Retinoic Acid Antagonizes Testis Development in Mice.

Mammalian sex determination depends on a complex interplay of signals that promote the bipotential fetal gonad to develop as either a testis or an ovary, but the details are incompletely understood. Here, we investigated whether removal of the signaling molecule retinoic acid (RA) by the degradative enzyme CYP26B1 is necessary for proper development of somatic cells of the testes. Gonadal organ culture experiments suggested that RA promotes expression of some ovarian markers and suppresses expression of some testicular markers, acting downstream of Sox9. XY Cyp26b1-null embryos, in which endogenous RA is not degraded, develop mild ovotestes, but more important, steroidogenesis is impaired and the reproductive tract feminized. Experiments involving purified gonadal cells showed that these effects are independent of germ cells and suggest the direct involvement of the orphan nuclear receptor DAX1. Our results reveal that active removal of endogenous RA is required for normal testis development in the mouse.

ALDH1A1 provides a source of meiosis-inducing retinoic acid in mouse fetal ovaries.

Substantial evidence exists that during fetal ovarian development in mammals, retinoic acid (RA) induces germ cells to express the pre-meiotic marker Stra8 and enter meiosis, and that these effects are prevented in the fetal testis by the RA-degrading P450 enzyme CYP26B1. Nonetheless, the role of RA has been disputed principally because germ cells in embryos lacking two major RA-synthesizing enzymes, ALDH1A2 and ALDH1A3, remain able to enter meiosis. Here we show that a third RA-synthesizing enzyme, ALDH1A1, is expressed in fetal ovaries, providing a likely source of RA in the absence of ALDH1A2 and ALDH1A3. In ovaries lacking ALDH1A1, the onset of germ cell meiosis is delayed. Our data resolve the conundrum posed by conflicting published data sets and reconfirm the model that meiosis is triggered by endogenous RA in the developing ovary.

Direct transcriptional reprogramming of adult cells to embryonic nephron progenitors.

Direct reprogramming involves the enforced re-expression of key transcription factors to redefine a cellular state. The nephron progenitor population of the embryonic kidney gives rise to all cells within the nephron other than the collecting duct through a mesenchyme-to-epithelial transition, but this population is exhausted around the time of birth. Here, we sought to identify the conditions under which adult proximal tubule cells could be directly transcriptionally reprogrammed to nephron progenitors. Using a combinatorial screen for lineage-instructive transcription factors, we identified a pool of six genes (SIX1, SIX2, OSR1, EYA1, HOXA11, and SNAI2) that activated a network of genes consistent with a cap mesenchyme/nephron progenitor phenotype in the adult proximal tubule (HK2) cell line. Consistent with these reprogrammed cells being nephron progenitors, we observed differential contribution of the reprogrammed population into the Six2(+) nephron progenitor fields of an embryonic kidney explant. Dereplication of the pool suggested that SNAI2 can suppress E-CADHERIN, presumably assisting in the epithelial-to-mesenchymal transition (EMT) required to form nephron progenitors. However, neither TGFß-induced EMT nor SNAI2 overexpression alone was sufficient to create this phenotype, suggesting that additional factors are required. In conclusion, these results suggest that reinitiation of kidney development from a population of adult cells by generating embryonic progenitors may be feasible, opening the way for additional cellular and bioengineering approaches to renal repair and regeneration.

Dissociation of embryonic kidney followed by re-aggregation as a method for chimeric analysis.

This chapter presents three methods for re-constructing mouse foetal kidney tissue from simple suspensions of cells. These techniques are very useful for a number of purposes: (1) they allow the production of fine-grained chimaeras in which cell autonomy of mutations can be tested, (2) they provide an environment that allows the renal differentiation potential of stem cells to be assessed, and (3) they are an excellent system in which to study the mechanisms of self-organization. Each of the methods described here begins with disaggregation of embryonic mouse kidneys, followed by re-aggregation and culture; the main differences are in the culture methods, each of which has advantages for particular purposes.

Somatic reactivation of expression of the silent maternal Mest allele and acquisition of normal reproductive behaviour in a colony of Peg1/Mest mutant mice.

Genomic imprinting confers allele-specific expression in less than 1% of genes, in a parent-of-origin specific fashion. In humans and mice the Peg1/Mest gene (Mest) is maternally repressed, and paternally expressed. Mest is expressed in embryogenic mesoderm-derived tissues and in adult brain, and paternal mutations in Mest lead to growth retardation and defective maternal behaviour. Despite our current understanding of mechanisms associated with the establishment of imprinting of Mest and other imprinted genes, it is unclear to what extent Mest imprinting needs to be maintained in adult tissues. Aberrations of imprinting are known to occur in certain rare syndromes, and involve either inherited mutations, or constitutive epigenetic alterations occurring soon after fertilization. Imprinting abnormalities may also occur in the aging somatic tissues of adult individuals. Here we report an occurrence of post-embryonic somatic variability of Mest allelic expression in a colony of mice where heterozygotes at the imprinted Mest locus for a mutation inherited from the father spontaneously expressed the normally silenced allele from the mother. In addition, a newly acquired ability to overcome the deficit in maternal reproductive behaviour had occurred in the mutant mice, but this appeared not to be directly linked to the Mest mutation. Our results suggest that at least one allele of Mest expression is required in the somatic tissues of adult individuals and that under certain conditions (such as in the presence of a Mest insertional mutation or in an altered genetic background), somatically acquired alterations of allelic expression at the Mest locus may occur.