Development of the B-Cell Progenitor Niche Is Dependent on Cells Derived from the Migratory Neural Crest

INTRODUCTION: The earliest stages of B-cell production occur in the bone marrow (BM), where hematopoietic stem cells (HSCs) differentiate into common lymphoid and myeloid progenitors. Common lymphoid progenitors then undergo progressive differentiation as B cell progenitors (BCPs) prior to exiting the BM and completing their maturation in the spleen and lymph nodes. BCP development is regulated by lymphotrophic cytokines produced by BM mesenchymal stromal cells (MSCs) and osteoprogenitors (OPs). While the factors that regulate the formation and activity of the BCP niche in the BM are unknown, other laboratories have determined that signals from the peripheral nervous system (PNS) direct the formation and activity of the HSC BM niche. In this study, we hypothesized that the PNS also coordinates the formation and activity of the BCP BM niche.

METHODS: To test this hypothesis, we targeted Schwann cells (SCs), which are required for peripheral nerve function, via a well-described mouse model of global peripheral nerve dysfunction (MP₀-Cre^+/-; Lamc1^fl/fl, hereafter referred to as mutant). MP₀ is expressed in the migratory neural crest from which SCs are derived, and the Lamc1 gene encodes an extracellular matrix protein (laminin-gamma-1) required for perinatal SC morphogenesis and differentiation. In mutant mice, SC development arrests at the 'immature' stage. Mutant SCs do not form myelinating or nonmyelinating SCs, which in mice takes place during the initial three weeks of postnatal development. Mutant mice die between 4-5 weeks of age; therefore 3-3.5 week old mice were used for all studies detailed below.

RESULTS: In comparison to controls, mutant mice were lymphopenic. There were no differences in number of red cells, neutrophils or platelets between control and mutant mice. In the bone marrow, Pre-B-cell but not myeloid colony formation was significantly reduced in mutant mice, as were the number of both Pre and Pro B cells. Insulin Like Growth Factor Type 1 (IGF-1), an osteoprogenitor-derived lymphopoietic cytokine required for BCP maturation, was reduced by 60% in mutant bone marrow supernatant. IGF-1 transcript was also reduced in osteoprogenitor/osteoblast cells (CD45^-Tert119^-CD31^-Sca1^-CD51⁺) from mutant BM. Control and mutant femurs were examined by µCT, which identified significant impairment in mutant cortical and trabecular bone structure and density.

A fluorescent reporter transgene (ROSA^mT/eGFP) was employed to facilitate isolation and characterization of those BM niche cells genetically targeted by MP₀-Cre. Using flow cytometry, we determined that less than four percent of osteoprogenitor/osteoblast cells expressed eGFP and were thus derived from the migratory neural crest. None of the MSCs (Tert119^-CD31^-Sca1⁺CD51⁺) expressed eGFP. The relative contribution of the migratory neural crest to each niche cell fraction did not vary between control and mutant BM. Lamc1 transcript from mutant eGFP⁺ cells was nearly undetectable and significantly less than from control eGFP⁺cells. Transcripts typically enriched in juvenile and nonmyelinating SCs (Erbb3, Sox10, GFAP, and s-100b) were enriched in eGFP⁺ but not eGFP^- BM niche cells from both control (MP₀-Cre^+/-; Lamc1^fl/+; ROSA^mT/eGFP) and mutant (MP₀-Cre^+/-; Lamc1^fl/fl) mice. There was no quantitative difference in IGF1 transcription between control and mutant eGFP⁺ cells.

CONCLUSIONS: From these data, we conclude that postnatal maturation of the migratory neural crest is required for establishment of the BCP niche. While the migratory neural crest cells found within the bone marrow are enriched with SC-transcripts, they also contribute a small fraction of BM stromal cells and potentially some neurons (sympathetic and parasympathetic). Since mutant mice are paretic, it is also possible that weight bearing-induced mechanical forces contribute to osteogenic BCP niche formation. Our work to date does not differentiate between these possibilities, but does provide significant preliminary evidence to support our hypothesis that 'neuronal' development contributes to formation and activity of the BCP niche within the BM.

No relevant conflicts of interest to declare.