Resolving the heterogeneity of diaphragmatic mesenchyme: a novel mouse model of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a relatively common developmental defect with considerable mortality and morbidity. Formation of the diaphragm is a complex process that involves several cell types, each with different developmental origins. Owing to this complexity, the aetiology of CDH is not well understood. The pleuroperitoneal folds (PPFs) and the posthepatic mesenchymal plate (PHMP) are transient structures that are essential during diaphragm development. Using several mouse models, including lineage tracing, we demonstrate the heterogeneous nature of the cells that make up the PPFs. The conditional deletion of Wilms tumor 1 homolog (Wt1) in the non-muscle mesenchyme of the PPFs results in CDH. We show that the fusion of the PPFs and the PHMP to form a continuous band of tissue involves movements of cells from both sources. The PPFs of mutant mice fail to fuse with the PHMP and exhibit increased RALDH2 (also known as ALDH1A2) expression. However, no changes in the expression of genes (including Snai1, Snai2, Cdh1 and Vim) implicated in epithelial-to-mesenchymal transition are observed. Additionally, the mutant PPFs lack migrating myoblasts and muscle connective tissue fibroblasts (TCF4+/GATA4+), suggesting possible interactions between these cell types. Our study demonstrates the importance of the non-muscle mesenchyme in development of the diaphragm.

Effects of CreERT2, 4-OH Tamoxifen, and Gender on CFU-F Assays.

Gene function in stem cell maintenance is often tested by inducing deletion via the Cre-loxP system. However, controls for Cre and other variables are frequently not included. Here we show that when cultured in the presence of 4-OH tamoxifen, bone and marrow cells containing the CreERT2 construct have a reduced colony forming ability. Inactive CreERT2 recombinase, however, has the opposite effect. Young female marrow cells containing the inactive CreERT2 construct grew more colonies than cells lacking the construct altogether. Young female control marrow cells (i.e., negative for CreERT2) also produced significantly greater colony numbers when cultured with 4-OH tamoxifen, compared with the ethanol vehicle control. In conclusion, we report that the use of the Cre-loxP system is inadvisable in combination with CFU-F assays, and that appropriate controls should be in place to extend the future use of Cre-loxP in alternate assays.

The pancreas is altered by in utero androgen exposure: implications for clinical conditions such as polycystic ovary syndrome (PCOS).

Using an ovine model of polycystic ovary syndrome (PCOS), (pregnant ewes injected with testosterone propionate (TP) (100 mg twice weekly) from day (d)62 to d102 of d147 gestation (maternal injection - MI-TP)), we previously reported female offspring with normal glucose tolerance but hyperinsulinemia. We therefore examined insulin signalling and pancreatic morphology in these offspring using quantitative (Q) RT-PCR and western blotting. In addition the fetal pancreatic responses to MI-TP, and androgenic and estrogenic contributions to such responses (direct fetal injection (FI) of TP (20 mg) or diethylstilbestrol (DES) (20 mg) at d62 and d82 gestation) were assessed at d90 gestation. Fetal plasma was assayed for insulin, testosterone and estradiol, pancreatic tissue was cultured, and expression of key ß-cell developmental genes was assessed by QRT-PCR. In female d62MI-TP offspring insulin signalling was unaltered but there was a pancreatic phenotype with increased numbers of ß-cells (P<0.05). The fetal pancreas expressed androgen receptors in islets and genes involved in ß-cell development and function (PDX1, IGF1R, INSR and INS) were up-regulated in female fetuses after d62MI-TP treatment (P<0.05-0.01). In addition the d62MI-TP pancreas showed increased insulin secretion under euglycaemic conditions (P<0.05) in vitro. The same effects were not seen in the male fetal pancreas or when MI-TP was started at d30, before the male programming window. As d62MI-TP increased both fetal plasma testosterone (P<0.05) and estradiol concentrations (P<0.05) we assessed the relative contribution of androgens and estrogens. FI-TP (commencing d62) (not FI-DES treatment) caused elevated basal insulin secretion in vitro and the genes altered by d62MI-TP treatment were similarly altered by FI-TP but not FI-DES. In conclusion, androgen over-exposure alters fetal pancreatic development and ß-cell numbers in offspring. These data suggest that that there may be a primary pancreatic phenotype in models of PCOS, and that there may be a distinct male and female pancreas.