Tracing ofher5progeny in zebrafish transgenics reveals the dynamics of midbrain-hindbrain neurogenesis and maintenance

Abstract

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, egfptranscription 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.

The serotonergic phenotype is acquired by converging genetic mechanisms within the zebrafish central nervous system

“…These results add to our previous observation that the early MHB progenitor pool generates cells populating r1 and r2 (and possibly also, to a low extent, r3 and r4; Tallafuss and Bally-Cuif, 2003). They also extend fate mapping data for Wnt1-positive cells in the embryonic day (E) 7.5 mouse embryo that gave rise to a few 5HT neurons of the anterior raphe (Zervas et al, 2004).…”

“…Because previous results from our laboratory also suggested that the MHB progenitor pool contributed neurons to the ventral anterior hindbrain (Tallafuss and Bally-Cuif, 2003), we specifically tested for the contribution of this pool to r1-2 serotonergic precursors. We used her5PAC:egfp transgenic embryos, which allow us to locate the early MHB pool by GFP expression at late gastrulation.…”

“…The origin of the pet1-expressing cells located in r1-2 after 24 hpf was traced using uncaging of DMNB-caged fluorescein (10 kDa; 5 mg/ml; Molecular Probes) injected into one-cell stage embryos from a her5PAC:egfp transgenic line (Tallafuss and Bally-Cuif, 2003). Injected GFP-positive embryos were uncaged at 90% epiboly to tail bud stage, when a distinct GFP expression could be seen identifying the MHB progenitor pool (Fig.…”

“…Surprisingly, we found that anterior and posterior clusters of 5HT neuronal precursors in the zebrafish hindbrain are separate at the level of r3, unlike the situation in mouse or chicken where the gap is in r4. We then used pet1 in combination with the her5PAC:egfp transgene (Tallafuss and Bally-Cuif, 2003) to trace the origin of anterior raphe serotonergic precursors. Our results demonstrate that the majority of the serotonergic precursors located in r1-2, but not those located further posteriorly (r4 and beyond), originate from a progenitor cell pool located at the midbrain-hindbrain boundary (MHB).…”

Retinoic acid is required for endodermal pouch morphogenesis and not for pharyngeal endoderm specification

“…Subsequently, we performed in situ hybridization with several genes known to be expressed in pouch endoderm, such as her5 (Fig. 6F,G ;Tallafuss and Bally-Cuif, 2003), foxa3, which is expressed in endoderm and axial mesendoderm (Warga and Kane, 2003), as well as sox17, a panendodermal marker (data not shown; Alexander and Stainier, 1999). We did not detect any obvious changes in the number of cells expressing these genes (Fig.…”