The interplay of atoh1 genes in the lower rhombic lip during hindbrain morphogenesis

Abstract

The Lower Rhombic Lip (LRL) is a transient neuroepithelial structure of the dorsal hindbrain, which expands from r2 to r7, and gives rise to deep nuclei of the brainstem, such as the vestibular and auditory nuclei and most posteriorly the precerebellar nuclei. Although there is information about the contribution of specific proneural-progenitor populations to specific deep nuclei, and the distinct rhombomeric contribution, little is known about how progenitor cells from the LRL behave during neurogenesis and how their transition into differentiation is regulated. In this work, we investigated the atoh1 gene regulatory network operating in the specification of LRL cells, and the kinetics of cell proliferation and behavior of atoh1a-derivatives by using complementary strategies in the zebrafish embryo. We unveiled that atoh1a is necessary and sufficient for specification of LRL cells by activating atoh1b, which worked as a differentiation gene to transition progenitor cells towards neuron differentiation in a Notch-dependent manner. This cell state transition involved the release of atoh1a-derivatives from the LRL: atoh1a progenitors contributed first to atoh1b cells, which are committed non-proliferative precursors, and to the lhx2b-neuronal lineage as demonstrated by cell fate studies and functional analyses. Using in vivo cell lineage approaches we revealed that the proliferative cell capacity, as well as the mode of division, relied on the position of the atoh1a progenitors within the dorsoventral axis. We showed that atoh1a may behave as the cell fate selector gene, whereas atoh1b functions as a neuronal differentiation gene, contributing to the lhx2b neuronal population. atoh1a-progenitor cell dynamics (cell proliferation, cell differentiation, and neuronal migration) relies on their position, demonstrating the challenges that progenitor cells face in computing positional information from a dynamic two-dimensional grid in order to generate the stereotyped neuronal structures in the embryonic hindbrain.

The Digital 3D-Atlas MAKER (DAMAKER): a dynamic and expandable digital 3D-tool for monitoring the temporal changes in tissue growth during hindbrain morphogenesis

“…As shown in Fig. 2, the NDD dramatically increased during this time period, consistent with previous qualitative descriptions (Belzunce et al, 2020; Lyons et al, 2003; Voltes et al, 2019). Only few cells underwent differentiation before 24 hpf (Fig.…”

“…Here, we made use of the zebrafish embryonic brainstem—the hindbrain, which undergoes a segmentation process along the anteroposterior (AP) axis leading to the formation of seven metamers called rhombomeres (Fraser et al, 1990; Jimenez-Guri and Pujades, 2011; Jimenez-Guri et al, 2010; Kiecker and Lumsden, 2005; Pujades, 2020). The first neuronal differentiation events in the hindbrain are observed just prior to 24 hours post-fertilization (hpf), with a pronounced increase of events from 30 hpf onwards, concomitant with dramatic morphogenetic changes (Belzunce et al, 2020). Neuronal differentiation in the hindbrain needs to be finely tuned, as it involves changes in proliferative capacity, an extraordinary displacement of neurons from their birth site and complex tissue morphogenetic movements over time.…”

The neurogenic fate of the hindbrain boundaries relies on Notch-dependent asymmetric cell divisions

“…As expected, we observed that boundary cells were devoid of Notch-activity at early stages (Figure 2D, and inserts; see mCherry cells with no GFP expression in (d)). In contrast, the rhombic lip and the rhombomeric territories were Notch-active (Figures 2D and S3A; Belzunce et al, 2020). However, by 48 hpf, most of the boundary progenitor cells were Notch-active (Figure 2E, and inserts; see in (e) transverse views and corresponding inserts, showing that most of the dorsal mCherry cells facing the ventricle expressed GFP).…”

“…The Tg[HuC:GFP] line labels differentiated neurons (Park et al, 2000). Tg[atoh1a:GFP] (Belzunce et al, 2020) labeling the rhombic lip was generated by crossing Tg[atoh1a:KalTA4] (Distel et al, 2009) and Tg[UAS:GFP].…”

“…KAEDE, mCherry , and notch3 probes were generated by PCR amplification adding the T7 promoter sequence to the Rv primer: KAEDE Fw 5’-GCT GCT TAT GGA AGG CAA TG −3’ and Rv 5’-CAA TCC AGA ATG GGC AAC AG −3’; mCherry Fw 5’-CCA AGC TGA AGG TGA CCA AG −3’ and Rv 5’-GCT CGT ACT GTT CCA CGA TG-3’; notch3 Fw 5’-ATG GGG AAT TAC AGC CTT TG −3’ and Rv 5’-GGC AAA CAA GCA CAT TCG TA −3’. sgca and rfng probes were generated as described (Letelier et al, 2018), ascl1, lhx1a and evx1 as described (Belzunce et al, 2020), and notch1a as described (Taberner et al, 2020). For fluorescent in situ hybridization, FLUO- and DIG-labeled probes were detected with TSA Fluorescein and Cy3, respectively.…”