Resumen de Unveiling the molecular and behavioral properties of hindbrain rhombomere centers
One of the fundamental questions in developmental biology is how from a unicellular zygote a multicellular and complex organism arises. One of the testimonies of this complexity is the Central Nervous System (CNS) which consists of the brain and the spinal cord. The primordium of the embryonic brain is the neural tube, which in order to transition towards an adult structure and generate a functional brain, it has to first undergo a phase of expansion, in which neural progenitors undergo symmetric divisions to expand the pool, to later on engage into de generation of specific lineages, first neuronal and later glial types by asymmetric or symmetric proliferation. This illustrates how progenitor’s potentiality to generate diverse fates gets restricted over time (1–3).
How does the brain organize and combine different potentialities in an extremely dynamic and complex tissue in an equipotent field of neural progenitors? To answer this question, we focus on the study of the embryonic hindbrain or rhombencephalon as a model system, since it is one of the most conserved embryonic brain structures in vertebrates. The hindbrain is transiently segmented into seven metamers or rhombomeres which constitute units of gene expression and lineage compartments (4). In the rhombencephalon, the precise regulation of neurogenesis is achieved by differentially localizing the neurogenic capacity along the territory and over time. At early stages, proneural gene expression restricts to boundary adjacent regions, being absent in both rhombomere centers and at the boundaries themselves (5,6). Thus, indicating the neurogenic activity mainly takes place in adjacent domains. Later in development, boundary cells change their molecular profile to eventually contribute to the neuronal differentiation domain as well (7,8). However, little is known about the molecular and cell behavior characteristics of the population at the center of the rhombomeres (6,9,10).
In order to answer how and why the rhombencephalon organizes its territory by differentially distributing neurogenesis over time and space, in this work, we have focused on this poorly explored population in rhombomere centers. We have performed a spatiotemporal molecular analysis and a cellular behavioral profile by a clonal analysis in vivo. The molecular study demonstrates that these cells are neural progenitors, not committed to the neuronal lineage. We have also shown that these cells acquire radial glia cells characteristics (11) in a sequential manner as development proceeds. 4D images acquired in vivo by clonal analysis have shown for the first time that these cells have proliferative capacity and that they can eventually give rise to neurons by asymmetric division. Yet, we also observed that most cells do not divide, remaining as progenitors. This suggests that, although these cells have the competence to generate neurons, this is not their main engagement, but rather keep in a non-committed progenitor state. By interrogating potential cellular mechanisms that could be regulating the behavior of these cells, we studied the Notch signaling pathway, which is involved in the acquisition of different cell fates, as well as in cell cycle control. By means of loss-of-function experiments we have demonstrated that Notch3 signaling is necessary to maintain these cells as progenitors since, upon loss of Notch3, cells in the center of the rhombomeres undergo differentiation, supporting our previous results regarding their potentiality. Notch3 signaling has been demonstrated in other systems upstream of the transcription factor hey1 (12,13). Our characterization of hey1 expression showed that, in the hindbrain, it is specifically expressed in the center of rhombomeres. However, functional hey1 experiments did not show any effect on the fate of these cells nor in their maintenance as progenitors, which opens several possibilities to explore in the immediate future about the role of this transcription factor in the control of the proliferative capacity of rhombomere center cells instead (13,14).
In this doctoral thesis, we propose that one of the possibilities that could explain the spatiotemporal distribution of the neurogenic activity in the hindbrain is the regionalization of capacities in this structure, meaning the center of the rhombomeres could harbor the gliogenic activity. A hypothesis that is not only supported by our molecular and cel behavioral studies, but that has also been raised by few studies in the past (6,9,10).
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