FOXO1 represses sprouty 2 and sprouty 4 expression to promote arterial specification and vascular remodeling in the mouse yolk sac.

Establishing a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss-of-function mouse models showed that FOXO1, a Forkhead family transcription factor, is required for yolk sac (YS) vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that at E8.25, loss of Foxo1 in Tie2-cre expressing cells resulted in increased sprouty 2 (Spry2) and Spry4 expression, reduced arterial gene expression and reduced Kdr (also known as Vegfr2 and Flk1) transcripts without affecting overall endothelial cell identity, survival or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, delta like 4, is significantly reduced in Foxo1 mutant YS without being substantially affected in the embryo proper. We show that FOXO1 binds directly to previously identified Spry2 gene regulatory elements (GREs) and newly identified, evolutionarily conserved Spry4 GREs to repress their expression. Furthermore, overexpression of Spry4 in transient transgenic embryos largely recapitulates the reduced expression of arterial genes seen in conditional Foxo1 mutants. Together, these data reveal a novel role for FOXO1 as a key transcriptional repressor regulating both pre-flow arterial specification and subsequent vessel remodeling within the murine YS.

Reprogramming of the Epigenome by MLL1 Links Early-Life Environmental Exposures to Prostate Cancer Risk.

Tissue and organ development is a time of exquisite sensitivity to environmental exposures, which can reprogram developing tissues to increase susceptibility to adult diseases, including cancer. In the developing prostate, even brief exposure to endocrine-disrupting chemicals (EDCs) can increase risk for developing cancer in adulthood, with disruption of the epigenome thought to play a key role in this developmental reprogramming. We find that EDC-induced nongenomic phosphoinositide 3-kinase; (PI3K) signaling engages the histone methyltransferase mixed-lineage leukemia 1 (MLL1), responsible for the histone H3 lysine 4 trimethylation (H3K4me3) active epigenetic mark, to increase cleavage and formation of active MLL1 dimers. In the developing prostate, EDC-induced MLL1 activation increased H3K4me3 at genes associated with prostate cancer, with increased H3K4me3 and elevated basal and hormone-induced expression of reprogrammed genes persisting into adulthood. These data identify a mechanism for MLL1 activation that is vulnerable to disruption by environmental exposures, and link MLL1 activation by EDCs to developmental reprogramming of genes involved in prostate cancer.

Identification of secretaglobin Scgb2a1 as a target for developmental reprogramming by BPA in the rat prostate.

Secretoglobins are a superfamily of secreted proteins thought to participate in inflammation, tissue repair, and tumorigenesis. Secretoglobin family 2A member 1 (Scgb2a1) is a component of prostatein, a major androgen-binding protein secreted by the rat prostate. Using a rat model for developmental reprogramming of susceptibility to prostate carcinogenesis, we identified, by RNA-seq, that Scgb2a1 is significantly upregulated (>100-fold) in the prostate of adult rats neonatally exposed to bisphenol A (BPA), with increased gene expression confirmed by quantitative RT-PCR and chromatin immunoprecipitation for histone H3 lysine 9 acetylation. Bisulfite analysis of both CpG islands located within 10 kb of the Scgb2a1 promoter identified significant hypomethylation of the CpG island upstream of the transcription start site of this gene in the reprogrammed prostate. These data suggest that expression of Scgb2a1 in the adult prostate could be epigenetically reprogrammed by BPA exposure during prostate development, with potential implications for cancer risk and response to chemotherapeutics associated with prostatein binding.

Molecular pathways: environmental estrogens activate nongenomic signaling to developmentally reprogram the epigenome.

Exposure to environmental xenoestrogens is a major health concern because of the ability of these compounds to perturb estrogen receptor (ER) signaling and act as endocrine disrupting compounds (EDC). Inappropriate exposure to EDCs during development, even at low doses, can predispose individuals to an increased lifetime risk of disease, including cancer. Recent data indicate that perinatal exposure to EDCs increases cancer risk by (re)programming the epigenome via alterations in DNA and histone methylation. We and others have begun to dissect the mechanisms by which xenoestrogens disrupt the epigenetic machinery to reprogram the epigenome and induce developmental reprogramming. Our studies revealed that xenoestrogens induce nongenomic ER signaling to activate PI3K/AKT, resulting in AKT phosphorylation and inactivation of the histone methyltransferase EZH2, thus providing a direct link to disruption of the epigenome. Other epigenetic "readers, writers, and erasers" may also be targeted by nongenomic signaling, suggesting this is a central mechanism by which xenoestrogens and other EDCs disrupt the epigenome to induce developmental reprogramming. Elucidating mechanisms of developmental reprogramming of the epigenome is important for understanding how environmental exposures increase cancer risk, and provides a rationale for developing epigenetic interventions that can reverse the effects of environmental exposures to reduce cancer risk.

Retinoic acid-dependent signaling pathways and lineage events in the developing mouse spinal cord.

Studies in avian models have demonstrated an involvement of retinoid signaling in early neural tube patterning. The roles of this signaling pathway at later stages of spinal cord development are only partly characterized. Here we use Raldh2-null mouse mutants rescued from early embryonic lethality to study the consequences of lack of endogenous retinoic acid (RA) in the differentiating spinal cord. Mid-gestation RA deficiency produces prominent structural and molecular deficiencies in dorsal regions of the spinal cord. While targets of Wnt signaling in the dorsal neuronal lineage are unaltered, reductions in Fibroblast Growth Factor (FGF) and Notch signaling are clearly observed. We further provide evidence that endogenous RA is capable of driving stem cell differentiation. Raldh2 deficiency results in a decreased number of spinal cord derived neurospheres, which exhibit a reduced differentiation potential. Raldh2-null neurospheres have a decreased number of cells expressing the neuronal marker ß-III-tubulin, while the nestin-positive cell population is increased. Hence, in vivo retinoid deficiency impaired neural stem cell growth. We propose that RA has separable functions in the developing spinal cord to (i) maintain high levels of FGF and Notch signaling and (ii) drive stem cell differentiation, thus restricting both the numbers and the pluripotent character of neural stem cells.

Mouse Fkbp8 activity is required to inhibit cell death and establish dorso-ventral patterning in the posterior neural tube.

Neural tube defects (NTDs) are birth defects that can be disabling or lethal and are second in their prevalence after cardiac defects among major human congenital malformations. Spina bifida is a NTD where the spinal cord is dysplastic, and the overlying spinal column is absent. At present, the molecular mechanisms underlying the spinal bifida development are largely unknown. In this study, we present a Fkbp8 mouse mutant that has an isolated and completely penetrant spina bifida, which is folate- and inositol-resistant. Fkbp8 mutants are not embryo lethal, but they display striking features of human spina bifida, including a dysplastic spinal cord, open neural canal and disability. The loss of Fkbp8 leads to increased apoptosis in the posterior neural tube, demonstrating that in vivo FKBP8 inhibits cell death. Gene expression analysis of Fkbp8 mutants revealed a perturbation of expression of neural tube patterning genes, suggesting that endogenous FKBP8 activity establishes dorso-ventral patterning of the neural tube. These studies demonstrate that Fkbp8 is not important for embryo survival, but is essential for spinal neural tube patterning, and to block apoptosis, in the developing neural tube. The mutant Fkbp8 allele is a new experimental model which will be useful in dissecting the pathogenesis of spinal NTDs, and enhance our understanding of the etiology of human NTDs.

Depletion of Mab21l1 and Mab21l2 messages in mouse embryo arrests axial turning, and impairs notochord and neural tube differentiation.

BACKGROUND: The nematode mab-21 gene specifies sensory ray cell identity and was first isolated because of its mutant sensory ray defects. Vertebrate Mab21 orthologs have since been identified in mammals and amphibians. In this report, we characterized in detail two Mab21 orthologs in mouse, Mab21l1 and Mab21l2. METHODS: We examined the genomic organizations of Mab21 genes and used northern blot and in situ hybridizations to assay their temporal-spatial expression pattern. Their embryonic functions were revealed by specific attenuation of Mab21 messages with antisense oligos in cultured embryos. RESULTS: Mab21l1 and Mab21l2 have very similar protein make-up and gene structures. Both genes were expressed in overlapping domains of actively differentiating embryonic tissues. In addition, Mab21l1 had unique expression in the lens vesicles and genital tubercle whereas Mab21l2 was expressed in the retinal epithelium and umbilical cord. Mab21l1 and Mab21l2 depleted embryos had severe defects in notochord, neural tube, organogenesis, vasculogenesis, and axial turning. CONCLUSIONS: The findings demonstrate that both Mab21 genes are required in developing embryos for embryonic turning, formation of the notochord, neural tube, and other organ tissues.