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.

Neurons share many features in common but are distinguished by expression of phenotypic characteristics that define their specific function, location, or connectivity. One aspect of neuronal fate determination that has been extensively studied is that of neurotransmitter choice. The generation of diversity of neuronal subtypes within the developing nervous system involves integration of extrinsic and intrinsic instructive cues resulting in the expression of a core set of regulatory molecules. This review focuses on mechanisms of growth and transcription factor regulation in the generation of peripheral neural crest-derived neurons. Although the specification and differentiation of noradrenergic neurons are the focus, I have tried to integrate these into a larger picture providing a general roadmap for development of autonomic neurons. There is a core of DNA binding proteins required for the development of sympathetic, parasympathetic, and enteric neurons, including Phox2 and MASH1, whose specificity is regulated by the recruitment of additional transcriptional regulators in a subtype-specific manner. For noradrenergic neurons, the basic helix-loop-helix DNA binding protein HAND2 (dHAND) appears to serve this function. The studies reviewed here support the notion that neurotransmitter identity is closely linked to other aspects of neurogenesis and reveal a molecular mechanism to coordinate expression of pan-neuronal genes with cell type-specific genes.

Background and Aims Bowel function requires coordinated activity of diverse enteric neuron subtypes. Our aim was to define gene expression in these neuron subtypes to facilitate development of novel therapeutic approaches to treat devastating enteric neuropathies, and to learn more about enteric nervous system function. Methods To identify subtype–specific genes, we performed single-nucleus RNA-seq on adult mouse and human colon myenteric plexus, and single-cell RNA-seq on E17.5 mouse ENS cells from whole bowel. We used immunohistochemistry, select mutant mice, and calcium imaging to validate and extend results. Results RNA-seq on 635 adult mouse colon myenteric neurons and 707 E17.5 neurons from whole bowel defined seven adult neuron subtypes, eight E17.5 neuron subtypes and hundreds of differentially expressed genes. Manually dissected human colon myenteric plexus yielded RNA-seq data from 48 neurons, 3798 glia, 5568 smooth muscle, 377 interstitial cells of Cajal, and 2153 macrophages. Immunohistochemistry demonstrated differential expression for BNC2, PBX3, SATB1, RBFOX1, TBX2, and TBX3 in enteric neuron subtypes. Conditional Tbx3 loss reduced NOS1-expressing myenteric neurons. Differential Gfra1 and Gfra2 expression coupled with calcium imaging revealed that GDNF and neurturin acutely and differentially regulate activity of ∼50% of myenteric neurons with distinct effects on smooth muscle contractions. Conclusion Single cell analyses defined genes differentially expressed in myenteric neuron subtypes and new roles for TBX3, GDNF and NRTN. These data facilitate molecular diagnostic studies and novel therapeutics for bowel motility disorders.

SUMMARYLower jaw development is a complex process in which multiple signaling cascades establish a proximal-distal organization. These cascades are regulated both spatially and temporally and are constantly refined through both induction of normal signals and inhibition of inappropriate signals. The connective tissue of the tongue arises from cranial neural crest cell-derived ectomesenchyme within the mandibular portion of the first pharyngeal arch and is likely to be impacted by this signaling. Although the developmental mechanisms behind later aspects of tongue development, including innervation and taste acquisition, have been elucidated, the early patterning signals driving ectomesenchyme into a tongue lineage are largely unknown. We show here that the basic helix-loop-helix transcription factor Hand2 plays key roles in establishing the proximaldistal patterning of the mouse lower jaw, in part through establishing a negative-feedback loop in which Hand2 represses Dlx5 and Dlx6 expression in the distal arch ectomesenchyme following Dlx5-and Dlx6-mediated induction of Hand2 expression in the same region. Failure to repress distal Dlx5 and Dlx6 expression results in upregulation of Runx2 expression in the mandibular arch and the subsequent formation of aberrant bone in the lower jaw along with proximal-distal duplications. In addition, there is an absence of lateral lingual swelling expansion, from which the tongue arises, resulting in aglossia. Hand2 thus appears to establish a distal mandibular arch domain that is conducive for lower jaw development, including the initiation of tongue mesenchyme morphogenesis.

The basic helix-loop-helix DNA binding protein Hand2 is expressed in neural crest-derived precursors of enteric neurons and has been shown to affect both neurogenesis and neurotransmitter specification of noradrenergic sympathetic ganglion neurons. In the current study, our goal was to determine whether