WT1 in heart and gonad development: a crucial gene for cell plasticity and differentiation

Abstract

Wt1 is a complex gene that encodes a protein whose best described function is to act as a transcription factor. Wt1 plays a crucial role in several organ developments, including kidneys, liver, heart or gonads. Moreover, Wt1 is implicated in the etiology of a set of syndromes and diseases ranging from cancer to disorders of sex development (DSD). Additionally, Wt1 has been described to play a role in adult homeostasis. In this thesis, I focused on Wt1 role in development, specifically in gonad and heart morphogenesis. For this purpose, I used two previously described Cre mouse models to generate two different Wt1KOs.

The heart is the first organ being formed and it is primarily composed of three layers: the endocardium, the myocardium and the epicardium. The epicardium is a mesothelial layer of cells covering the vertebrate heart surface. Epicardial cells give rise to epicardial derived cells (EPDCs), progenitors of crucial cell types in heart development like vascular smooth muscle cells and cardiac fibroblasts. Moreover, the epicardium contributes to heart morphogenesis by secreting paracrine factors. After myocardial infarction, the epicardium embryonic genetic signature is reactivated. Thus, understanding the mechanisms behind epicardial development constitutes a topic of general interest.

The epicardial genetic program has been previously characterized and the Wt1 role in the regulation of this program has been established. One of the pathways modulated during epicardium development is the BMP4 pathway. The results of this thesis demonstrate that WT1 directly regulates Bmp4 expression. Additionally, exposed results demonstrate how the BMP4 pathway is crucial for epicardial cell maturation, through the regulation of cell morphology, from a cuboidal to a squamous cell shape, epicardial proliferation, and transcriptomic changes. Finally, the data shown in this work indicated that changes and mechanisms described for epicardium may be a phenomenon extrapolated to other mesotheliums like the lung mesothelium.

To better study Wt1 role in the development of other organs, we have characterized the recombination activity of a Wt1Cre mouse model using two different reporter mouse models, the R26RmTmG and the R26RtdRFP. Results demonstrated that Wt1Cre is efficiently activated in hearts and gonads but not in other organs generated from the genital ridge, like the kidney or the adrenal gland. Taking advantage of this, the Wt1Cre mouse model was used to generate a new Wt1KO: the Wt1Cre; Wt1Loxp/GFP. Wt1Cre; Wt1Loxp/GFP mice show a partial embryonic lethality, potentially caused by defects in heart development, but some of them reach adulthood, becoming an ideal model to investigate Wt1 role from embryonic to adult stages. Therefore, we decided to study Wt1role in gonad development in Wt1Cre; Wt1Loxp/GFP mice.

Sex development is a complex and coordinated process that starts with the differentiation of the bipotential gonads. WT1 mutations or haploinsufficiency have been reported in different syndromes and conditions linked to DSD. The study of WT1 role in embryonic development and the impact on adult sex development has been hampered by the complete gonadal agenesis or embryonic lethality presented by other Wt1KO mouse models.

The results presented demonstrate a sharp reduction in Wt1 levels in Wt1Cre; Wt1Loxp/GFP gonads since the bipotential stage. This causes, in the adult, the formation of small, atrophic gonads, a hermaphroditism of the genital tract and external ambiguous genitalia. Besides, the data reported in this thesis demonstrates that Wt1Cre; Wt1Loxp/GFP mice present an impaired gonad embryonic development due to the lack of differentiation of the main cell lineages responsible for gonad development and function: supportive cells, steroidogenic cells and primordial germ cells (PGCs).

Finally, the observed sub-lethality in Wt1Cre; Wt1Loxp/GFP mice is explained by the observation of a severe heart developmental impairment in a portion of Wt1Cre; Wt1Loxp/GFP embryos, whereas others show no apparent heart defects at embryonic stages. Equally, an histological study performed on Wt1Cre; Wt1Loxp/GFP mice also described changes in the spleen and brown adipose tissue.

In summary, our results indicate the importance of morphological changes epicardial cells undergo during development, a process regulated by Wt1 modulation of the BMP4 pathway. In addition, the generation and characterization of the Wt1Cre; Wt1Loxp/GFP mouse renders an informative and useful tool to study Wt1 role beyond embryonic stages and provide a more accurate frame for the understanding of results previously obtained when using the Wt1Cre mouse model.