Conditional deletion of Hand2 reveals critical functions in neurogenesis and cell type-specific gene expression for development of neural crest-derived noradrenergic sympathetic ganglion neurons

Neural crest-derived structures that depend critically upon expression of the basic helix-loop-helix DNA binding protein Hand2 for normal development include craniofacial cartilage and bone, the outflow tract of the heart, cardiac cushion, and noradrenergic sympathetic ganglion neurons. Loss of Hand2 is embryonic lethal by E9.5, obviating a genetic analysis of its in-vivo function. We have overcome this difficulty by specific deletion of Hand2 in neural crest-derived cells by crossing our line of floxed Hand2 mice with Wnt1-Cre transgenic mice. Our analysis of Hand2 knock-out in neural crest-derived cells reveals effects on development in all neural crest-derived structures where Hand2 is expressed. In the autonomic nervous system, conditional disruption of Hand2 results in a significant and progressive loss of neurons as well as a significant loss of TH expression. Hand2 affects generation of the neural precursor pool of cells by affecting both the proliferative capacity of the progenitors as well as affecting expression of Phox2a and Gata3, DNA binding proteins important for the cell autonomous development of noradrenergic neurons. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting differentiation and cell type-specific gene expression in neural crest-derived noradrenergic sympathetic ganglion neurons. Hand2 has a pivotal function in a non-linear cross-regulatory network of DNA binding proteins that affect cell autonomous control of differentiation and cell type-specific gene expression.     

Development of the cholinergic neurotransmitter phenotype in postganglionic sympathetic neurons

Sympathetic ganglia are composed of noradrenergic neurons and cholinergic neurons that differ in the expression of neurotransmitter-synthesizing enzymes, neurotransmitter transporters and neuropeptides. The analysis of the cholinergic differentiation during development revealed important principles involved in the generation of neuronal diversity, in particular the importance of signals from the innervated target. Some peripheral targets, such as the sweat glands in the mammalian footpads, are purely cholinergically innervated in the adult, whereas skeletal muscle arteries receive both noradrenergic and cholinergic innervation. For sympathetic neurons innervating sweat glands there is convincing evidence that these neurons are initially noradrenergic and that the interaction of innervating fibers and target tissue induces a shift in the neurotransmitter phenotype from noradrenergic to cholinergic. In addition to this target-dependent differentiation, an earlier expression of cholinergic characters was observed in sympathetic ganglia that occurs before target contact. These data raise the possibility that different subpopulations of cholinergic sympathetic neurons, innervating distinct peripheral targets, may develop along distinct schedules. In vitro studies suggest that growth factors of the family of neuropoietic cytokines are involved in the specification of the cholinergic sympathetic phenotype. Recent in vivo studies that interfered with cytokine receptor expression in developing avian sympathetic ganglia indicate that only the late, target-dependent differentiation depends on cytokine signaling. The signals involved in the early, target-independent expression of cholinergic properties remain to be determined, as well as the identity of the target-derived cytokine. Thus, cholinergic sympathetic differentiation seems to be more complex than expected, involving either both target-independent and target-dependent control or only target-induced differentiation, according to the specific neuronal subpopulation and target.     

Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

The establishment of correct neurotransmitter characteristics is an essential step of neuronal fate specification in CNS development. However, very little is known about how a battery of genes involved in the determination of a specific type of chemical-driven neurotransmission is coordinately regulated during vertebrate development. Here, we investigated the gene regulatory networks that specify the cholinergic neuronal fates in the spinal cord and forebrain, specifically, spinal motor neurons (MNs) and forebrain cholinergic neurons (FCNs). Conditional inactivation of Isl1, a LIM homeodomain factor expressed in both differentiating MNs and FCNs, led to a drastic loss of cholinergic neurons in the developing spinal cord and forebrain. We found that Isl1 forms two related, but distinct types of complexes, the Isl1-Lhx3-hexamer in MNs and the Isl1-Lhx8-hexamer in FCNs. Interestingly, our genome-wide ChIP-seq analysis revealed that the Isl1-Lhx3-hexamer binds to a suite of cholinergic pathway genes encoding the core constituents of the cholinergic neurotransmission system, such as acetylcholine synthesizing enzymes and transporters. Consistently, the Isl1-Lhx3-hexamer directly coordinated upregulation of cholinergic pathways genes in embryonic spinal cord. Similarly, in the developing forebrain, the Isl1-Lhx8-hexamer was recruited to the cholinergic gene battery and promoted cholinergic gene expression. Furthermore, the expression of the Isl1-Lhx8-complex enabled the acquisition of cholinergic fate in embryonic stem cell-derived neurons. Together, our studies show a shared molecular mechanism that determines the cholinergic neuronal fate in the spinal cord and forebrain, and uncover an important gene regulatory mechanism that directs a specific neurotransmitter identity in vertebrate CNS development.     

The development of the noradrenergic transmitter phenotype in postganglionic sympathetic neurons

Here we review recent data on molecular aspects of the differentiation of the noradrenergic neurotransmitter phenotype in postganglionic sympathetic neurons during avian and mammalian embryogenesis. By experimental manipulation of the chick embryo, it has been shown that neural tube and notochord are important for noradrenergic differentiation which occurs when migrating neural crest cells, the precursors of sympathetic ganglion cells, reach the dorsal aorta. Bone morphogenetic proteins expressed in the dorsal aorta before and during the time of noradrenergic differentiation are likely candidates for growth factors involved in induction of noradrenergic differentiation in vivo. To analyze noradrenergic differentiation, enzymes of the noradrenaline biosynthesis pathway and catecholamine stores have been used as differentiation markers. The molecules involved in neurotransmitter release which are as important for a functional noradrenergic neuron as those required for transmitter synthesis and storage are only recently being studied in this context. For a comprehensive view of the embryonic development of the noradrenergic neurotransmitter phenotype, it will be necessary to understand how the systems for synthesis, storage and release of noradrenaline are assembled during neuronal differentiation. Special issue dedicated to Dr. Hans Thoenen.     

Developmental interactions between sweat glands and the sympathetic neurons which innervate them: effects of delayed innervation on neurotransmitter plasticity and gland maturation

The neurotransmitter properties of the sympathetic innervation of sweat glands in rat footpads have previously been shown to undergo a striking change during development. When axons first reach the developing glands, they contain catecholamine histofluorescence and immunoreactivity for catecholamine synthetic enzymes. As the glands and their innervation mature, catecholamines disappear and cholinergic and peptidergic properties appear. Final maturation of the sweat glands, assayed by secretory competence, is correlated temporally with the development of cholinergic function in the innervation. To determine if the neurotransmitter phenotype of sympathetic neurons developing in vivo is plastic, if sympathetic targets can play a role in determining neurotransmitter properties of the neurons which innervate them, and if gland maturation is dependent upon its innervation, the normal developmental interaction between sweat glands and their innervation was disrupted. This was accomplished by a single injection of 6-hydroxy-dopamine (6-OHDA) on Postnatal Day 2. Following this treatment, the arrival of noradrenergic sympathetic axons at the developing glands was delayed 7 to 10 days. Like the gland innervation of normal rats, the axons which innervated the sweat glands of 6-OHDA-treated animals acquired cholinergic function and their expression of endogenous catecholamines declined. The change in neurotransmitter properties, however, occurred later in development than in untreated animals and was not always complete. Even in adult animals, some fibers continued to express endogenous catecholamines and many nerve terminals contained a small proportion of small granular vesicles after permanganate fixation. The gland innervation in the 6-OHDA-treated animals also differed from that of normal rats in that immunoreactivity for VIP was not expressed in the majority of glands. It seems likely that following treatment with 6-OHDA sweat glands were innervated both by neurons that would normally have done so and by neurons that would normally have innervated other, noradrenergic targets in the footpads, such as blood vessels. Contact with sweat glands, therefore, appears to suppress noradrenergic function and induce cholinergic function not only in the neurons which normally innervate the glands but also in neurons which ordinarily innervate other targets. Effects of delayed innervation were also observed on target development. The appearance of sensitivity to cholinergic agonists by the sweat glands was coupled with the onset of cholinergic transmission.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calmodulin Suppresses Synaptotagmin-2 Transcription in Cortical Neurons

Calmodulin (CaM) is a ubiquitous Ca²⁺ sensor protein that plays a pivotal role in regulating innumerable neuronal functions, including synaptic transmission. In cortical neurons, most neurotransmitter release is triggered by Ca²⁺ binding to synaptotagmin-1; however, a second delayed phase of release, referred to as asynchronous release, is triggered by Ca²⁺ binding to an unidentified secondary Ca²⁺ sensor. To test whether CaM could be the enigmatic Ca²⁺ sensor for asynchronous release, we now use in cultured neurons short hairpin RNAs that suppress expression of ∼70% of all neuronal CaM isoforms. Surprisingly, we found that in synaptotagmin-1 knock-out neurons, the CaM knockdown caused a paradoxical rescue of synchronous release, instead of a block of asynchronous release. Gene and protein expression studies revealed that both in wild-type and in synaptotagmin-1 knock-out neurons, the CaM knockdown altered expression of >200 genes, including that encoding synaptotagmin-2. Synaptotagmin-2 expression was increased several-fold by the CaM knockdown, which accounted for the paradoxical rescue of synchronous release in synaptotagmin-1 knock-out neurons by the CaM knockdown. Interestingly, the CaM knockdown primarily activated genes that are preferentially expressed in caudal brain regions, whereas it repressed genes in rostral brain regions. Consistent with this correlation, quantifications of protein levels in adult mice uncovered an inverse relationship of CaM and synaptotagmin-2 levels in mouse forebrain, brain stem, and spinal cord. Finally, we employed molecular replacement experiments using a knockdown rescue approach to show that Ca²⁺ binding to the C-lobe but not the N-lobe of CaM is required for suppression of synaptotagmin-2 expression in cortical neurons. Our data describe a previously unknown, Ca²⁺/CaM-dependent regulatory pathway that controls the expression of synaptic proteins in the rostral-caudal neuraxis.     

Norepinephrine transporter expression and function in noradrenergic cell differentiations

The classical view of norepinephrine transporter (NET) function is the re-uptake of released norepinephrine (NE) by mature sympathetic neurons and noradrenergic neurons of the locus ceruleus (LC; [1-3]). In this report we review previous data and present new results that show that NET is expressed in the young embryo in a wide range of neuronal and non-neuronal tissues and that NET has additional functions during embryonic development. Sympathetic neurons are derived from neural crest stem cells. Fibroblast growth factor-2 (FGF-2), neurotrophin-3 (NT-3) and transforming growth factor-1 (TGF-1) regulate NET expression in cultured quail neural crest cells by causing an increase in NET mRNA levels. They also promote NET function in both neural crest cells and presumptive noradrenergic cells of the LC. The growth factors are synthesized by the neural crest cells and therefore are likely to have autocrine function. In a subsequent stage of development, NE transport regulates differentiation of noradrenergic neurons in the peripheral nervous system and the LC by promoting expression of tyrosine hydroxylase (TH) and dopamine--hydroxylase (DBH). Conversely, uptake inhibitors, such as the tricyclic antidepressant, desipramine, and the drug of abuse, cocaine, inhibit noradrenergic differentiation in both tissues. Taken together, our data indicate that NET is expressed early in embryonic development, NE transport is involved in regulating expression of the noradrenergic phenotype in the peripheral and central nervous systems, and norepinephrine uptake inhibitors can disturb noradrenergic cell differentiation in the sympathetic ganglion (SG) and LC.