Neuronal differentiation is regulated by proneural genes that promote neurogenesis and inhibitory mechanisms that maintain progenitors. This raises the question of how the up-regulation of proneural genes required to initiate neurogenesis occurs in the presence of such inhibition. We carried out loss and gain of gene function, an interaction screen for binding partners, and biochemical analyses to uncover the regulation, developmental role, and mechanism of action of a ubiquitination adaptor protein, Btbd6a (BTB domain containing 6a). We find that the proneural gene neurog1 up-regulates btbd6a, which in turn is required for up-regulation of neurog1. Btbd6a is an adaptor for the Cul3 ubiquitin ligase complex, and we find that it binds to the transcriptional repressor Plzf (promyelocytic leukemia zinc finger). Btbd6a promotes the relocation of Plzf from nucleus to cytoplasm and targets Plzf for ubiquitination and degradation. plzfa is expressed widely in the neural epithelium; when overexpressed, it inhibits neurogenesis, and this inhibition is reversed by btbd6a. The antagonism of endogenous plzfa by btbd6a is required for neurogenesis, since the block in neuronal differentiation caused by btbd6a knockdown is alleviated by plzfa knockdown. These findings reveal a feedback loop mediated by degradation of an inhibitor that is essential for progenitors to undergo the transition to neuronal differentiation.[Keywords: Neurogenesis; ubiquitination; Plzf; Cul3 adaptor] Supplemental material is available at http://www.genesdev.org.

Within the developing vertebrate head, the migration of neural tube-derived neural crest cells (NCCs) through the cranial mesenchyme is patterned into three streams, with mesenchyme adjacent to rhombomeres (r)3 and r5 maintained NCC-free. The receptor tyrosine kinase erbB4 is expressed within r3 and r5 and is required to maintain the r3-adjacent NCC-free zone in mouse embryos. In this study, we demonstrate that the extent of r3 involvement in patterning mouse NCC migration is restricted to the same dorsolateral region regulated by erbB4. In chick embryos, we show that erbB4 signaling similarly maintains the r3-adjacent NCC-free zone. However, although r5 expresses erbB4, this is insufficient to maintain the r3-adjacent NCC-free zone in grafting experiments where r5 replaced r3, indicating that erbB4 requires additional factors at the A-P level of r3 to pattern NCC migration. Furthermore, we show that the r5-adjacent NCC-free zone is maintained independently of r5, but requires surface ectoderm. Finally, we demonstrate that avian cranial surface ectoderm is patterned molecularly, with dorsolateral surface ectoderm at the levels of r2/3 and r7 expressing the sulfatase QSulf1 in quail, or the orthologue CSulf1 in chick. Aberrant NCC migration into r3-adjacent mesenchyme correlated with more focused QSulf1 expression in r2/3 surface ectoderm.

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Cranial neural crest cells are a pluripotent population of cells derived from the neural tube that migrate into the branchial arches to generate the distinctive bone, connective tissue and peripheral nervous system components characteristic of the vertebrate head. The highly conserved segmental organisation of the vertebrate hindbrain plays an important role in pattering the pathways of neural crest cell migration and in generating the distinct or separate streams of crest cells that form unique structures in each arch. We have used focal injections of DiI into the developing mouse hindbrain in combination with in vitro whole embryo culture to map the patterns of cranial neural crest cell migration into the developing branchial arches. Our results show that mouse hindbrain-derived neural crest cells migrate in three segregated streams adjacent to the even-numbered rhombomeres into the branchial arches, and each stream contains contributions of cells from three rhombomeres in a pattern very similar to that observed in the chick embryo. There are clear neural crest-free zones adjacent to r3 and r5. Furthermore, using grafting and lineage-tracing techniques in cultured mouse embryos to investigate the differential ability of odd and even-numbered segments to generate neural crest cells, we find that odd and even segments have an intrinsic ability to produce equivalent numbers of neural crest cells. This implies that inter-rhombomeric signalling is less important than combinatorial interactions between the hindbrain and the adjacent arch environment in specific regions, in the process of restricting the generation and migration of neural crest cells. This creates crest-free territories and suggests that tissue interactions established during development and patterning of the branchial arches may set up signals that the neural plate is primed to interpret during the progressive events leading to the delamination and migration of neural crest cells. Using interspecies grafting experiments between mouse and chick embryos, we have shown that this process forms part of a conserved mechanism for generating neural crest-free zones and contributing to the separation of migrating crest populations with distinct Hox expression during vertebrate head development.

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