Tracing of her5 progeny in zebrafish transgenics reveals the dynamics of midbrain-hindbrain neurogenesis and maintenance.

The midbrain-hindbrain domain (MH) of the vertebrate embryonic neural tube develops in response to the isthmic organizer (IsO), located at the midbrain-hindbrain boundary (MHB). MH derivatives are largely missing in mutants affected in IsO activity; however, the potentialities and fate of MH precursors in these conditions have not been directly determined. To follow the dynamics of MH maintenance in vivo, we used artificial chromosome transgenesis in zebrafish to construct lines where egfp transcription is driven by the complete set of regulatory elements of her5, the first known gene expressed in the MH area. In these lines, egfp transcription faithfully recapitulates her5 expression from its induction phase onwards. Using the stability of GFP protein as lineage tracer, we first demonstrate that her5 expression at gastrulation is a selective marker of MH precursor fate. By comparing GFP protein and her5 transcription, we further reveal the spatiotemporal dynamics of her5 expression that conditions neurogenesis progression towards the MHB over time. Finally, we trace the molecular identity of GFP-positive cells in the acerebellar (ace) and no-isthmus (noi) mutant backgrounds to analyze directly fgf8 and pax2.1 mutant gene activities for their ultimate effect on cell fate. We demonstrate that most MH precursors are maintained in both mutants but express abnormal identities, in a manner that strikingly differs between the ace and noi contexts. Our observations directly support a role for Fgf8 in protecting anterior tectal and metencephalic precursors from acquiring anterior identities, while Pax2.1 controls the choice of MH identity as a whole. Together, our results suggest a model where an ordered MH pro-domain is identified at gastrulation, and where cell identity choices within this domain are subsequently differentially controlled by Fgf8 and Pax2.1 functions

Genetic and cellular analyses of zebrafish atrioventricular cushion and valued development.

Defects in cardiac valve morphogenesis and septation of the heart chambers constitute some of the most common human congenital abnormalities. Some of these defects originate from errors in atrioventricular (AV) endocardial cushion development. Although this process is being extensively studied in mouse and chick, the zebrafish system presents several advantages over these models, including the ability to carry out forward genetic screens and study vertebrate gene function at the single cell level. In this paper, we analyze the cellular and subcellular architecture of the zebrafish heart during stages of AV cushion and valve development and gain an unprecedented level of resolution into this process. We find that endocardial cells in the AV canal differentiate morphologically before the onset of epithelial to mesenchymal transformation, thereby defining a previously unappreciated step during AV valve formation. We use a combination of novel transgenic lines and fluorescent immunohistochemistry to analyze further the role of various genetic (Notch and Calcineurin signaling) and epigenetic (heart function) pathways in this process. In addition, from a large-scale forward genetic screen we identified 55 mutants, defining 48 different genes, that exhibit defects in discrete stages of AV cushion development. This collection of mutants provides a unique set of tools to further our understanding of the genetic basis of cell behavior and differentiation during AV valve development

The zebrafish buttonhead-like factor Bts1 is an early regulator of pax2.1 expression during mid-hindbrain development.

Little is known about the factors that control the specification of the mid-hindbrain domain (MHD) within the vertebrate embryonic neural plate. Because the head-trunk junction of the Drosophila embryo and the MHD have patterning similarities, we have searched for vertebrate genes related to the Drosophila head gap gene buttonhead (btd), which in the fly specifies the head-trunk junction. We report here the identification of a zebrafish gene which, like btd, encodes a zinc-finger transcriptional activator of the Sp-1 family (hence its name, bts1 for btd/Sp-related-1) and shows a restricted expression in the head. During zebrafish gastrulation, bts1 is transcribed in the posterior epiblast including the presumptive MHD, and precedes in this area the expression of other MHD markers such as her5, pax2.1 and wnt1. Ectopic expression of bts1 combined to knock-down experiments demonstrate that Bts1 is both necessary and sufficient for the induction of pax2.1 within the anterior neural plate, but is not involved in regulating her5, wnt1 or fgf8 expression. Our results confirm that early MHD development involves several genetic cascades that independently lead to the induction of MHD markers, and identify Bts1 as a crucial upstream component of the pathway selectively leading to pax2.1 induction. In addition, they imply that flies and vertebrates, to control the development of a boundary embryonic region, have probably co-opted a similar strategy: the restriction to this territory of the expression of a Btd/Sp-like factor

Requirements for endoderm and BMP signaling in sensory neurogenesis in zebrafish.

Cranial sensory neurons largely derive from neurogenic placodes (epibranchial and dorsolateral), which are ectodermal thickenings that form the sensory ganglia associated with cranial nerves, but the molecular mechanisms of placodal development are unclear. Here, we show that the pharyngeal endoderm induces epibranchial neurogenesis in zebrafish, and that BMP signaling plays a crucial role in this process. Using a her5:egfp transgenic line to follow endodermal movements in living embryos, we show that contact between pharyngeal pouches and the surface ectoderm coincides with the onset of neurogenesis in epibranchial placodes. By genetic ablation and reintroduction of endoderm by cell transplantation, we show that these contacts promote neurogenesis. Using a genetic interference approach we further identify bmp2b and bmp5 as crucial components of the endodermal signals that induce epibranchial neurogenesis. Dorsolateral placodes (trigeminal, auditory, vestibular, lateral line) develop independently of the endoderm and BMP signaling, suggesting that these two sets of placodes are under separate genetic control. Our results show that the endoderm regulates the differentiation of cranial sensory ganglia, which coordinates the cranial nerves with the segments that they innervate

bHLH transcription factor Her5 links patterning to regional inhibition of neurogenesis at the midbrain-hindbrain boundary.

The midbrain-hindbrain (MH) domain of the vertebrate embryonic neural plate displays a stereotypical profile of neuronal differentiation, organized around a neuron-free zone ('intervening zone', IZ) at the midbrain-hindbrain boundary (MHB). The mechanisms establishing this early pattern of neurogenesis are unknown. We demonstrate that the MHB is globally refractory to neurogenesis, and that forced neurogenesis in this area interferes with the continued expression of genes defining MHB identity. We further show that expression of the zebrafish bHLH Hairy/E(spl)-related factor Her5 prefigures and then precisely delineates the IZ throughout embryonic development. Using morpholino knock-down and conditional gain-of-function assays, we demonstrate that Her5 is essential to prevent neuronal differentiation and promote cell proliferation in a medial compartment of the IZ. We identify one probable target of this activity, the zebrafish Cdk inhibitor p27Xic1. Finally, although the her5 expression domain is determined by anteroposterior patterning cues, we show Her5 does not retroactively influence MH patterning. Together, our results highlight the existence of a mechanism that actively inhibits neurogenesis at the MHB, a process that shapes MH neurogenesis into a pattern of separate neuronal clusters and might ultimately be necessary to maintain MHB integrity. Her5 appears as a partially redundant component of this inhibitory process that helps translate early axial patterning information into a distinct spatiotemporal pattern of neurogenesis and cell proliferation within the MH domain