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.

Regional variation in health is predominantly driven by lifestyle rather than genetics.

Regional differences in health-related phenotypes have been detected between and within countries. In Scotland, regions differ for a variety of health-related traits and display differences in mean lifespan of up to 7.5 years. Both genetics and lifestyle differences are potential causes of this variation. Using data on obesity-related traits of ~11,000 Scottish individuals with genome-wide genetic information and records of lifestyle and socioeconomic factors, we explored causes of regional variation by using models that incorporate genetic and environmental information jointly. We found that variation between individuals within regions showed substantial influence of both genetic variation and family environment. Regional variation for most obesity traits was associated with lifestyle and socioeconomic variables, such as smoking, diet and deprivation which are potentially modifiable. There was limited evidence that regional differences were of genetic origin. This has important implications for healthcare policies, suggesting that inequalities can be tackled with appropriate social and economic interventions.Health-related traits are known to vary geographically. Here, Amador and colleagues show that regional variation of obesity-related traits in a Scottish population is influenced more by lifestyle differences than it is by genetic differences.

Transcription factor Wilms' tumor 1 regulates developmental RNAs through 3' UTR interaction.

Wilms' tumor 1 (WT1) is essential for the development and homeostasis of multiple mesodermal tissues. Despite evidence for post-transcriptional roles, no endogenous WT1 target RNAs exist. Using RNA immunoprecipitation and UV cross-linking, we show that WT1 binds preferentially to 3' untranslated regions (UTRs) of developmental targets. These target mRNAs are down-regulated upon WT1 depletion in cell culture and developing kidney mesenchyme. Wt1 deletion leads to rapid turnover of specific mRNAs. WT1 regulates reporter gene expression through interaction with 3' UTR-binding sites. Combining experimental and computational analyses, we propose that WT1 influences key developmental and disease processes in part through regulating mRNA turnover.

Wt1, the mesothelium and the origins and heterogeneity of visceral fat progenitors.

One major gap in adipocyte biology has been a lack of understanding of the developmental origins of the different visceral white adipose tissue (WAT) depots and subcutaneous WAT. In a recent study we showed that most visceral WAT but no subcutaneous WAT arises from cells expressing the Wilms' tumor 1 (Wt1) gene late in mouse gestation.(1) Wt1 continues to be expressed in visceral WAT progenitors into adult life. We also showed that visceral WAT is lined by a mesothelium and provided evidence that this structure is the source of adipocytes. Our study also adds to the growing body of evidence that there is heterogeneity in the visceral progenitors, such that there are Wt1-expressing and non-expressing subsets, the relative proportions of which vary between depots. This raises the enticing prospect that the adipocytes arising from these progenitor subsets may have different properties and our preliminary data support this notion. Finally, evidence from our study and one from Spiegelman's group(2) suggests that Wt1 is not just a marker but regulates visceral WAT identity and the progenitor population. We discuss the implications of this work and some of the questions and future directions that arise from it.

In vivo imaging of the tumor and its associated microenvironment using combined CARS / 2-photon microscopy.

The use of confocal and multi-photon microscopy for intra-vital cancer imaging has impacted on our understanding of cancer cell behavior and interaction with the surrounding tumor microenvironment in vivo. However, many studies to-date rely on the use fluorescent dyes or genetically encoded probes that enable visualization of a structure or cell population of interest, but do not illuminate the complexity of the surrounding tumor microenvironment. Here, we show that multi-modal microscopy combining 2-photon fluorescence with CARS can begin to address this deficit, enabling detailed imaging of the tumor niche without the need for additional labeling. This can be performed on live tumor-bearing animals through optical observation windows, permitting real-time and longitudinal imaging of dynamic processes within the tumor niche.

Visceral and subcutaneous fat have different origins and evidence supports a mesothelial source.

Fuelled by the obesity epidemic, there is considerable interest in the developmental origins of white adipose tissue (WAT) and the stem and progenitor cells from which it arises. Whereas increased visceral fat mass is associated with metabolic dysfunction, increased subcutaneous WAT is protective. There are six visceral fat depots: perirenal, gonadal, epicardial, retroperitoneal, omental and mesenteric, and it is a subject of much debate whether these have a common developmental origin and whether this differs from that for subcutaneous WAT. Here we show that all six visceral WAT depots receive a significant contribution from cells expressing Wt1 late in gestation. Conversely, no subcutaneous WAT or brown adipose tissue arises from Wt1-expressing cells. Postnatally, a subset of visceral WAT continues to arise from Wt1-expressing cells, consistent with the finding that Wt1 marks a proportion of cell populations enriched in WAT progenitors. We show that all visceral fat depots have a mesothelial layer like the visceral organs with which they are associated, and provide several lines of evidence that Wt1-expressing mesothelium can produce adipocytes. These results reveal a major ontogenetic difference between visceral and subcutaneous WAT, and pinpoint the lateral plate mesoderm as a major source of visceral WAT. They also support the notion that visceral WAT progenitors are heterogeneous, and suggest that mesothelium is a source of adipocytes.

ErbB4 modulates tubular cell polarity and lumen diameter during kidney development.

ErbB4 receptor tyrosine kinase contributes to the development of the heart, the central nervous system, and the lactating mammary gland, but whether it has a role in the development of the kidney epithelium is unknown. Here, we found that expression of Erbb4 isoforms JM-a CYT-1 and JM-a CYT-2 was first detectable around embryonic day 13 in the mouse, mainly in the collecting ducts and both the proximal and distal tubules. In vitro, overexpression of a relevant ErbB4 isoform promoted proliferation and disturbed polarization of kidney epithelial cells when cultured as three-dimensional structures. We examined ErbB4 function in developing kidney tubules in vivo with Pax8-Cre-mediated conditional overexpression of Rosa26 locus-targeted ERBB4 and with conditional Erbb4 knock-out mice. The Pax8-Cre-driven ERBB4 overexpression enhanced proliferation in the collecting ducts, reduced the size of epithelial duct lumens, and promoted formation of cortical tubular cysts. These defects were associated with changes in the subcellular distribution of markers of epithelial cell polarity. Similarly, the Pax8-Cre-mediated Erbb4 knock-out mice manifested dysfunctional kidneys with larger duct lumens and epithelial cell mispolarization. Taken together, these data suggest that ErbB4 signaling modulates proliferation and polarization, cellular functions critical for the development of epithelial ducts in the kidney.

A murine model of Denys-Drash syndrome reveals novel transcriptional targets of WT1 in podocytes.

The Wilms tumor-suppressor gene WT1, a key player in renal development, also has a crucial role in maintenance of the glomerulus in the mature kidney. However, molecular pathways orchestrated by WT1 in podocytes, where it is highly expressed, remain unknown. Their defects are thought to modify the cross-talk between podocytes and other glomerular cells and ultimately lead to glomerular sclerosis, as observed in diffuse mesangial sclerosis (DMS) a nephropathy associated with WT1 mutations. To identify podocyte WT1 targets, we generated a novel DMS mouse line, performed gene expression profiling in isolated glomeruli and identified excellent candidates that may modify podocyte differentiation and growth factor signaling in glomeruli. Scel, encoding sciellin, a protein of the cornified envelope in the skin, and Sulf1, encoding a 6-O endosulfatase, are shown to be expressed in wild-type podocytes and to be strongly down-regulated in mutants. Co-expression of Wt1, Scel and Sulf1 was also found in a mesonephric cell line, and siRNA-mediated knockdown of WT1 decreased Scel and Sulf1 mRNAs and proteins. By ChIP we show that Scel and Sulf1 are direct WT1 targets. Cyp26a1, encoding an enzyme involved in the degradation of retinoic acid, is shown to be up-regulated in mutant podocytes. Cyp26a1 may play a role in the development of glomerular lesions but does not seem to be regulated by WT1. These results provide novel clues in our understanding of normal glomerular function and early events involved in glomerulosclerosis.

Common variants in the JAZF1 gene associated with height identified by linkage and genome-wide association analysis.

Genes for height have gained interest for decades, but only recently have candidate genes started to be identified. We have performed linkage analysis and genome-wide association for height in approximately 4000 individuals from five European populations. A total of five chromosomal regions showed suggestive linkage and in one of these regions, two SNPs (rs849140 and rs1635852) were associated with height (nominal P = 7.0 x 10(-8) and P = 9.6 x 10(-7), respectively). In total, five SNPs across the genome showed an association with height that reached the threshold of genome-wide significance (nominal P < 1.6 x 10(-7)). The association with height was replicated for two SNPs (rs1635852 and rs849140) using three independent studies (n = 31 077, n=1268 and n = 5746) with overall meta P-values of 9.4 x 10(-10) and 5.3 x 10(-8). These SNPs are located in the JAZF1 gene, which has recently been associated with type II diabetes, prostate and endometrial cancer. JAZF1 is a transcriptional repressor of NR2C2, which results in low IGF1 serum concentrations, perinatal and early postnatal hypoglycemia and growth retardation when knocked out in mice. Both the linkage and association analyses independently identified the JAZF1 region affecting human height. We have demonstrated, through replication in additional independent populations, the consistency of the effect of the JAZF1 SNPs on height. Since this gene also has a key function in the metabolism of growth, JAZF1 represents one of the strongest candidates influencing human height identified so far.

Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumour suppressor is required for nephron differentiation.

Wt1 is a tumour suppressor gene, mutation of which is a cause of Wilms' tumour, a childhood renal nephroblastoma. Wt1 is expressed in a rich pattern during renal development suggesting that it acts at three stages: determination of the kidney area, the differentiation of nephrons and maturation of glomeruli. Wt1-/- mice confirm that Wt1 is essential for the inception of kidney development; cells that ought to form kidneys die by apoptosis instead. Specific human WT1 mutations cause defects of glomerular maturation (Denys-Drash and Frasier syndromes), providing circumstantial evidence for action of Wt1 during glomerular maturation. There is, however, no genetic evidence for a function during nephron differentiation because this stage is never reached in Wt1-/- mice. We have therefore developed a novel technique, based on small interfering RNA (siRNA), to repress the expression of Wt1 and other specific genes at different stages of kidney development in culture. We find that early repression of Wt1 phenocopies the Wt1-/- mouse, but later repression prevents cells differentiating into nephrons and causes them instead to proliferate abnormally, possibly mimicking aspects of Wilms' tumour. In line with established hypotheses about genetic pathways that control kidney development, we find that repressing Pax2 using siRNAs represses Wt1 expression and blocks both bud growth and nephron differentiation, but that repressing Wnt4 blocks nephron differentiation without affecting Wt1 expression. As well as illuminating previously inaccessible aspects of Wt1 biology, our results suggest that siRNA in organ culture will be a powerful method for analyzing other developmental pathways and testing the effects of stage-specific loss of tumour suppressor genes.

Expression in Xenopus oocytes shows that WT1 binds transcripts in vivo, with a central role for zinc finger one.

The Wilms' tumour suppressor gene WT1 encodes a protein involved in urogenital development and disease. The salient feature of WT1 is the presence of four 'Krüppel'-type C(2)-H(2) zinc fingers in the C-terminus. Uniquely to WT1, an evolutionarily conserved alternative splicing event inserts three amino acids (KTS) between the third and fourth zinc fingers, which disrupts DNA binding. The ratio of +KTS:-KTS isoforms is crucial for normal development. Previous work has shown that WT1 (+KTS) interacts with splice factors and that WT1 zinc fingers, particularly zinc finger one, bind to RNA in vitro. In this study we investigate the role of zinc finger one and the +KTS splice in vivo by expressing tagged proteins in mammalian cells and Xenopus oocytes. We find that both full-length +/-KTS isoforms and deletion constructs that include zinc finger one co-sediment with ribonucleoprotein particles (RNP) on density gradients. In Xenopus oocytes both isoforms located to the lateral loops of lampbrush chromosomes. Strikingly, only the +KTS isoform was detected in B-snurposomes, but not when co-expressed with -KTS. However, co-expression of the C-terminus (amino acids 233-449, +KTS) resulted in snurposome staining, which is consistent with an in vivo interaction between isoforms via the N-terminus. Expressed WT1 was also detected in the RNA-rich granular component of nucleoli and co-immunoprecipitated with oocyte transcripts. Full-length WT1 was most stably bound to transcripts, followed by the C-terminus; the least stably bound was CTDeltaF1 (C-terminus minus zinc finger one). Expression of the transcription factor early growth response 1 (EGR1), whose three zinc fingers correspond to WT1 zinc fingers 2-4, caused general chromosomal loop retraction and transcriptional shut-down. However, a construct in which WT1 zinc finger one was added to EGR1 mimicked the properties of WT1 (-KTS). We suggest that in evolution, WT1 has acquired the ability to interact with transcripts and splice factors because of the modification of zinc finger one and the +KTS alternative splice.