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Significance Immunoglobulin heavy chain checkpoint (IgHCC) is a critical step during early B cell development. The role of immunoglobulin heavy chain (IgHC) at this step is well established. However, with the expanding knowledge of RNA in regulating central biological processes, there could be a noncoding contribution of IgHC mRNA (IgHR) in controlling the IgHCC. Here, we generated a novel mouse model that enabled us to determine a potential role of IgHR in the IgHCC, independent of IgHC signaling. Our data indicate that IgHR has no role in IgHCC and the latter is predominantly controlled by IgHC, as proposed earlier. Furthermore, this study highlights the sensitivity of progenitor B cells to low amounts of IgHC., Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged Igh allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive Igh allele is expressed, a phenomenon known as Igh allelic exclusion. In contrast to a productively rearranged Igh allele, the Igh messenger RNA (mRNA) (IgHR) from a nonproductively rearranged Igh allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable IgHR to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the IghTer5H∆TM mouse model from IghTer5H mice having a premature termination codon at position +5 in leader exon of IghTer5H allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of IghTer5H message, indicating that previous conclusions regarding a role of IgHR in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the IghTer5H∆TM knock-in allele, which generated stable IgHR but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not IgHR expression.

Summary Colorectal cancer stem cells (CSCs) express Lgr5 and display extensive stem cell-like multipotency and self-renewal and are thought to seed metastatic disease. Here, we used a mouse model of colorectal cancer (CRC) and human tumor xenografts to investigate the cell of origin of metastases. We found that most disseminated CRC cells in circulation were Lgr5− and formed distant metastases in which Lgr5+ CSCs appeared. This plasticity occurred independently of stemness-inducing microenvironmental factors and was indispensable for outgrowth, but not establishment, of metastases. Together, these findings show that most colorectal cancer metastases are seeded by Lgr5− cells, which display intrinsic capacity to become CSCs in a niche-independent manner and can restore epithelial hierarchies in metastatic tumors., Graphical Abstract, Highlights • The majority of disseminating cells of colorectal cancer are Lgr5− • Lgr5− cancer cells are the main seeds of colorectal cancer metastatic lesions • Long-term metastatic growth from Lgr5− cells requires appearance of Lgr5+ cells • Lgr5− metastases have the intrinsic capacity to re-establish the cellular hierarchy, Van Rheenen and colleagues study Lgr5+ cancer stem cells during colorectal cancer metastasis. They demonstrate that the majority of metastases are seeded by Lgr5− cells, which upon arrival seed metastases in which Lgr5+ cells appear. This plasticity can occur independently of stemness-inducing factors and is indispensable for long-term metastatic growth.