Inhibition of Let-7 Maturation By Lin28b Controls Timing of Embryonic and Adult Myeloid Progenitor Phenotypes during Development

Within the mammalian fetal liver (FL) or adult bone marrow (ABM), terminally differentiated neutrophils, monocytes, and red blood cells emerge from a pool of multipotent common myeloid progenitors (CMPs). At steady state, the output of these cells is finally balanced but highly adaptable to the pathophysiologic requirements of the host. In the FL, erythroid output predominates to generate adequate oxygen transport for the metabolic demands of rapid organism growth in a hypoxic intrauterine environment; while granulocytic production comprises a larger balance of myelopoietic output in the ABM to provide innate immunity. The genetic regulators that specify timing of developmental stage-specific granulocytic/erythrocytic output from the myeloid progenitor pool have not been described.

Initially defined as regulators of developmental timing in C. elegans, the mammalian Lin28a and Lin28b genes encode RNA binding-proteins capable of contributing to pluripotency induction in fully differentiated cells. Lin28 proteins exert their effects in part through inhibition of the maturation of the let-7 family of microRNAs - of which there are eight forms in mammals - that collectively drive cell terminal differentiation. Therefore, Lin28a/b expression is largely confined to embryonic tissues and is associated with the undifferentiated pluripotent or multipotent state in vivo. Given this role in regulation of multipotent cell function, we assessed the relative roles of theLin28/let-7 axis in developmental timing of myeloid progenitor phenotypes.

We first analyzed patterns of let-7 expression during FL and ABM myelopoiesis. We found that several mature let-7 forms accumulated in CMPs during the transition from FL to ABM hematopoiesis (up to 30-fold induction as observed in let-7b in ABM compared to FL), coincident with 5-fold downregulation of the let-7 target and fetal-specific hematopoietic regulator Hmga2, indicating that the presence of mature let-7species may specify ABM-like myelopoiesis.

To investigate this concept, we utilized a model of ectopic expression of LIN28B to block maturation of all let-7 species in ABM. Mice with activated LIN28B expression had a FL-like erythroid-dominant hematopoietic phenotype compared to controls (megakaryocyte-erythroid progenitor [MEP]:granulocyte-macrophage progenitor [GMP] ratio 3.9 ± 0.9 vs 0.8 ± 0.1, P = 0.005), along with blunted output of mature neutrophils in the ABM (P = 0.0001). Expression of LIN28B decreased formation of mature let-7 species in CMPs. Taken together, these results suggest that LIN28B expression reprograms ABM CMPs to a FL-like phenotype.

To further define the Lin28-let-7 interaction in myelopoiesis, we analyzed the expression patterns of Lin28a and Lin28b as well as precursor let-7 microRNAs and their host genes during myeloid development. We observed up to 30-fold activation of precursor let-7 microRNAs and 4-fold activation of the polycistron encoding let-7b and let-7c2 coincident with 5-fold downregulation of Lin28b in CMPs during the transition from FL to ABM myelopoiesis. These results are consistent with mechanisms regulating levels of mature let-7 microRNAs at both the transcriptional and post-transcriptional level.

We next characterized the effects of dysregulation of the Lin28b-let-7 interaction in the FL. Enforced expression of let-7g in mouse embryos drove increased granulocytic output in FL myeloid progenitors in culture (P = 0.001). While ectopic expression of Lin28 has been shown to confer fetal hematopoietic programs in other systems, the impact of loss of Lin28 genes on fetal hematopoiesis has not been reported. Fetal loss of Lin28b skewed the myeloid progenitor pool toward an ABM-like phenotype compared to wild-type animals (MEP:GMP 2.8 ± 0.5 vs. 4± 0.4, P = 0.1), though this phenotype is blunted due to the relative absence of let-7 precursors in the FL compared to the ABM, resulting in deregulated maturation of only a subset of mature let-7 forms. However, Lin28b-null FLs contained increased numbers of mature neutrophils (P = 0.005). Together, these data identify a Lin28/let-7 genetic switch with multiple levels of regulation that controls timing of the developmental myelopoietic phenotype.