Lin28B and Let-7 in the Control of Sympathetic Neurogenesis and Neuroblastoma Development.

The RNA binding protein Lin28B is expressed in developing tissues and sustains stem and progenitor cell identity as a negative regulator of the Let-7 family of microRNAs, which induces differentiation. Lin28B is activated in neuroblastoma (NB), a childhood tumor in sympathetic ganglia and adrenal medulla. Forced expression of Lin28B in embryonic mouse sympathoadrenal neuroblasts elicits postnatal NB formation. However, the normal function of Lin28B in the development of sympathetic neurons and chromaffin cells and the mechanisms involved in Lin28B-induced tumor formation are unclear. Here, we demonstrate a mirror-image expression of Lin28B and Let-7a in developing chick sympathetic ganglia. Lin28B expression is not restricted to undifferentiated progenitor cells but, is observed in proliferating noradrenergic neuroblasts. Lin28 knockdown in cultured sympathetic neuroblasts decreases proliferation, whereas Let-7 inhibition increases the proportion of neuroblasts in the cell cycle. Lin28B overexpression enhances proliferation, but only during a short developmental period, and it does not reduce Let-7a. Effects of in vivo Lin28B overexpression were analyzed in the LSL-Lin28B(DBHiCre) mouse line. Sympathetic ganglion and adrenal medulla volume and the expression level of Let-7a were not altered, although Lin28B expression increased by 12- to 17-fold. In contrast, Let-7a expression was strongly reduced in LSL-Lin28B(DbhiCre) NB tumor tissue. These data demonstrate essential functions for endogenous Lin28 and Let-7 in neuroblast proliferation. However, Lin28B overexpression neither sustains neuroblast proliferation nor affects let-7 expression. Thus, in contrast to other pediatric tumors, Lin28B-induced NB is not due to expansion of proliferating embryonic neuroblasts, and Let-7-independent functions are implicated during initial NB development. SIGNIFICANCE STATEMENT: Lin28A/B proteins are highly expressed in early development and maintain progenitor cells by blocking the biogenesis and differentiation function of Let-7 microRNAs. Lin28B is aberrantly upregulated in the childhood tumor neuroblastoma (NB). NB develops in sympathetic ganglia and adrenal medulla and is elicited by forced Lin28B expression. We demonstrate that Lin28A/B and Let-7 are essential for sympathetic neuroblast proliferation during normal development. Unexpectedly, Lin28B upregulation in a mouse model does not affect neuroblast proliferation, ganglion size, and Let-7 expression during early postnatal development. Lin28B-induced NB, in contrast to other pediatric cancers, does not evolve from neuroblasts that continue to divide and involves Let-7-independent functions during initial development.

Prox1 identifies proliferating neuroblasts and nascent neurons during neurogenesis in sympathetic ganglia.

Neurogenesis in embryonic sympathetic ganglia involves neuroblasts that resume proliferation following neuronal differentiation. As cell cycle exit is not associated with neuronal differentiation, the identity of proliferating neuroblasts is incompletely understood. Here, we use sympathetic ganglia of chick embryos to define the timing of neurogenesis and neuroblast identity focusing on the expression and function of the transcription factor Prox1. We show that a large fraction of neuroblasts has initially withdrawn from the cell cycle at embryonic day 3 (E3), which is reflected by a high proportion of p27(+)/Islet1(+) neuroblasts (63%) and low numbers of EdU(+)/Islet1(+) cells (12%). The proportion of proliferating Islet1(+) neuroblasts, identified by EdU pulse labeling and by the absence of the postmitotic marker p27 increases to reach maximal levels at E5, when virtually all neuroblasts are in the cell cycle (95%). Subsequently, the proportion of EdU-labeled and p27(-) neuroblasts is reduced to reach low levels at E11. Interestingly, the expression of the transcription factor Prox1 is restricted to the neuronal lineage, that is, Sox10(+)/Phox2b(+) neuron progenitors, proliferating p27(-)/Islet1(+) neuroblasts and nascent neurons but is rapidly lost in postmitotic neurons. In vitro and in vivo knockdown and overexpression experiments demonstrate effects of Prox1 in the support of neuroblast proliferation and survival. Taken together, these results define the neurogenesis period in the chick paravertebral sympathetic ganglia including an initial cell cycle withdrawal and identify Prox1 as a marker and regulator of proliferating sympathetic neuroblasts.

Lineage and stage specific requirement for Dicer1 in sympathetic ganglia and adrenal medulla formation and maintenance.

The development of sympathetic neurons and chromaffin cells is differentially controlled at distinct stages by various extrinsic and intrinsic signals. Here we use conditional deletion of Dicer1 in neural crest cells and noradrenergic neuroblasts to identify stage specific functions in sympathoadrenal lineages. Conditional Dicer1 knockout in neural crest cells of Dicer1(Wnt1Cre) mice results in a rapid reduction in the size of developing sympathetic ganglia and adrenal medulla. In contrast, Dicer1 elimination in noradrenergic neuroblasts of Dicer1(DbhiCre) animals affects sympathetic neuron survival starting at late embryonic stages and chromaffin cells persist at least until postnatal week 1. A differential function of Dicer1 signaling for the development of embryonic noradrenergic and cholinergic sympathetic neurons is demonstrated by the selective increase in the expression of Tlx3 and the cholinergic marker genes VAChT and ChAT at E16.5. The number of Dbh, Th and TrkA expressing noradrenergic neurons is strongly decreased in Dicer1-deficient sympathetic ganglia at birth, whereas Tlx3(+)/ Ret(+) cholinergic neurons cells are spared from cell death. The postnatal death of chromaffin cells is preceded by the loss of Ascl1, mir-375 and Pnmt and an increase in the markers Ret and NF-M, which suggests that Dicer1 is required for the maintenance of chromaffin cell differentiation and survival. Taken together, these findings demonstrate distinct stage and lineage specific functions of Dicer1 signaling in differentiation and survival of sympathetic neurons and adrenal chromaffin cells.

Dendrite complexity of sympathetic neurons is controlled during postnatal development by BMP signaling.

Dendrite development is controlled by the interplay of intrinsic and extrinsic signals affecting initiation, growth, and maintenance of complex dendrites. Bone morphogenetic proteins (BMPs) stimulate dendrite growth in cultures of sympathetic, cortical, and hippocampal neurons but it was unclear whether BMPs control dendrite morphology in vivo. Using a conditional knock-out strategy to eliminate Bmpr1a and Smad4 in immature noradrenergic sympathetic neurons we now show that dendrite length, complexity, and neuron cell body size are reduced in adult mice deficient of Bmpr1a. The combined deletion of Bmpr1a and Bmpr1b causes no further decrease in dendritic features. Sympathetic neurons devoid of Bmpr1a/1b display normal Smad1/5/8 phosphorylation, which suggests that Smad-independent signaling paths are involved in dendritic growth control downstream of BMPR1A/B. Indeed, in the Smad4 conditional knock-out dendrite and cell body size are not affected and dendrite complexity and number are increased. Together, these results demonstrate an in vivo function for BMPs in the generation of mature sympathetic neuron dendrites. BMPR1 signaling controls dendrite complexity postnatally during the major dendritic growth period of sympathetic neurons.