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.

Low-frequency variation in TP53 has large effects on head circumference and intracranial volume.

Cranial growth and development is a complex process which affects the closely related traits of head circumference (HC) and intracranial volume (ICV). The underlying genetic influences shaping these traits during the transition from childhood to adulthood are little understood, but might include both age-specific genetic factors and low-frequency genetic variation. Here, we model the developmental genetic architecture of HC, showing this is genetically stable and correlated with genetic determinants of ICV. Investigating up to 46,000 children and adults of European descent, we identify association with final HC and/or final ICV + HC at 9 novel common and low-frequency loci, illustrating that genetic variation from a wide allele frequency spectrum contributes to cranial growth. The largest effects are reported for low-frequency variants within TP53, with 0.5 cm wider heads in increaser-allele carriers versus non-carriers during mid-childhood, suggesting a previously unrecognized role of TP53 transcripts in human cranial development.

DNA Polymerase Epsilon Deficiency Causes IMAGe Syndrome with Variable Immunodeficiency.

During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins.

Mutations in TOP3A Cause a Bloom Syndrome-like Disorder.

Bloom syndrome, caused by biallelic mutations in BLM, is characterized by prenatal-onset growth deficiency, short stature, an erythematous photosensitive malar rash, and increased cancer predisposition. Diagnostically, a hallmark feature is the presence of increased sister chromatid exchanges (SCEs) on cytogenetic testing. Here, we describe biallelic mutations in TOP3A in ten individuals with prenatal-onset growth restriction and microcephaly. TOP3A encodes topoisomerase III alpha (TopIIIα), which binds to BLM as part of the BTRR complex, and promotes dissolution of double Holliday junctions arising during homologous recombination. We also identify a homozygous truncating variant in RMI1, which encodes another component of the BTRR complex, in two individuals with microcephalic dwarfism. The TOP3A mutations substantially reduce cellular levels of TopIIIα, and consequently subjects' cells demonstrate elevated rates of SCE. Unresolved DNA recombination and/or replication intermediates persist into mitosis, leading to chromosome segregation defects and genome instability that most likely explain the growth restriction seen in these subjects and in Bloom syndrome. Clinical features of mitochondrial dysfunction are evident in several individuals with biallelic TOP3A mutations, consistent with the recently reported additional function of TopIIIα in mitochondrial DNA decatenation. In summary, our findings establish TOP3A mutations as an additional cause of prenatal-onset short stature with increased cytogenetic SCEs and implicate the decatenation activity of the BTRR complex in their pathogenesis.

Fifty shades of white: Understanding heterogeneity in white adipose stem cells.

The excessive expansion of white adipose tissue underlies the global obesity epidemic. However, not all fat is equal, and the impact of heterogeneity on the development and expansion of different adipose depots is becoming increasingly apparent. Two mechanisms are responsible for the growth of adipose tissue: hyperplasia (increasing adipocyte number) and hypertrophy (increasing adipocyte size). The former relies on the differentiation of adipocyte stem cells, which reside within the adipose stromal vascular fraction. Many differences in gene expression, adipogenesis, and the response to obesogenic stimuli have been described when comparing adipose stem cells from different depots. Considering that there is disparity in the pathogenicity of the depots, understanding this heterogeneity has clinically relevant implications. Here we review the current knowledge surrounding such differences, in the context of development, expansion and therapeutics. Moreover, given the importance of these differences, we suggest that careful consideration for the precise methodologies used, is essential if we are to truly understand the physiologically relevant consequences of this heterogeneity.

Isolation and Fluorescence-Activated Cell Sorting of Murine WT1-Expressing Adipocyte Precursor Cells.

The current global obesity epidemic has triggered increased interest in adipose tissue biology. A major area of attention for many is adipose tissue development. A greater understanding of adipocyte ontogeny could be highly beneficial in answering questions about obesity-associated disease. Recent work has shown that a proportion of mature adipocytes in visceral white adipose tissue are derived from Wt1-expressing adipocyte precursor cells. These adipocyte precursor cells reside within the adipose tissue itself, and are a constituent of the stromal vascular fraction (SVF), along with other, non-adipogenic, cell types. Crucially, heterogeneity exists within the adipocyte precursor population, with only a proportion of cells expressing Wt1. Moreover, it appears that this difference in the precursor cells may influence the mature adipocytes, with Wt1-lineage-positive adipocytes having fewer, larger lipid droplets than the Wt1-lineage negative. Using fluorescence-activated cell sorting, based on specific marker profiles, it is possible to isolate the adipocyte precursor cells from the SVF. Subsequently, this population can be divided into Wt1-expressing and non-expressing fractions, therefore permitting further analysis of the two cell populations, and the mature adipocytes derived from them. In this chapter we outline a method by which adipocyte precursor cells can be isolated, and how, using a specific mouse model, Wt1-expressing and non-expressing cells can be separated.