The role of atoh1 genes in the development of the lower rhombic lip during zebrafish hindbrain morphogenesis

BACKGROUND 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. RESULTS 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 showed that the proliferative cell capacity, as well as their mode of division, relied on the position of the atoh1a progenitors within the dorsoventral axis. CONCLUSIONS Our data demonstrates that the zebrafish provides an excellent model to study the in vivo behavior of distinct progenitor populations to the final neuronal differentiated pools, and to reveal the subfunctionalization of ortholog genes. Here, we unveil that atoh1a behaves 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.


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The most anterior region of the RL, which coincides with the dorsal pole of r1, is known as Upper Rhombic Lip (UPL) and produces all granule cells of the external and internal granular layers of the cerebellum [14,15]. The rest of the RL, which expands from r2 to r7, is known as Lower Rhombic Lip (LRL) and gives rise to deep nuclei of the brainstem, such as the vestibular and auditory nuclei and most posteriorly the precerebellar nuclei [16,17]. The genetic program for cerebellum development is largely conserved among vertebrates [16]; as an example, zebrafish and mouse use similar mechanisms to control cerebellar neurogenesis with a crucial role of atoh1 and ptf1 genes [17,18]. For the LRL, we know both the contribution of ptf1a/atoh1a proneural progenitor populations to specific deep nuclei [19], and the distinct rhombomeric identity [20]. However, little is known about how progenitor cells from the LRL behave during neurogenesis and how their transition into differentiation is regulated, in order to balance the rate of differentiation and proliferation to produce the proper neuronal numbers.
In this work, we sought to understand the role of atoh1 genes in the generation of the neuronal derivatives of LRL. We used complementary strategies in the zebrafish embryos to provide information about the gene regulatory network operating in the specification of LRL cells, and the kinetics of cell proliferation and behavior of atoh1aderivatives. 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 neuronal 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 showed that the proliferative cell as well as their mode of division, relied on the position of the atoh1a progenitors within the dorsoventral axis.

Expression of proneural genes within the zebrafish hindbrain
We first analyzed the formation of molecularly distinct neural progenitor domains, each of them able to generate particular neuronal cell types, during hindbrain embryonic development. We performed a comprehensive spatiotemporal analysis of the expression of distinct proneural genes along the anteroposterior (AP) and dorsoventral (DV) axes within the hindbrain and defined the DV order of proneural gene expression.
The expression profiles of atoh1a, ptf1a, ascl1a, ascl1b, and neurog1 indicated that their onset of expression differed along the AP axis ( Figure S1). The dorsal most progenitor cells express atoh1a all along the AP axis from 18hpf onwards, which remained  Figure S2E-H') [21]. Thus, by double in situ hybridization experiments we could assess the organization of the different proneural progenitor pools along the DV axis as following: atoh1a, ptf1a/ascl1a, ascl1b, neurog1, being atoh1a-cells the dorsal most progenitor cell population ( Figure S1P-S). Interestingly, this was not the same order than proneural gene expression in the zebrafish spinal cord, where a second domain of neurog1 progenitors positioned just underneath the atoh1a domain [23]. Proneural genes were expressed in non-differentiated progenitors, and accordingly, nonoverlapping expression was observed with HuC-staining ( Figure S2A'-H', Figure S3A', B-C). Interestingly, progenitors located in the dorsal most domain, became placed more lateral upon morphogenesis (see atoh1a-expressing cells in Figure 1E The three atoh1 paralogs -atoh1a, atoh1b and atoh1cwere shown to be expressed within the hindbrain and to contribute to the development of the cerebellum, with the expression of atoh1c restricted to the upper rhombic lip [17,18]. Since our main interest was understanding the development of the lower rhombic lip (LRL), we focused on the study of atoh1a and atoh1b and compared their onset of expression. atoh1a preceded the expression of atoh1b in the most dorsal progenitor cells of the hindbrain at 14hpf ( Figure 1A-A'). This was in contrast with the onset in the otic epithelium, where atoh1b was expressed earlier than atoh1a (see magenta in the otic placode in Figure 1A; [24]).
At 18hpf, atoh1a expression remained in the dorsal most cells, whereas atoh1b expression domain was more lateral, overlapping with atoh1a-cells and mostly contained within this expression domain ( Figure 1B-B', C-C'). Upon the opening of the neural tube, the atoh1a/b domains were laterally displaced and atoh1a remained medial whereas atoh1b positioned lateral ( Figure 1D-D'), and by 42hpf -when the fourth ventricle was already formed-atoh1b expression was completely lateral, and atoh1a remained dorsal and medial ( Figure 1E-E'). Thus, atoh1a and atoh1b were dorsally expressed but they differed in their mediolateral (apicobasal) position. To demonstrate that they were kept as progenitor cells, we stained Tg[HuC:GFP] embryos with atoh1a/b and observed that neither atoh1a nor atoh1b were expressed in differentiated neurons ( Figure 1F-H, F'-H'). Their differential apicobasal distribution and the fact that progenitor cell divisions always happened in the apical domains, suggested that atoh1b-progenitor cells might have experienced an apical displacement of their cell body before undergoing differentiation. To demonstrate this, we stained embryos with atoh1a/b and anti-pH3, a marker for mitotic figures, and observed that more atoh1a than atoh1b cells seemed to undergo mitosis ( Figure 1I-I', J-J'). In this same line, the analyses of single mitotic cells in the transgenic Tg[atoh1a:GFP] fish line ( Figure 1K-O) that labeled atoh1aexpressing cells and their derivatives [18], showed that mitotic atoh1a:GFP cells where always located in the ventricular domain (see white asterisks in Figure 1L whereas the ones that did not divide were laterally displaced just above the neuronal differentiation domain (see black asterisks in Figure 1L-O, M'-O'). Thus, atoh1b cells may derive from atoh1a progenitors that diminished their proliferative capacity and behaved as committed progenitors transitioning towards differentiation.

atoh1a progenitors gave rise to atoh1b cells and lhx2b neurons
Next, we sought to unravel whether indeed atoh1b cells derived from atoh1a progenitors and which were the atoh1a neuronal derivatives. For this we used the same Tg[atoh1a:GFP] fish line than before [18], which allows to label the cell derivatives of atoh1a progenitors due the stability of GFP, and combined in situ hybridization experiments with immunostaining using atoh1 probes, specific neuronal differentiation genes such as lhx2b, lhx1a, and pan-neuronal differentiation markers such as HuC  Figure 2D). By 48hpf, atoh1a-derivatives already populated the basal domain of the hindbrain (at this morphogenetic stage ventrally located), generating arched-like structures that coincided with rhombomeric boundaries (see yellow arrowhead in Figure 2G-L, see white arrowheads in Figure S4), implying that once the dorsal progenitors commit, they undergo cellular migration during differentiation.
In summary, atoh1a progenitors gave rise to atoh1b cells and to the lateral domain of lhx2b neurons. First differentiated atoh1a cells placed between rhombomeres to finally populate the basal hindbrain and generate arched-liked structures.

Reconstruction of the atoh1a lineage
Next question was to address how the rate of differentiation and proliferation of atoh1a cells was balanced to achieve the needed cell diversity. For this, we used genetic lineages that allowed to delineate cell types arising from atoh1a subsets. To trace the  Figure 4C, F, I, L; Table 1).
To address the possibility that the decrease in the number of neurons in atoh1a fh282 mutants was the result of a smaller number of atoh1a progenitor cells, we quantified the number of LRL atoh1a:GFP cells undergoing mitosis ( Figure 5A), and the overall number of atoh1a:GFP cells ( Figure 5B), both in atoh1a WT and atoh1a fh282 embryos. No significative differences were observed, suggesting that loss of atoh1a function did not affect the original number of LRL progenitors ( Figure 5A; LRL atoh1a:GFP cells displaying PH3-staining: atoh1a WT 17.9 ± 3.6 cells n = 15 vs. atoh1a fh282 15.9 ± 3.1 cells, n = 8; Figure   5B; total atoh1a:GFP cells: atoh1a WT 69.5 ± 6.4 cells n = 15 vs. atoh1a fh282 68.4 ± 7.5 cells, n = 8; see Table 2). Since the domains of neural bHLH gene expression are established and/or maintained by cross-repression resulting in the control of specific neuronal populations [1], we sought whether this neuronal loss was due to a change in cell fate rather than to a reduction of the number of progenitor cells. Thus, we analyzed proneural gene expression changes both in wild type and mutant context (Figure 5C-F; atoh1a WT n = 8, atoh1a fh282 n = 10). We observed that upon atoh1a mutation, atoh1a expression dramatically increased as previously reported [18] (compare Figure 5C and F) and the GFP-expressing progenitor cells did not die ( Figure 5D To further demonstrate the requirement of atoh1a in atoh1b expression and lhx2b neuronal differentiation, and better dissect the proneural gene hierarchy, we performed conditional gain of function experiments. We injected Mu4127 embryos expressing Gal4 in r3 and r5 with H2B-citrine:UAS vectors carrying either atoh1a or atoh1b genes, and analyzed the effects in atoh1 genes and lhx2b neurons ( Figure 6, Table 3 We showed that atoh1a cycling cells gave rise to atoh1b post-mitotic committed precursors. Since this commitment is suspected to be irreversible and leading towards neuronal differentiation, we thought the Notch signaling pathway as a reasonable candidate to be regulating this transition. Thus, we explored the Notch activity within the LRL to understand how atoh1b expression was restricted to a given atoh1a-domain in the neural tube. First, we assessed Notch activity by the use of the Tg[tp1:d2GFP] transgenic line, which is a readout of Notch-active cells [25]. Indeed, Notch-activity was restricted to the most dorsomedial atoh1a cell population ( Figure 7A-A'), whereas the more laterally located atoh1b cells were devoid of it ( Figure 7B-B'). This suggested that Notch activity was responsible of preventing atoh1a progenitor cells to transition to atoh1b and therefore modulating neuronal differentiation. To demonstrate this, we conditionally inhibited Notch activity by incubating Tg[atoh1a:GFP] embryos with the gamma-secretase inhibitor LY411575, and asked whether atoh1a/b expression domains were altered. Upon inhibition of Notch activity, there was an increase of atoh1bexpression at expense of atoh1a ( Figure 7C-D, F-G): atoh1b expression was expanded more medially, and atoh1a expression dramatically decreased (compare the border of the atoh1b expression in Figure 7D' with G'). As expected, the atoh1b cells did not arise de novo but derived from atoh1a:GFP progenitors ( Figure 7E-E', H-H'), supporting the idea that Notch-pathway regulated the transition of atoh1a progenitors towards differentiation.

DISCUSSION
Progenitor cell populations undergo important changes in their relative spatial distribution upon morphogenesis, which need to be precisely coordinated with the balance between progenitor cells vs. differentiated neurons. Here, we have defined the role of atoh1 genes along the development of the LRL population, and how this progenitor cell population behaves during the early neurogenic phase.
The spatiotemporal activation of proneural genes in the hindbrain shows that the neurogenic capacity is regionalized along the AP axis, such as that hindbrain boundaries and rhombomere centers remain devoid of neurogenesis [22]. This is valid for most of proneural genes except for atoh1 genes, because these are expressed all along the AP axis in the dorsal most hindbrain; however, RL derivatives delaminate from the dorsal epithelium, migrate and transitorily locate in the boundary regions. Interestingly, our results demonstrate that the function of different atoh1 genes depends on the context.
In the inner ear, atoh1a and atoh1b cross-regulate each other but are differentially required during distinct developmental periods: atoh1b activates atoh1a early, whereas in a late phase atoh1a maintains atoh1b [24]. In the URL, atoh1a and atoh1c have equivalent function in the generation of granular cells progenitors [18], whereas we argue that in the LRL atoh1a and atoh1b are not interchangeable, since they work directionally and have distinct functions. Although in the URL atoh1a activates the expression of neurod1 in intermediate, non-proliferative precursors [26], neurod1 expression is not detected in the zebrafish LRL before the 48hpf, implying that atoh1b is the one defining LRL intermediate precursors rather than neurod1 during early LRLderived neurogenesis.
Zebrafish has three atoh1 genes, atoh1a, atoh1b and atoh1c, which are expressed in overlapping but distinct progenitor domains within the rhombic lip [17,18]. Although Recently, it has been shown that nuclei in the hindbrain start migrating from variable apicobasal positions and move toward the apical surface in a directed and smooth manner, and this movement is controlled by Rho-ROCK-dependent myosin contractility [27]. However, the mechanisms by which actin generates the forces required for apical nuclear movement and the link between forces and atoh1b are not understood. however, signals participating in this particular context are unknown. Nonetheless, boundary cells are signalling centres instructing the neuronal allocation in the neighbouring tissue [9]; thus, one plausible hypothesis is that boundary cells might dictate the allocation of newly-differentiated neurons.
Balancing the rate of differentiation and proliferation in developing neural tube is essential for the production of appropriate numbers and achieving the needed cell diversity to form a functional central nervous system (CNS). This requires a finely tuned balance between the different modes of division that neural progenitor cells undergo [29]. Three distinct modes of divisions occur during vertebrate CNS development: selfexpanding (symmetric proliferative, PP) divisions ensure the expansion of the progenitor pool by generating two daughter cells with identical progenitor potential, self-renewing (asymmetric, PN) divisions generate one daughter cell with the same developmental potential than the parental cell and another with a more restricted potential, and self-consuming (symmetric terminal neurogenic, NN) divisions generate two cells committed to differentiation, thereby depleting the progenitor pool [29,30]. Our in vivo cell lineage studies shed light into this specific question in respect to the atoh1a cell population. We reveal the importance of the initial allocation of atoh1a progenitors: dorsal most atoh1a progenitors display more neurogenic capacity than ventral ones, since they give rise only to NN divisions upon the early neurogenic phase, whereas atoh1a progenitors located just underneath undergo the three distinct modes of division ensuring the expansion of the atoh1a-pool and providing committed progenitors. Most probably, the originally located dorsal progenitors will quickly become atoh1b and transition towards differentiation allocating more laterally. Interestingly, in the amniote spinal cord the modes of progenitor division are coordinated over time [31], instead of space. Why such a difference? One explanation is that in the LRL, where the position of progenitor cells changes dramatically over time, the most efficient way to provide fast neuronal production without exhausting the pool of progenitors could be regionalising the proliferative capacity. On the other hand, in vivo experiments in the chick spinal cord showed that an endogenous gradient of SMAD1/5 activity dictated the mode of division of spinal interneuron progenitors, in such a way that high levels of SMAD1/5 signalling promoted PP divisions, whereas a reduction in SMAD1/5 activity forced spinal progenitors to reduce self-expanding divisions in favour of self-consuming divisions [32].
This would suggest that dorsal most atoh1a cells would respond less to BMP signalling than ventral atoh1a cells. However, during hindbrain morphogenesis there is an important change in the position of atoh1a progenitors, and therefore their relative position in respect to the gradient sources. Since morphogen gradients quickly decrease with distance [33,34], it is difficult to apply the same rationale here than in the spinal cord. Still very little is known about how these gradients are established within the hindbrain [35], and how hindbrain progenitors interpret the quantitative information encoded by the concentration and duration of exposure to gradients. An alternative explanation is that different E proteins may control the ability of atoh1a to instruct dorsal or ventral neural progenitor cells to produce specific, specialized neurons, and thus ensure that the distinct types of neurons are produced in appropriate amounts as it happens in the chick spinal cord [36].
The loss of atoh1a function clearly affects the formation of the lateral column of lhx2b differentiated neurons and decreases the number of overall differentiated neurons. But what are the derivatives of these atoh1a-derived lhx2b cells? It has been described that the hindbrain displays a striking organization into transmitter stripes reflecting a broad patterning of neurons by cell type, morphology, age, projections, cellular properties, and activity patterns [37]. According to this pattern, the lateral lhx2b column would correspond to glutamatergic neurons expressing the barhl2 transcription factor [37], which in turn is an atoh1a target [38,39].
Notch has been extensively studied as a regulator of proneural gene expression by a process called lateral inhibition, in which cells expressing higher levels of proneural genes are selected as "neuroblasts" for further commitment and differentiation, while concomitantly maintaining their neighbors as proliferating neural precursors available for a later round of neuroblast selection [40]. Indeed, in the LRL the transition atoh1a to atoh1b seems to be regulated by Notch-activity, since upon Notch-inhibition most of the atoh1a cells disappear and they become atoh1b, and therefore are ready to undergo differentiation. Thus, although atoh1a is the upstream factor in LRL cell specification, several mechanisms seem to be in place to precisely coordinate acquisition of the neurogenic capacity and progenitor vs. differentiation transitions.

CONCLUSIONS
Our data demonstrates that the zebrafish provides an excellent model to study the contribution of distinct progenitor populations to the final neuronal differentiated pools, and to reveal the subfunctionalization of ortholog genes. We unveil that atoh1a
In order to quantify the number of proliferating LRL-cells in atoh1a WT and atoh1a fh282 embryos in the Tg[atoh1a:GFP] background, the number of mitotic figures within the atoh1a:GFP progenitor domain was assessed ( Figure 5A; see Table 2 for numbers and statistics).
For the quantification of the total number of LRL atoh1a:cells in atoh1a WT and atoh1a fh282 embryos in the Tg[atoh1a:GFP] background, embryos were stained with Draq5 and the total number of nuclei of atoh1a:GFP cells was assessed in r5 ( Figure 5B; see Table 2 for numbers and statistics). Cell tracking was performed using the MaMuT software (Fiji plug-in) [49].

Conditional overexpression
The full-length coding sequences of zebrafish atoh1aand atoh1b [24] were cloned into the MCS of a custom dual vector that expressed Citrine from one side of 5xUAS sequence and the cDNA of interest from the opposite side [21]. Mu4127 embryos (expressing KalT4 in r3 and r5) were injected either with H2B-citrine:UAS, H2B-citrine:UAS:atoh1a or H2B-citrine:UAS:atoh1b constructs at the one-cell stage, grown at 28.5°C and analyzed at 24hpf for atoh1a/b and lhxb2 in situ hybridization and Citrine expression.

Pharmacological treatments
Tg[atoh1a:GFP] embryos were treated either with 10µM of the gamma-secretase inhibitor LY411575 (Stemgent) or DMSO for control. The treatment was applied into the swimming water at 28.5°C from 24hpf to 30hpf. After treatment, embryos were fixed in 4%PFA for further analysis.

Ethics approval and consent to participate
Zebrafish (Dario rerio) were treated according to the Spanish/European regulations for the handling of animals in research. All protocols were approved by the Institutional Animal Care and Use Ethic Committees and implemented according to European regulations. Experiments were carried out in accordance with the principles of the 3Rs.
Embryos were obtained by mating of adult fish using standard methods.

Consent for publication
Not applicable.

Availability of data and materials
Most of the data generated or analyzed during this study are included in this published article [and its supplementary information files]. However, the imaging datasets used and/or analyzed in Figure 3-5 are available from the corresponding author on reasonable request.

Competing interests
The authors IB and CP declare no competing financial interests.    TABLE 3 Analysis of the phenotypes in gain-of-function experiments ( Figure 6).
Numbers indicate the of embryos displaying a phenotype as the one shown in Figure 6, over the total number of analyzed embryos (X/Y).     (Table 1 for values and statistical analysis).
Note the reduction in the number of atoh1a:GFP differentiated neurons in atoh1a fh282 embryos. ov, otic vesicle; r, rhombomere. Scale bars correspond to 50µm. embryos. Note that no differences between wild type and mutant embryos was observed ( Table 2 for  Note that cells from in wild types exit the LRL much earlier than in mutants. Since the atoh1a fh282 mutant allele only caused a deleterious phenotype in homozygosity, wild type and heterozygous conditions showed identical phenotypes and they were displayed as single wild type condition. nt; neural tube lumen; ov, otic vesicle. Scale bars correspond to 50µm. ns, non-statistically significant; *** p<0.001.  Table 3 for numbers of analyzed embryos. r, rhombomere. Scale bars correspond to 50µm.