Resumen de Uncovering the interplay between cell fate specification and progenitor dynamics during the development of the lower rhombic lip
A single fertilized egg holds the potential to generate the vast array of cellular phenotypes that populate an adult multicellular organism. Besides, in order to generate function, the embryo not only faces the challenge of specializing functional cells, but also to coordinate growth with the generation of shapes that will ultimately generate organs. In short, we can say that embryonic development is the most important period in the life of any animal.
The adult rhomboencephalon or hindbrain, which is the most posterior part of the brain, serves as the perfect example of such developmental challenge. It houses the neuronal circuits implicated in key autonomous processes, such as heartbeat, respiration and wakefulness cycles. Moreover, it also serves as a relay station for sensory information that allows the fine tuning of motor coordination. Each one of the circuits aforementioned are constituted by neurons that bear unique characteristics in terms of morphology and gene expression, being specialized for the function they are meant to fulfil. After characterizing the function of the neuronal circuits that populate the adult hindbrain, we keep stumbling upon the same fundamental question: how are such unique and diverse neuronal identities generated during embryogenesis? In this work, we wanted to elucidate the genetic players behind the generation of diverse neuronal lineages within the hindbrain. In the past decades, many efforts have been devoted at understanding the molecular and genetic codes that confer specific identities to progenitor cells within the embryo, restricting the potential of said progenitors and instructing cell fate choices. Thanks to all the work that came before us, most of such molecular mechanisms are well defined today. However, there is still a gap between our knowledge on the gene regulatory networks active during neurogenesis and the dynamic cellular events that progenitor cells undergo during the process of differentiation. Once a progenitor cell has been recruited to a given progenitor population through patterning cues, how does this progenitor domain cope with cellular proliferation, differentiation and apoptosis? How do the dynamics of progenitor cells shape the neural tissue during neurogenic stages? And, most importantly, how are these processes coordinated with the changing neural tube architecture upon morphogenesis? To shed some light into these questions, it was of special interest to us to investigate the proneural gene code instructing neurogenesis within the very dynamic context that is the developing hindbrain. In this regard, a particular hindbrain lineage caught our attention: the Lower Rhombic Lip (LRL), which presented the challenge of being a neurogenically active progenitor population at the time of extensive hindbrain morphogenesis. Thus, we thought of LRL-progenitors as the suitable cellular candidates in which to study proneural function along with cellular dynamics. However, in order to grasp the dynamism that is so characteristic to neural progenitors at the onset of hindbrain neurogenesis, we needed to quantify cellular kinetics of proliferation and differentiation. It was in this thought process that it became evident how important in vivo imaging was for the fulfilment of these work and for modern developmental neurobiology in general. Hence, the zebrafish embryo felt as the more natural model in which to perform our research.
In all, the work presented in this thesis combines the use of classical developmental biology approaches as well as more advanced imaging techniques. Such procedures allowed us to identify different modes of clonal growth according to position of progenitor cells, an early exploratory work that already revealed that the LRL behaved in different ways than the rest of hindbrain progenitors. Moreover, we inform of the sub-functionalization of the atoh1 genes driving neurogenesis within the LRL, which define different progenitor states within the neurogenic process. Lastly, we provide information on the different modes of division that LRL-progenitors undergo, as well as the location preferences of LRL-derived neurons within the differentiation domain of the developing hindbrain.
The Lower Rhombic Lip (LRL) is a transient neuroepithelial structure of dorsal hindbrain that gives rise to deep brainstem nuclei like the vestibular, auditory and precerebellar nuclei. In this work, we have followed the LRL-progenitor cell population through early steps of neurogenesis and hindbrain morphogenesis to understand proneural function and progenitor dynamics during neuronal specification. We provide information about the atoh1 gene regulatory network operating in the specification of LRL cells, and the kinetics of cell proliferation and behaviour of atoh1a-derivatives by using functional and in vivo imaging strategies in the zebrafish embryo.
We propose that atoh1a and atoh1b have subfunctionalized: atoh1a acts as the fate selector gene in LRL-progenitors, whereas atoh1b acts as the downstream neuronal differentiation gene carrying out the neurogenic program. Moreover, our in vivo cell lineage approaches revealed a regionalization of modes of division within the LRL, orchestrating the balance between neuronal differentiation and progenitor cell self-renewal.
