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.

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.

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.

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.

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 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.

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.

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.

Why serology just is not enough: Strategic parvovirus risk assessment using a novel qPCR assay.

Health monitoring of laboratory rodents not only improves animal health but also enhances the validity of animal experiments. In particular, infections of laboratory animals with murine parvoviruses influence biomedical research data. Despite strict barrier housing, prevalence remains high in animal facilities, leading to increased risk of parvovirus introduction after the import of contaminated mice. Unfortunately, hygienic rederivation can be challenging, since gametes often contain residual virus material. Consequently, the process has to be closely monitored with highly sensitive diagnostic methods to verify parvovirus decontamination of the rederived progeny. However, diagnostic sensitivity of traditional methods is often low and requires testing of large animal cohorts. Therefore, we aimed to develop a powerful quantitative real-time polymerase chain reaction (qPCR) assay for the fast and reliable detection of murine parvoviruses in different sample materials. We validated the assay within an infection experiment and systematically analysed various animal-derived and environmental sample materials. We further developed a strategic risk assessment procedure for parvovirus monitoring after embryo transfer. Our novel qPCR assay reliably detected parvovirus DNA in a broad variety of sample materials, with environmental samples dominating in the acute phase of infection, whereas animal-derived samples were more suitable to detect low virus loads in the chronic phase. Here, the assay served as a highly sensitive screening method for parvovirus contamination in mouse colonies, requiring significantly lower sample sizes than traditional methods like conventional PCR and serology. Thus, the use of our novel qPCR assay substantially improves parvovirus diagnostics, enhancing research validity according to the 6Rs.

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.

Pathogenesis of Anorectal Malformations in Retinoic Acid Receptor Knockout Mice Studied by HREM.

Anorectal malformations (ARMs) are relatively common congenital abnormalities, but their pathogenesis is poorly understood. Previous gene knockout studies indicated that the signalling pathway mediated by the retinoic acid receptors (RAR) is instrumental to the formation of the anorectal canal and of various urogenital structures. Here, we show that simultaneous ablation of the three RARs in the mouse embryo results in a spectrum of malformations of the pelvic organs in which anorectal and urinary bladder ageneses are consistently associated. We found that these ageneses could be accounted for by defects in the processes of growth and migration of the cloaca, the embryonic structure from which the anorectal canal and urinary bladder originate. We further show that these defects are preceded by a failure of the lateral shift of the umbilical arteries and propose vascular abnormalities as a possible cause of ARM. Through the comparisons of these phenotypes with those of other mutant mice and of human patients, we would like to suggest that morphological data may provide a solid base to test molecular as well as clinical hypotheses.

Meiosis occurs normally in the fetal ovary of mice lacking all retinoic acid receptors.

Gametes are generated through a specialized cell differentiation process, meiosis, which, in ovaries of most mammals, is initiated during fetal life. All-trans retinoic acid (ATRA) is considered as the molecular signal triggering meiosis initiation. In the present study, we analyzed female fetuses ubiquitously lacking all ATRA nuclear receptors (RAR), obtained through a tamoxifen-inducible cre recombinase-mediated gene targeting approach. Unexpectedly, mutant oocytes robustly expressed meiotic genes, including the meiotic gatekeeper STRA8. In addition, ovaries from mutant fetuses grafted into adult recipient females yielded offspring bearing null alleles for all Rar genes. Thus, our results show that RAR are fully dispensable for meiotic initiation, as well as for the production of functional oocytes. Assuming that the effects of ATRA all rely on RAR, our study goes against the current model according to which meiosis is triggered by endogenous ATRA in the developing ovary. It therefore revives the search for the meiosis-inducing substance.

Spermatogonia differentiation requires retinoic acid receptor γ.

Vitamin A is instrumental to mammalian reproduction. Its metabolite, retinoic acid (RA), acts in a hormone-like manner through binding to and activating three nuclear receptor isotypes, RA receptor (RAR)α (RARA), RARß, and RARγ (RARG). Here, we show that 1) RARG is expressed by A aligned (A(al)) spermatogonia, as well as during the transition from A(al) to A(1) spermatogonia, which is known to require RA; and 2) ablation of Rarg, either in the whole mouse or specifically in spermatogonia, does not affect meiosis and spermiogenesis but impairs the A(al) to A(1) transition in the course of some of the seminiferous epithelium cycles. Upon ageing, this phenomenon yields seminiferous tubules containing only spermatogonia and Sertoli cells. Altogether, our findings indicate that RARG cell-autonomously transduces, in undifferentiated spermatogonia of adult testes, a RA signal critical for spermatogenesis. During the prepubertal spermatogenic wave, the loss of RARG function can however be compensated by RARA, as indicated by the normal timing of appearance of meiotic cells in Rarg-null testes. Accordingly, RARG- and RARA-selective agonists are both able to stimulate Stra8 expression in wild-type prepubertal testes. Interestingly, inactivation of Rarg does not impair expression of the spermatogonia differentiation markers Kit and Stra8, contrary to vitamin A deficiency. This latter observation supports the notion that the RA-signaling pathway previously shown to operate in Sertoli cells also participates in spermatogonia differentiation.

Retinoic acid drives aryl hydrocarbon receptor expression and is instrumental to dioxin-induced toxicity during palate development.

BACKGROUND: Palate development depends on complex events and is very sensitive to disruption. Accordingly, clefts are the most common congenital malformations worldwide, and a connection is proposed with fetal exposure to toxic factors or environmental contaminants, such as dioxins. There is increasing evidence that dioxin interferes with all-trans-retinoic acid (atRA), a hormone-like signal derived from vitamin A, which plays an essential role during embryonic development. Although similarities have been described between dioxin-induced toxicity and the outcome of altered atRA signaling during palate development, their relationship needs to be clarified. OBJECTIVES: We used a genetic approach to understand the interaction between atRA and dioxin and to identify the cell type targeted by dioxin toxicity during secondary palate formation in mice. METHODS: We analyzed the phenotype of mouse embryos harboring an atRA-sensitive reporter transgene or bearing null mutations for atRA-synthesizing enzymes (RALDH) or atRA receptors (RAR) and maternally exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at gestation day 10.5. RESULTS: We found that an intact atRA signal was required to enable TCDD to induce cleft palate. This mandatory atRA signal was generated through the activity of RALDH3 in the nasal epithelium and was transduced by RARγ (RARG) in the nasal mesenchyme, where it notably controlled aryl hydrocarbon receptor (Ahr) transcript levels. TCDD also did not alter the developmental pattern of atRA signaling during palate formation. CONCLUSIONS: TCDD-induced alteration of secondary palate development in the mouse appears to depend on atRA signaling, which controls AHR expression. This mechanism is likely conserved throughout vertebrate evolution and may therefore be relevant in humans.

STRA8-deficient spermatocytes initiate, but fail to complete, meiosis and undergo premature chromosome condensation.

We analysed the phenotypic outcome of a Stra8-null mutation on male meiosis. Because the mutant spermatocytes (1) underwent premeiotic DNA replication, (2) displayed cytological features attesting initiation of recombination and of axial-element assembly, and (3) expressed Spo11 and numerous other meiotic genes, it was concluded that STRA8 is dispensable for meiotic initiation. The few mutant spermatocytes that progressed beyond leptonema showed a prolonged bouquet-stage configuration, asynapsis and heterosynapsis, suggesting function(s) of STRA8 in chromosome pairing. Most importantly, a large number of mutant leptotene spermatocytes underwent premature chromosome condensation, within 24 hours following the meiotic S phase. This phenomenon yielded aberrant metaphase-like cells with 40 univalent chromosomes, similar to normal mitotic metaphases. From these latter observations and from the wild-type pattern of Stra8 expression, we propose that, in preleptotene spermatocytes, STRA8 is involved in the process that leads to stable commitment to the meiotic cell cycle.