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.

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.

MiR-124 is differentially expressed in derivatives of the sympathoadrenal cell lineage and promotes neurite elongation in chromaffin cells.

The neural-crest-derived sympathoadrenal cell lineage gives rise to sympathetic neurons and to endocrine chromaffin cells of the adrenal medulla. Both cell types express a largely overlapping set of genes, including those coding for the molecular machinery related to the synthesis and exocytotic release of catecholamines. During their early development, sympathetic neurons and chromaffin cells rely on a shared transcription factor network that controls the establishment of these common features. Despite many similarities, mature sympathetic neurons and chromaffin cells significantly differ regarding their morphology and function. Most prominently, sympathetic neurons possess axons that are absent in mammalian adrenal chromaffin cells. The molecular mechanism underlying the divergent development of sympathoadrenal cells into neuronal and endocrine cells remains elusive. Mutational inactivation of the ribonuclease dicer hints at the importance of microRNAs in this diversification. We show here that miR-124 is detectable in developing sympathetic neurons but absent in chromaffin cell precursors. We further demonstrate that miR-124 promotes neurite elongation when transfected into cultured chromaffin cells indicating its capability to support the establishment of a neuronal morphology in non-neuronal sympathoadrenal cells. Our results also show that treatment of PC12 cells with the neurotrophin nerve growth factor leads to an upregulation of miR-124 expression and that inhibition of miR-124 reduces nerve-growth-factor-induced neurite outgrowth in PC12 cells. Thus, our data indicate that miR-124 contributes to the establishment of specific neuronal features in developing sympathoadrenal 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.

HoxB8 in noradrenergic specification and differentiation of the autonomic nervous system.

Different prespecification of mesencephalic and trunk neural crest cells determines their response to environmental differentiation signals and contributes to the generation of different autonomic neuron subtypes, parasympathetic ciliary neurons in the head and trunk noradrenergic sympathetic neurons. The differentiation of ciliary and sympathetic neurons shares many features, including the initial BMP-induced expression of noradrenergic characteristics that is, however, subsequently lost in ciliary but maintained in sympathetic neurons. The molecular basis of specific prespecification and differentiation patterns has remained unclear. We show here that HoxB gene expression in trunk neural crest is maintained in sympathetic neurons. Ectopic expression of a single HoxB gene, HoxB8, in mesencephalic neural crest results in a strongly increased expression of sympathetic neuron characteristics like the transcription factor Hand2, tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) in ciliary neurons. Other subtype-specific properties like RGS4 and RCad are not induced. HoxB8 has only minor effects in postmitotic ciliary neurons and is unable to induce TH and DBH in the enteric nervous system. Thus, we conclude that HoxB8 acts by maintaining noradrenergic properties transiently expressed in ciliary neuron progenitors during normal development. HoxC8, HoxB9, HoxB1 and HoxD10 elicit either small and transient or no effects on noradrenergic differentiation, suggesting a selective effect of HoxB8. These results implicate that Hox genes contribute to the differential development of autonomic neuron precursors by maintaining noradrenergic properties in the trunk sympathetic neuron lineage.

Generation of the tamoxifen-inducible DBH-Cre transgenic mouse line DBH-CT.

We generated transgenic mice bearing a tamoxifen-dependent Cre recombinase expressed under the control of the dopamine-ß-hydroxylase promoter. By crossing to the ROSA26 reporter mice we show that tamoxifen-induced Cre recombinase in adult mice specifically activates ß-galactosidase expression in differentiated noradrenergic neurons of the central and peripheral nervous system. Tamoxifen application in adult mice did not induce ß-galactosidase activity in parasympathetic neurons that transiently express DBH during development. Thus, this transgenic mouse line represents a valuable tool to study gene function in mature noradrenergic neurons by conditional inactivation.

The Gata3 transcription factor is required for the survival of embryonic and adult sympathetic neurons.

The transcription factor Gata3 is essential for the development of sympathetic neurons and adrenal chromaffin cells. As Gata3 expression is maintained up to the adult stage, we addressed its function in differentiated sympathoadrenal cells at embryonic and adult stages by conditional Gata3 elimination. Inactivation of Gata3 in embryonic DBH-expressing neurons elicits a strong reduction in neuron numbers due to apoptotic cell death and reduced proliferation. No selective effect on noradrenergic gene expression (TH and DBH) was observed. Interestingly, Gata3 elimination in DBH-expressing neurons of adult animals also results in a virtually complete loss of sympathetic neurons. In the Gata3-deficient population, the expression of anti-apoptotic genes (Bcl-2, Bcl-xL, and NFkappaB) is diminished, whereas the expression of pro-apoptotic genes (Bik, Bok, and Bmf) was increased. The expression of noradrenergic genes (TH and DBH) is not affected. These results demonstrate that Gata3 is continuously required for maintaining survival but not differentiation in the sympathetic neuron lineage up to mature neurons of adult animals.

In vivo role for CREB signaling in the noradrenergic differentiation of sympathetic neurons.

Signaling pathways involving cAMP and CREB have been implicated in several aspects of sympathetic neuron differentiation. Here, we used in vivo loss-of-function approaches in both mouse and chick embryos to characterize the physiological role of cAMP/CREB. Whereas sympathetic neuron development proceeds normally in CREB-deficient mouse embryos, a decrease in noradrenergic differentiation (TH, DBH) was observed in chick sympathetic ganglia in response to ACREB, a dominant-negative CREB variant which interferes with the function of all CREB family members. In contrast, expression of the generic neuronal marker SCG10 was not affected by ACREB. As the decrease in noradrenergic gene expression is compensated at later stages of development and TH expression in differentiated neurons is not CREB-dependent, a transient role for CREB is proposed, accelerating noradrenergic but not generic neuronal differentiation of sympathetic neurons.

Absence of gp130 in dopamine beta-hydroxylase-expressing neurons leads to autonomic imbalance and increased reperfusion arrhythmias.

Inflammatory cytokines that act through glycoprotein (gp)130 are elevated in the heart after myocardial infarction and in heart failure. These cytokines are potent regulators of neurotransmitter and neuropeptide production in sympathetic neurons but are also important for the survival of cardiac myocytes after damage to the heart. To examine the effect of gp130 cytokines on cardiac nerves, we used gp130(DBH-Cre/lox) mice, which have a selective deletion of the gp130 cytokine receptor in neurons expressing dopamine beta-hydroxylase (DBH). Basal sympathetic parameters, including norepinephrine (NE) content, tyrosine hydroxylase expression, NE transporter expression, and sympathetic innervation density, appeared normal in gp130(DBH-Cre/lox) compared with wild-type mice. Likewise, basal cardiovascular parameters measured under isoflurane anesthesia were similar in both genotypes, including mean arterial pressure, left ventricular peak systolic pressure, dP/dt(max), and dP/dt(min). However, pharmacological interventions revealed an autonomic imbalance in gp130(DBH-Cre/lox) mice that was correlated with an increased incidence of premature ventricular complexes after reperfusion. Stimulation of NE release with tyramine and infusion of the beta-agonist dobutamine revealed blunted adrenergic transmission that correlated with decreased beta-receptor expression in gp130(DBH-Cre/lox) hearts. Due to the developmental expression of the DBH-Cre transgene in parasympathetic ganglia, gp130 was eliminated. Cholinergic transmission was impaired in gp130(DBH-Cre/lox) hearts due to decreased parasympathetic drive, but tyrosine hydroxylase immunohistochemistry in the brain stem revealed that catecholaminergic nuclei appeared grossly normal. Thus, the apparently normal basal parameters in gp130(DBH-Cre/lox) mice mask an autonomic imbalance that includes alterations in sympathetic and parasympathetic transmission.

Distinct roles of hand2 in developing and adult autonomic neurons.

The bHLH transcription factor Hand2 is essential for the acquisition and maintenance of noradrenergic properties of embryonic sympathetic neurons and controls neuroblast proliferation. Hand2 is also expressed in embryonic and postnatal parasympathetic ganglia and remains expressed in sympathetic neurons up to the adult stage. Here, we address its function in developing parasympathetic and adult sympathetic neurons. We conditionally deleted Hand2 in the parasympathetic sphenopalatine ganglion by crossing a line of floxed Hand2 mice with DbhiCre transgenic mice, taking advantage of the transient Dbh expression in parasympathetic ganglia. Hand2 elimination does not affect Dbh expression and sphenopalatine ganglion size at E12.5 and E16.5, in contrast to sympathetic ganglia. These findings demonstrate different functions for Hand2 in the parasympathetic and sympathetic lineage. Our previous Hand2 knockdown in postmitotic, differentiated chick sympathetic neurons resulted in decreased expression of noradrenergic marker genes but it was unclear whether Hand2 is required for maintaining noradrenergic neuron identity in adult animals. We now show that Hand2 elimination in adult Dbh-expressing sympathetic neurons does not decrease the expression of Th and Dbh, in contrast to the situation during development. However, gene expression profiling of adult sympathetic neurons identified 75 Hand2-dependent target genes. Interestingly, a notable proportion of down-regulated genes (15%) encode for proteins with synaptic and neurotransmission functions. These results demonstrate a change in Hand2 target genes during maturation of sympathetic neurons. Whereas Hand2 controls genes regulating noradrenergic differentiation during development, Hand2 seems to be involved in the regulation of genes controlling neurotransmission in adult sympathetic neurons. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1111-1124, 2016.

Synaptic protein and pan-neuronal gene expression and their regulation by Dicer-dependent mechanisms differ between neurons and neuroendocrine cells.

BACKGROUND: Neurons in sympathetic ganglia and neuroendocrine cells in the adrenal medulla share not only their embryonic origin from sympathoadrenal precursors in the neural crest but also a range of functional features. These include the capacity for noradrenaline biosynthesis, vesicular storage and regulated release. Yet the regulation of neuronal properties in early neuroendocrine differentiation is a matter of debate and the developmental expression of the vesicle fusion machinery, which includes components found in both neurons and neuroendocrine cells, is not resolved. RESULTS: Analysis of synaptic protein and pan-neuronal marker mRNA expression during mouse development uncovers profound differences between sympathetic neurons and adrenal chromaffin cells, which result in qualitatively similar but quantitatively divergent transcript profiles. In sympathetic neurons embryonic upregulation of synaptic protein mRNA follows early and persistent induction of pan-neuronal marker transcripts. In adrenal chromaffin cells pan-neuronal marker expression occurs only transiently and synaptic protein messages remain at distinctly low levels throughout embryogenesis. Embryonic induction of synaptotagmin I (Syt1) in sympathetic ganglia and postnatal upregulation of synaptotagmin VII (Syt7) in adrenal medulla results in a cell type-specific difference in isoform prevalence. Dicer 1 inactivation in catecholaminergic cells reduces high neuronal synaptic protein mRNA levels but not their neuroendocrine low level expression. Pan-neuronal marker mRNAs are induced in chromaffin cells to yield a more neuron-like transcript pattern, while ultrastructure is not altered. CONCLUSIONS: Our study demonstrates that remarkably different gene regulatory programs govern the expression of synaptic proteins in the neuronal and neuroendocrine branch of the sympathoadrenal system. They result in overlapping but quantitatively divergent transcript profiles. Dicer 1-dependent regulation is required to establish high neuronal mRNA levels for synaptic proteins and to maintain repression of neurofilament messages in neuroendocrine cells.

The cortistatin gene PSS2 rather than the somatostatin gene PSS1 is strongly expressed in developing avian autonomic neurons.

Somatostatin and cortistatin are neuromodulators with divergent expression patterns and biological roles. Whereas expression and function of genes encoding somatostatin (PSS1) and the related peptide cortistatin (PSS2) have been studied in detail for the central nervous system (CNS) and immune system, relatively little is known about their expression patterns in the peripheral nervous system (PNS). We compare the expression patterns of PSS1 and PSS2 in chicken embryos. At E14, PSS1 is higher in the CNS versus PNS, whereas PSS2 is higher in the PNS. During early development, PSS1 is transiently expressed in lumbar sympathetic ganglia and is detectable at low levels throughout the development of dorsal root and ciliary ganglia. In contrast, PSS2 expression increases as development progresses in sympathetic and dorsal root ganglia, whereas levels in ciliary ganglia by E8 are more than 100-fold higher than in sympathetic ganglia. Activin, which induces somatostatin-like immunoreactivity in ciliary ganglion neurons in vivo and in vitro, controls PSS2 expression by stabilizing PSS2 but not PSS1 mRNA. We conclude that much of the somatostatin-like immunoreactivity in the developing avian peripheral nervous system is actually cortistatin, the PSS2 product, as opposed to true somatostatin, which is the PSS1 product. The identification of PSS2 as the predominantly expressed somatostatin gene family member in avian autonomic neurons provides a molecular basis for further functional and pharmacological studies.

Interaction of Mash1 and Phox2b in sympathetic neuron development.

The transcription factors Mash1 and Phox2b are both essential for sympathetic neuron development. To understand in more detail their function and interaction, Phox2b and Mash1 were ectopically expressed in vivo, in peripheral nerve precursors. Here, we demonstrate that the Phox2b-induced generation of ectopic noradrenergic neurons in chick peripheral nerve involves the induction of Cash1, the chick homolog of Mash1. All Phox2-induced neurons coexpress the noradrenergic marker genes TH and DBH. Conversely, Mash1 induces neuronal differentiation characterized by the expression of generic neuronal genes SCG10, Hu and NF160; however, only a subpopulation of these neurons also displays an autonomic, noradrenergic phenotype. This context-dependent action of Mash1 implicates autonomic codeterminants, required for noradrenergic differentiation in response to Mash1. In contrast, Phox2b coordinates generic and noradrenergic gene expression, recruiting Mash1/Cash1, which may have a major function in the control of pan-neuronal gene expression during noradrenergic neuron development.

Essential role of Gata transcription factors in sympathetic neuron development.

Sympathetic neurons are specified during their development from neural crest precursors by a network of crossregulatory transcription factors, which includes Mash1, Phox2b, Hand2 and Phox2a. Here, we have studied the function of Gata2 and Gata3 zinc-finger transcription factors in autonomic neuron development. In the chick, Gata2 but not Gata3 is expressed in developing sympathetic precursor cells. Gata2 expression starts after Mash1, Phox2b, Hand2 and Phox2a expression, but before the onset of the noradrenergic marker genes Th and Dbh, and is maintained throughout development. Gata2 expression is affected in the chick embryo by Bmp gain- and loss-of-function experiments, and by overexpression of Phox2b, Phox2a, Hand2 and Mash1. Together with the lack of Gata2/3 expression in Phox2b knockout mice, these results characterize Gata2 as member of the Bmp-induced cluster of transcription factors. Loss-of-function experiments resulted in a strong reduction in the size of the sympathetic chain and in decreased Th expression. Ectopic expression of Gata2 in chick neural crest precursors elicited the generation of neurons with a non-autonomic, Th-negative phenotype. This implies a function for Gata factors in autonomic neuron differentiation, which, however, depends on co-regulators present in the sympathetic lineage. The present data establish Gata2 and Gata3 in the chick and mouse, respectively, as essential members of the transcription factor network controlling sympathetic neuron development.