Developmental & Stem Cell Biology

We are interested in the regulatory mechanisms underlying vertebrate pattern formation during embryogenesis and tissue homeostasis in adults. Our current focus is mouse small intestine. The adult small intestine is a highly organized structure consisting of two compartments: villi and crypts. Homeostasis of the intestinal epithelium requires rapid and continuous regeneration. The intestinal stem cells (ISCs) residing in the crypts constantly replenish differentiated enterocytes, goblet and entero-endocrine cells, which move upward along the villi structure. The other differentiated cells, Paneth cells, are located in the crypts and form the stem cell niche. The generation of specific cell types from the ISCs is determined by the combinatorial actions of diverse signals that impose developmental restrictions on progenitor cells. Transcription factors and histone-modifying complexes are instrumental for the maintenance of stem cell potential and for a stable determination of cell fate. On the other hand, dynamic changes in DNA and histone modifications at critical gene loci are required during differentiation of stem cells to more specialized cell types.

We are investigating when and how the ISCs are defined and integrated into the future crypt compartment during embryogenesis. In contrast to postnatal and adult periods, the embryonic small intestine is a simple tube containing cytologically identical epithelial cells. Little is known about which and how many cell types are within the embryonic intestinal epithelium. To functionally characterize different epithelial cell populations within the embryonic small intestine we are using both mouse genetics and genomics tools. We are applying tamoxifen-inducible genetic tools to evaluate how these specific embryonic cell populations contribute to the adult intestine, as well as to test their potential function in patterning this tissue in vivo. To elucidate the molecular pathways regulating formation, maintenance and differentiation the ISCs we are conducting ex-vivo based screens using crypt-villus organoid cultures. We are also interested in how environmental cues (microbiota) may influence the maintenance and differentiation of the adult ISCs in health and after induced injury. A better understanding of these processes may lead to development of novel stem cell and regenerative medicine therapies.

Figure 1: An overview of the tools used in our laboratory. (A) Schematic of the workflow aiming to reveal the distribution of chromatin marks, including DNA methylation (MBD-seq) and histone modifications (ChIP-seq), accompanying changes in transcription (RNA-seq) during maintenance and differentiation of the mouse embryonic (E12.5) or adult intestinal stem cells isolated by FACS. (B) Repression of Sonic hedgehog (Shh) transcription in adult ISCs (RNA, green) correlates with an increase in DNA methylation over the gene body (MBD, black). The spatial distribution of Shh transcripts is assessed by in situ hybridization on tissue sections of mouse embryonic (E12.5) and adult small intestine. (C) An example of a lineage tracing analysis at different times of embryonic development. Confocal images of the small intestine from mouse embryos administered tamoxifen at E11.5 and analyzed at E13.5 (left panel) or E17.5 (right panel). A specific cell population is labeled by EGFP (green), whereas the rest of the cells are expressing tdTomato (red). (D) Video (below): An example of the crypt-villi organoid culture from Lgr5-GFP-ires-CreERT mice. The ISCs are GFP positive (green).