation of Sox9, whereas female development involves R-spondin1 (RSPO1), an activator of the WNT/beta-catenin signaling pathway. Genetic analyses in mice have demonstrated Sry and Sox9 to be both required and sufficient to induce testicular development. These genes are therefore considered as master regulators of the male pathway. Indeed, female-to-male sex reversal in XX Rspo1 mutant mice correlates with Sox9 expression, suggesting that this transcription factor induces testicular differentiation in pathological conditions. Unexpectedly, here we show that testicular differentiation can occur in XX mutants lacking both Rspo1 and Sox9 (referred to as XX Rspo1(KO)Sox9(cKO) ()), indicating that Sry and Sox9 are dispensable to induce female-to-male sex reversal. Molecular analyses show expression of both Sox8 and Sox10, suggesting that activation of Sox genes other than Sox9 can induce male differentiation in Rspo1(KO)Sox9(cKO) mice. Moreover, since testis development occurs in XY Rspo1(KO)Sox9(cKO) mice, our data show that Rspo1 is the main effector for male-to-female sex reversal in XY Sox9(cKO) mice. Thus, Rspo1 is an essential activator of ovarian development not only in normal situations, but also in sex reversal situations. Taken together these data demonstrate that both male and female sex differentiation is induced by distinct, active, genetic pathways. The dogma that considers female differentiation as a default pathway therefore needs to be definitively revised.

Retinoic acid induces Sertoli cell paracrine signals for spermatogonia differentiation but cell autonomously drives spermatocyte meiosis.

Direct evidence for a role of endogenous retinoic acid (RA), the active metabolite of vitamin A in the initial differentiation and meiotic entry of spermatogonia, and thus in the initiation of spermatogenesis is still lacking. RA is synthesized by dedicated enzymes, the retinaldehyde dehydrogenases (RALDH), and binds to and activates nuclear RA receptors (RARA, RARB, and RARG) either within the RA-synthesizing cells or in the neighboring cells. In the present study, we have used a combination of somatic genetic ablations and pharmacological approaches in vivo to show that during the first, prepubertal, spermatogenic cycle (i) RALDH-dependent synthesis of RA by Sertoli cells (SC), the supporting cells of the germ cell (GC) lineage, is indispensable to initiate differentiation of A aligned into A1 spermatogonia; (ii) RARA in SC mediates the effects of RA, possibly through activating Mafb expression, a gene whose Drosophila homolog is mandatory to GC differentiation; (iii) RA synthesized by premeiotic spermatocytes cell autonomously induces meiotic initiation through controlling the RAR-dependent expression of Stra8. Furthermore, we show that RA of SC origin is no longer necessary for the subsequent spermatogenic cycles but essential to spermiation. Altogether, our data establish that the effects of RA in vivo on spermatogonia differentiation are indirect, via SC, but direct on meiotic initiation in spermatocytes, supporting thereby the notion that, contrary to the situation in the female, RA is necessary to induce meiosis in the male.

The STRA6 receptor is essential for retinol-binding protein-induced insulin resistance but not for maintaining vitamin A homeostasis in tissues other than the eye.

The plasma membrane protein STRA6 is thought to mediate uptake of retinol from its blood carrier retinol-binding protein (RBP) into cells and to function as a surface receptor that, upon binding of holo-RBP, activates a JAK/STAT cascade. It was suggested that STRA6 signaling underlies insulin resistance induced by elevated serum levels of RBP in obese animals. To investigate these activities in vivo, we generated and analyzed Stra6-null mice. We show that the contribution of STRA6 to retinol uptake by tissues in vivo is small and that, with the exception of the eye, ablation of Stra6 has only a modest effect on retinoid homeostasis and does not impair physiological functions that critically depend on retinoic acid in the embryo or in the adult. However, ablation of Stra6 effectively protects mice from RBP-induced suppression of insulin signaling. Thus one biological function of STRA6 in tissues other than the eye appears to be the coupling of circulating holo-RBP levels to cell signaling, in turn regulating key processes such as insulin response.

Genetic disruption of aurora B uncovers an essential role for aurora C during early mammalian development.

Mitosis is controlled by multiple kinases that drive cell cycle progression and prevent chromosome mis-segregation. Aurora kinase B interacts with survivin, borealin and incenp to form the chromosomal passenger complex (CPC), which is involved in the regulation of microtubule-kinetochore attachments and cytokinesis. Whereas genetic ablation of survivin, borealin or incenp results in early lethality at the morula stage, we show here that aurora B is dispensable for CPC function during early cell divisions and aurora B-null embryos are normally implanted. This is due to a crucial function of aurora C during these early embryonic cycles. Expression of aurora C decreases during late blastocyst stages resulting in post-implantation defects in aurora B-null embryos. These defects correlate with abundant prometaphase figures and apoptotic cell death of the aurora B-deficient inner cell mass. Conditional deletion of aurora B in somatic cells that do not express aurora C results in chromosomal misalignment and lack of chromosome segregation. Re-expression of wild-type, but not kinase-dead, aurora C rescues this defect, suggesting functional overlap between these two kinases. Finally, aurora B-null cells partially arrest in the presence of nocodazole, suggesting that this kinase is not essential for the spindle assembly checkpoint.

Timeline of Developmental Defects Generated upon Genetic Inhibition of the Retinoic Acid Receptor Signaling Pathway.

It has been established for almost 30 years that the retinoic acid receptor (RAR) signalling pathway plays essential roles in the morphogenesis of a large variety of organs and systems. Here, we used a temporally controlled genetic ablation procedure to precisely determine the time windows requiring RAR functions. Our results indicate that from E8.5 to E9.5, RAR functions are critical for the axial rotation of the embryo, the appearance of the sinus venosus, the modelling of blood vessels, and the formation of forelimb buds, lung buds, dorsal pancreatic bud, lens, and otocyst. They also reveal that E9.5 to E10.5 spans a critical developmental period during which the RARs are required for trachea formation, lung branching morphogenesis, patterning of great arteries derived from aortic arches, closure of the optic fissure, and growth of inner ear structures and of facial processes. Comparing the phenotypes of mutants lacking the 3 RARs with that of mutants deprived of all-trans retinoic acid (ATRA) synthesising enzymes establishes that cardiac looping is the earliest known morphogenetic event requiring a functional ATRA-activated RAR signalling pathway.

Corneal epithelial cell fate is maintained during repair by Notch1 signaling via the regulation of vitamin A metabolism.

Integrity and preservation of a transparent cornea are essential for good vision. The corneal epithelium is stratified and nonkeratinized and is maintained and repaired by corneal stem cells. Here we demonstrate that Notch1 signaling is essential for cell fate maintenance of corneal epithelium during repair. Inducible ablation of Notch1 in the cornea combined with mechanical wounding show that Notch1-deficient corneal progenitor cells differentiate into a hyperplastic, keratinized, skin-like epithelium. This cell fate switch leads to corneal blindness and involves cell nonautonomous processes, characterized by secretion of fibroblast growth factor-2 (FGF-2) through Notch1(-/-) epithelium followed by vascularization and remodeling of the underlying stroma. Vitamin A deficiency is known to induce a similar corneal defect in humans (severe xerophthalmia). Accordingly, we found that Notch1 signaling is linked to vitamin A metabolism by regulating the expression of cellular retinol binding protein 1 (CRBP1), required to generate a pool of intracellular retinol.

A transcriptionally silent RXRalpha supports early embryonic morphogenesis and heart development.

Retinoic acid (RA) receptors (RARs) alpha, beta, and gamma heterodimerized with rexinoid receptors (RXRs) alpha, beta, and gamma mediate the RA signal. To analyze the contribution of the transcriptional activity of RXRalpha, the main RXR during embryogenesis, we have engineered a mouse line harboring a transcriptionally silent RXRalpha mutant that lacks the activation functions AF1 and AF2. All homozygous mutants (Rxra(afo)) display the ocular defects previously observed in compound Rar-null and Rxra/Rar-null mutants, thus demonstrating that a transcriptionally active RXRalpha is required during eye development. In contrast, the vast majority of Rxra(afo) fetuses do not display the Rxra-null mutant hypoplasia of the myocardium, thus demonstrating that RXRalpha can act as a transcriptionally silent heterodimerization partner. Similarly, a transcriptionally silent RXRalpha mutant can support early embryogenesis, as Rxra(afo)/Rxrb-null embryos display a normal morphology, contrasting with the severe malformations exhibited by compound Rxra/Rxrb-null embryos. Along the same line, we show that a silent RXRalpha mutant is sufficient to allow the initial formation of the placental labyrinth, whereas later steps of trophoblast cell differentiation critically requires the AF2, but not the AF1, function of RXRalpha.

Retinoic Acid Receptor Alpha Is Essential in Postnatal Sertoli Cells but Not in Germ Cells.

Retinoic acid signaling is indispensable for the completion of spermatogenesis. It is known that loss of retinoic acid nuclear receptor alpha (RARA) induces male sterility due to seminiferous epithelium degeneration. Initial genetic studies established that RARA acts in Sertoli cells, but a recent paper proposed that RARA is also instrumental in germ cells. In the present study, we have re-assessed the function of RARA in germ cells by genetically ablating the Rara gene in spermatogonia and their progenies using a cell-specific conditional mutagenesis approach. We show that loss of Rara in postnatal male germ cells does not alter the histology of the seminiferous epithelium. Furthermore, RARA-deficient germ cells differentiate normally and give rise to normal, living pups. This establishes that RARA plays no crucial role in germ cells. We also tested whether RARA is required in Sertoli cells during the fetal period or after birth. For this purpose, we deleted the Rara gene in Sertoli cells at postnatal day 15 (PN15), i.e., after the onset of the first spermatogenic wave. To do so, we used temporally controlled cell-specific mutagenesis. By comparing the testis phenotypes generated when Rara is lost either at PN15 or at embryonic day 13, we show that RARA exerts all of its functions in Sertoli cells not at the fetal stage but from puberty.

Multilayer omics analysis reveals a non-classical retinoic acid signaling axis that regulates hematopoietic stem cell identity.

Hematopoietic stem cells (HSCs) rely on complex regulatory networks to preserve stemness. Due to the scarcity of HSCs, technical challenges have limited our insights into the interplay between metabolites, transcription, and the epigenome. In this study, we generated low-input metabolomics, transcriptomics, chromatin accessibility, and chromatin immunoprecipitation data, revealing distinct metabolic hubs that are enriched in HSCs and their downstream multipotent progenitors. Mechanistically, we uncover a non-classical retinoic acid (RA) signaling axis that regulates HSC function. We show that HSCs rely on Cyp26b1, an enzyme conventionally considered to limit RA effects in the cell. In contrast to the traditional view, we demonstrate that Cyp26b1 is indispensable for production of the active metabolite 4-oxo-RA. Further, RA receptor beta (Rarb) is required for complete transmission of 4-oxo-RA-mediated signaling to maintain stem cells. Our findings emphasize that a single metabolite controls stem cell fate by instructing epigenetic and transcriptional attributes.