Intestinal crypts display robust regeneration upon injury. The relatively rare secretory precursors can replace lost stem cells, but it is unknown if the abundant enterocyte progenitors that express the Alkaline phosphate intestinal (Alpi) gene also have this capacity. We created an Alpi-IRES-CreERT2 (Alpi(CreER)) knockin allele for lineage tracing. Marked clones consist entirely of enterocytes and are all lost from villus tips within days. Genetic fate-mapping of Alpi(+) cells before or during targeted ablation of Lgr5-expressing stem cells generated numerous long-lived crypt-villus "ribbons," indicative of dedifferentiation of enterocyte precursors into Lgr5(+) stems. By single-cell analysis of dedifferentiating enterocytes, we observed the generation of Paneth-like cells and proliferative stem cells. We conclude that the highly proliferative, short-lived enterocyte precursors serve as a large reservoir of potential stem cells during crypt regeneration.

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Edited by Peter CresswellThe pre-T cell receptor (pre-TCR) is a pT␣-␤ heterodimer functioning in early ␣␤ T cell development. Although once thought to be ligand-autonomous, recent studies show that preTCRs participate in thymic repertoire formation through recognition of peptides bound to major histocompatibility molecules (pMHC). Using optical tweezers, we probe pre-TCR bonding with pMHC at the single molecule level. Like the ␣␤TCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the C␤ FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCR␣␤ and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic V␤ patch apposition. This patch functions in the pre-TCR as a surrogate V␣ domain, fostering ligand promiscuity to favor development of ␤ chains with self-reactivity but is occluded by ␣ subunit replacement of pT␣ upon ␣␤TCR formation. At the double negative 3 thymocyte stage where the pre-TCR is first expressed, pre-TCR interaction with self-pMHC ligands imparts growth and survival advantages as revealed in thymic stromal cultures, imprinting fundamental self-reactivity in the T cell repertoire. Collectively, our data imply the existence of sequential mechanosensor ␣␤TCR repertoire tuning via the pre-TCR.The mammalian adaptive immune system protects its host against infectious diseases as well as tumors in a highly specific manner. At the core of ␣␤ T lymphocyte recognition is self-versus non-self-discrimination, a functionality endowed by clonal cell-surface T cell receptors (TCRs) 4 (1-3). In the mammalian thymus, the millions of distinct TCRs expressed create a repertoire that is refined to eliminate unwanted autoreactive specificities prior to export into the peripheral lymphoid compartment (Ref. 4 and references therein).The earliest thymocytes, termed double negative (DN1-4), lack both CD4 and CD8 and expression of ␣␤TCR complexes (hereafter termed ␣␤TCRs) (5). Within the DN3 stage, a pre-TCR complex is generated comprised of a variable TCR␤ chain disulfide-linked to the invariant pT␣ subunit. In turn, the pT␣-␤ heterodimer is noncovalently complexed with the same CD3 dimers as found in the ␣␤TCR, namely CD3⑀␥, CD3⑀␦, and CD3 (1, 2). This pre-TCR complex triggers cellular survival and expansion and, importantly, induces expression of CD4 and CD8 co-receptors so that the thymocytes transit to the DP (CD4 ϩ CD8 ϩ ) thymocyte stage where rearrangement of the TCR␣ gene occurs. Only at the DP stage is the ␣␤TCR expressed. The pre-TCR signaling process, termed ␤ selection, also controls allelic exclusion of the TCR␤ locus in a given cell (6). Pre-TCR signaling components include tyrosine kinases Lck, Fyn, with Notch-1, Notch-1 ligand DL4, interleukin 7, and CXCR4 supporting pre-TCR function (5, 10). Although ␣␤TCR DP thymocyte selection processes involve pMHC-dependent posit...