The neural crest (NC) lineage gives rise to a wide array of cell types ranging from neurons and glia of the peripheral nervous system to skeletal elements of the head. The mechanisms regulating NC differentiation into such a large number of cell types remains largely unknown. MicroRNAs (miRNAs) play key roles in regulating developmental events suggesting they may also play a role during NC differentiation. To determine what roles miRNAs play in differentiation of NC-derived tissues, we deleted the miRNA processing gene Dicer in NC cells using the Wnt1-Cre deleter line. We show that deletion of Dicer soon after NC cells have formed does not affect their migration and colonization of their targets in the embryo. However, the post-migratory NC is dependent on Dicer for survival. In the head, loss of Dicer leads to a loss of NC derived craniofacial bones while in the trunk, cells of the enteric, sensory and sympathetic nervous systems are lost during development. We found that loss of Dicer does not prevent the initial differentiation of NC but as development progresses, NC derivatives are lost due to apoptotic cell death. When Dicer is deleted, both Caspase-dependent and -independent apoptotic pathways are activated in the sensory ganglia but only the Caspase-dependent apoptotic program was activated in the sympathetic nervous system showing that the specific endogenous apoptotic programs are turned on by loss of Dicer. Our results show that Dicer and miRNAs, are required for survival of NC-derived tissues by preventing apoptosis during differentiation.

Induction of the sympathetic nervous system (SNS) from its neural crest (NC) precursors is dependent on BMP signaling from the dorsal aorta. To determine the roles of BMP signaling and the pathways involved in SNS development, we conditionally knocked out components of the BMP pathways. To determine if BMP signaling is a cell-autonomous requirement of SNS development, the Alk3 (BMP receptor IA) was deleted in the NC lineage. The loss of Alk3 does not prevent NC cell migration, but the cells die immediately after reaching the dorsal aorta. The paired homeodomain factor Phox2b, known to be essential for survival of SNS precursors, is downregulated, suggesting that Phox2b is a target of BMP signaling. To determine if Alk3 signals through the canonical BMP pathway, Smad4 was deleted in the NC lineage. Loss of Smad4 does not affect neurogenesis and ganglia formation; however, proliferation and noradrenergic differentiation are reduced. Analysis of transcription factors regulating SNS development shows that the basic helix-loop-helix factor Ascl1 is downregulated by loss of Smad4 and that Ascl1 regulates SNS proliferation but not noradrenergic differentiation. To determine if the BMP-activated Tak1 (Map3k7) pathway plays a role in SNS development, Tak1 was deleted in the NC lineage. We show that Tak1 is not involved in SNS development. Taken together, our results suggest multiple roles for BMP signaling during SNS development. The Smad4-independent pathway acts through the activation of Phox2b to regulate survival of SNS precursors, whereas the Smad4-dependent pathway controls noradrenergic differentiation and regulates proliferation by maintaining Ascl1 expression.

SUMMARY Hand1 regulates development of numerous tissues within the embryo, extraembryonic mesoderm and trophectoderm. Systemic loss of Hand1 results in early embryonic lethality but the cause has remained unknown. To determine if Hand1 expression in extraembryonic mesoderm is essential for embryonic survival, Hand1 was conditionally deleted using the HoxB6-Cre mouse line that expresses Cre in extraembryonic and lateral mesoderm. Deletion of Hand1 using HoxB6-Cre resulted in embryonic lethality identical to systemic knockout. To determine if lethality is due to Hand1 function in extraembryonic mesoderm or lateral mesoderm, we generated a Tlx2-Cre mouse line expressing Cre in lateral mesoderm but not extraembryonic tissues. Deletion of Hand1 using the Tlx2-Cre line results in embryonic survival with embryos exhibiting herniated gut and thin enteric smooth muscle. Our results show that Hand1 regulates development of lateral mesoderm derivatives and its loss in extraembryonic mesoderm is the primary cause of lethality in Hand1-null embryos.

Sonic hedgehog (Shh) plays critical roles during nervous system development, yet little is known about its function in the sympathetic nervous system. Using a mouse Shh null line, we examined the roles of Shh during SNS development. Loss of Shh did not prevent formation of the sympathetic trunk, but the ganglia are hypoplastic and misspatterned. Neuronal differentiation was delayed in Shh mutant embryos showing that Shh is required for correct developmental timing in addition to its role in sympathetic nervous system patterning. Immunohistochemical analyses of the ganglia for expression of the pan-neuronal marker beta3-tubulin, the noradrenergic biosynthetic enzyme tyrosine hydroxylase and the glial marker B-FABP showed that Shh is not required for differentiation of sympathetic neurons or glia.

The sympathetic nervous system (SNS) and enteric nervous system (ENS) employ many of the same signaling pathways and transcription circuits to regulate their development. However, while the SNS is a relatively simple nervous system, with neurons predominantly noradrenergic, the ENS is composed of neurons that are as diverse as those found in the central nervous system. This suggests that molecules regulating development of these two nervous systems function in both unique and in overlapping roles. The Hand2 basic helix‐loop‐helix transcription factor is expressed in both the SNS and ENS during development and our analysis of Hand2 function in mice shows that it regulates similar and unique events during their development. In the SNS, Hand2 is required for proliferation, survival and noradrenergic differentiation of neurons. Analysis of the transcriptional circuit regulating catecholaminergic differentiation showed that it does not affect expression of GATA3. Phox2a or Phox2b suggesting that it may directly regulate genes encoding the norepinephrine biosynthetic enzymes. In the ENS, loss of Hand2 leads to changes in the ratios of neurons expressing different neuronal transmitters and in ENS patterning. We will present a comparative analysis of how Hand2 regulates different aspects of SNS and ENS development.