Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium.

BACKGROUND: Methods for the long-term in situ transduction of the unperturbed murine intestinal epithelium have not been developed in past research. Such a method could speed up functional studies and screens to identify genetic factors influencing intestinal epithelium biology. Here, we developed an efficient method achieving this long-sought goal. RESULTS: We used ultrasound-guided microinjections to transduce the embryonic endoderm at day 8 (E8.0) in utero. The injection procedure can be completed in 20 min and had a 100% survival rate. By injecting a small volume (0.1-0.2 µl) of concentrated virus, single shRNA constructs as well as lentiviral libraries can successfully be transduced. The new method stably and reproducibly targets adult intestinal epithelium, as well as other endoderm-derived organs such as the lungs, pancreas, liver, stomach, and bladder. Postnatal analysis of young adult mice indicates that single transduced cells at E8.0 gave rise to crypt fields that were comprised of 20-30 neighbouring crypts per crypt-field at 90 days after birth. Lentiviral targeting of ApcMin/+ mutant and wildtype mice revealed that heterozygous loss of Apc function suppresses the developmental normal growth pattern of intestinal crypt fields. This suppression of crypt field sizes did not involve a reduction of the crypt number per field, indicating that heterozygous Apc loss impaired the growth of individual crypts within the fields. Lentiviral-mediated shRNA knockdown of p53 led to an approximately 20% increase of individual crypts per field in both Apc+/+ and ApcMin/+ mice, associating with an increase in crypt size in ApcMin/+ mice but a slight reduction in crypt size in Apc+/+ mice. Overall, p53 knockdown rescued the reduction in crypt field size in Apc-mutant mice but had no effect on crypt field size in wildtype mice. CONCLUSIONS: This study develops a novel technique enabling robust and reproducible in vivo targeting of intestinal stem cells in situ in the unperturbed intestinal epithelium across different regions of the intestine. In vivo somatic gene editing and genetic screening of lentiviral libraries has the potential to speed up discoveries and mechanistic understanding of genetic pathways controlling the biology of the intestinal epithelium during development and postnatal life. The here developed method enables such approaches.

Interplay of opposing fate choices stalls oncogenic growth in murine skin epithelium.

Skin epithelium can accumulate a high burden of oncogenic mutations without morphological or functional consequences. To investigate the mechanism of oncogenic tolerance, we induced HrasG12V in single murine epidermal cells and followed them long term. We observed that HrasG12V promotes an early and transient clonal expansion driven by increased progenitor renewal that is replaced with an increase in progenitor differentiation leading to reduced growth. We attribute this dynamic effect to emergence of two populations within oncogenic clones: renewing progenitors along the edge and differentiating ones within the central core. As clone expansion is accompanied by progressive enlargement of the core and diminishment of the edge compartment, the intraclonal competition between the two populations results in stabilized oncogenic growth. To identify the molecular mechanism of HrasG12V-driven differentiation, we screened known Ras-effector in vivo and identified Rassf5 as a novel regulator of progenitor fate choice that is necessary and sufficient for oncogene-specific differentiation.

Oncogenic activation of PI3K induces progenitor cell differentiation to suppress epidermal growth.

Oncogenic lesions are surprisingly common in morphologically and functionally normal human skin. However, the cellular and molecular mechanisms that suppress their cancer-driving potential to maintain tissue homeostasis are unknown. By employing assays for the direct and quantitative assessment of cell fate choices in vivo, we show that oncogenic activation of PI3K-AKT, the most commonly activated oncogenic pathway in cancer, promotes the differentiation and cell cycle exit of epidermal progenitors. As a result, oncogenic PI3K-AKT-activated epidermis exhibits a growth disadvantage even though its cells are more proliferative. We then sought to uncover the underlying mechanism behind oncogene-induced differentiation via a series of genetic screens in vivo. An AKT substrate, SH3RF1, is identified as a specific promoter of epidermal differentiation that has no effect on proliferation. Our study provides evidence for a direct, cell autonomous mechanism that can suppresses progenitor cell renewal and block clonal expansion of epidermal cells bearing a common and activating mutation in Pik3ca.