Resumen de Hindbrain boundaries: Addressing the crossroad between tissue segmentation and cell fate regulation
MOTIVATION: The architectural and functional complexity of the nervous system is without a shadow of a doubt of crucial importance for understanding biodiversity. The nervous system harbors the toolkit for biological roles encompassing from the regulation of basic physiological functions such as motor coordination or breathing to the scaffolding of sentience and self-awareness.
The generation of cell diversity is one of the keystones in developmental biology. This intellectual aspiration is inevitably bound to questions such as how cell fates are regulated, how cell behavior and cell fate are intertwined and how the spatiotemporal dynamics of morphogenesis conditions but also relies on the orchestration and regulation of progenitors and differentiated cells.
Developmental neurobiology aims at unveiling the nuts and bolts of the ontogeny of the nervous system. Moreover, this discipline, at its intersection with evolutionary biology, allows addressing how the genetic programs for the generation of neurophysiological and neuroanatomical plans are distributed in the phylogeny. Interestingly, the posterior brain, also known as rhombencephalon or hindbrain, is the most conserved brain region along evolution, with tissue segmentation as probably the most determinant event in the developmental story of this territory.
Splitting up the posterior brain into compartments during embryonic development highlights the relevance of the challenge of gradually increasing the refinement of gene expression patterns, which goes hand by hand with the establishment of cell lineage restriction boundaries. Importantly, and possibly due to its strategic location within tissues, compartment boundaries display several roles involved in the making of tissue and organ architecture.
My thesis work has combined classical developmental biology tools and recently developed methods on in vivo imaging and genome edition to depict an integrative view of the progenitor biology of hindbrain interhombomeric boundary cells. Thus, from the spatiotemporal characterization to the molecular regulation of boundary cell behavior and cell fate we provide important insights on the principles of boundary progenitor homeostasis. Importantly, this work addresses the interconnection between the tissue microenvironment and the downstream biochemical responses and cell behaviors. Furthermore, this thesis also contains novel results regarding hindbrain boundary cell lineage that set a framework for future mechanistic interrogations.
FRAMEWORK: Embryonic development is a crucial period in the life of a multicellular organism, during which limited sets of progenitors are responsible for producing all the cellular variability in the adult body. In this sense, complexity displays an increasing progression during ontogeny. At its dawn, vertebrates and higher invertebrates are a single cell that, by way of a highly dynamic developmental process, will result into an entirely functional multicellular organism. Tens of thousands to trillions of cells comprise the resulting organism, and these cellular building blocks are arranged in specialized tissues and organs able to perform highly elaborate tasks. Thus, the timely delimitation of specific territories and their arrangement in particular shapes become defining features governing embryo development (Meinhardt, 2009; Lander, 2011).
Understanding the mechanics of segmentation is central to determining the link between biophysical creation of form and structure and the mechanotransduction consequences at molecular and cellular scales. One overarching question has to do with the interplay between actomyosin assembly, tissue tension, cell behavior and cell fate decisions. In other words, how tissue segmentation and morphogenesis impacts on cell position, and therefore on cell identity, cell survival and proliferative capacity? In addition, tissue segmentation generates compartment boundaries that also act as signaling centers. Thus, compartment boundaries can play a role in dictating spatiotemporal coordinates for cell specification in adjacent territories. In this regard, formation and maintenance of boundaries are key events in the final architectural output of a tissue. The depiction of the spatial and temporal profile of boundary-dependent pattern formation and the eventual consequences in terms of cell population specification and distribution awaits further insight. Finally, a primary ambition in the field seeks the integration between local behaviors and global properties. In line with this, the questions as to how the dynamism of morphogenetic events is in register with the developmental history of cell lineages and how these processes reciprocally affect each other posit a thrilling quest for developmental biologists.
Tissue segmentation results in the subdivision of the embryo into spatially segregated compartments confined by boundaries. Both compartments and boundaries are built up by cells and, noteworthy, understanding the complex dynamics of the formation of embryonic compartments, tissues, organs and even entire organisms as a function of the substratal cell behavior is a central goal of developmental biology (Keller et al., 2008; Khairy and Keller, 2011; Amat and Keller, 2013). Cell lineages, namely the developmental history of positions, movements and divisions of cells, become of crucial importance when it comes to depicting a systematic characterization of functional relationships during embryogenesis and to provide key insights into the quantitative rules underlying developmental building plans (Amat and Keller, 2013).
The hindbrain offers an evolutionary conserved scenario with dynamic temporal and spatial distribution of cells during morphogenesis. Hindbrain boundaries, specified at the interface between adjacent rhombomeres, are cell populations endowed with unique characteristics when compared to the rest of hindbrain cells. On the one side, zebrafish hindbrain boundaries are kept undifferentiated at early stages when most hindbrain progenitors are engaged into neurogenic and gliogenic programs (Esain et al., 2010; Gonzalez-Quevedo et al., 2010). On the other side, it is important to note the characteristic mechanical juncture in the BCP. Hindbrain boundary cells display specific triangular cell shape (Gutzman and Sive, 2010) and they contain an apically located cable-like cytoskeletal arrangement involved in avoiding cell intermingling between cells from different compartments, being the forces generated by mitotic rounding in the boundary vicinities the main challenge to cell-lineage restriction (Calzolari, Terriente and Pujades, 2014; Letelier et al., 2018). Thus, BCP biology and its tentacular mechanical framework establish a mindset in which hypothesizing a potential relationship between progenitor biology and tissue microenvironment was tempting at the very least.
Considering the mechanical microenvironment in the BCP and its identity specificities, we propose YAP/TAZ-TEAD activity as the molecular scaffold that underpins the crossroad between hindbrain segmentation and proliferative capacity modulation. In this work we show that mechanical stimuli in the BCP trigger YAP/TAZ-TEAD activity (Voltes et al., 2018). In turn, this activity is responsible for transiently modulating the proliferative capacity of boundary cells, which eventually differentiate into neurons (Voltes et al., 2018).
CONCLUSIONS: [1] The hindbrain boundary cell population occupies two cell rows along the AP axis at rhombomeric interfaces. Boundary cells display rhombomeric markers; however, each cell row displays different markers corresponding to each of the two contacting compartments.
[2] In zebrafish, hindbrain boundary cells do proliferate and, contrary to other systems such as chick or mouse, no specific spatial distribution of proliferation capacity is detected.
[3] A hindbrain boundary cell subpopulation of Sox2-positive progenitors displays YAP/TAZ-TEAD activity from around 22 hpf up to 48 hpf.
[4] Both YAP and TAZ independently contribute to TEAD-dependent activity.
[5] Mechanical microenvironment is responsible for triggering YAP/TAZ-TEAD activity in hindbrain boundaries; however, it is not necessary for the maintenance of the activity.
[6] YAP/TAZ-TEAD-active hindbrain boundary cells do proliferate up to around 40-48 hpf, coinciding with YAP/TAZ-TEAD activity shutdown. At that time, there is a behavioral switch and proliferative capacity is lost in this cell population.
[7] YAP/TAZ-TEAD-active progenitors produce neuronal derivatives that allocate in the boundary mantle zone and express neuronal differentiation markers.
[8] YAP and TAZ control proliferative behavior but not cell survival in hindbrain boundary cells.
BIBLIOGRAPHY: Meinhardt, H. (2009) ‘Models for the generation and interpretation of gradients.’, Cold Spring Harbor perspectives in biology, 1(4), p. a001362. doi: 10.1101/cshperspect.a001362.
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Khairy, K. and Keller, P. J. (2011) ‘Reconstructing embryonic development’, Genesis, 49(7), pp. 488–513. doi: 10.1002/dvg.20698.
Amat, F. and Keller, P. J. (2013) ‘Towards comprehensive cell lineage reconstructions in complex organisms using light-sheet microscopy’, Development Growth and Differentiation, 55(4), pp. 563–578. doi: 10.1111/dgd.12063.
Esain, V. et al. (2010) ‘FGF-receptor signalling controls neural cell diversity in the zebrafish hindbrain by regulating olig2 and sox9’, Development, 137(1), pp. 33–42. doi: 10.1242/dev.038026.
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Calzolari, S., Terriente, J. and Pujades, C. (2014) ‘Cell segregation in the vertebrate hindbrain relies on actomyosin cables located at the interhombomeric boundaries’, EMBO Journal, 33(7), pp. 686–701. doi: 10.1002/embj.201386003.
Letelier, J. et al. (2018) ‘Evolutionary emergence of the rac3b/rfng/sgca regulatory cluster refined mechanisms for hindbrain boundaries formation’, Proceedings of the National Academy of Sciences, 115(16), p. E3731 LP-E3740.
Voltes, A. et al. (2018) ‘YAP/TAZ-TEAD activity links mechanical cues to specific cell fate within hindbrain boundaries’, bioRxiv 366351; doi: https://doi.org/10.1101/366351
