The assembly of functional neural circuits requires the specification of neuronal identities and the execution of developmental programs that establish precise neural network wiring. The regional plan of the vertebrate Central Nervous System, and therefore its future functional specializations, is beautifully orchestrated by restricted expression of crucial genes during embryonic development. As development proceeds, neurogenesis leads to the formation of distinct neuronal groups that upon maturation they migrate to their final destination, form synaptic connections and integrate into functional circuits. Noteworthy, the generation of this brain cell diversity takes place during embryogenesis, and one of the main unsolved questions is how multiple cell types are generated and maintained in highly organized spatial patterns upon brain morphogenesis.
Our goal is to understand how spatiotemporally coordinated cell progenitor specification and differentiation occur during morphogenesis to construct a functional brain. While incorporating time as a missing-yet-crucial factor, we want to provide a holistic view of how cell fate decisions are taken in the brain in order to gain biological insight into: i) how tissue growth is intertwined with cell proliferation and differentiation; ii) how the heterogeneity of neural progenitors is generated; and iii) how individual progenitors behave during patterning and morphogenesis.
We focus on the embryonic brainstem, which controls life-sustaining functions and is extremely conserved in vertebrates, using cutting-edge complementary approaches such as: CRISPR-Cas9 genome editing, high-resolution 4D-imaging paired with cell tracking tools, and analyses of gene regulatory landscapes.
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RECONSTRUCTING CELL LINEAGES
Reconstructing the lineage relationships and dynamic event histories of individual cells within their native context is central to understanding how the wide diversity of cell types develops during the construction of an organ. This is a long-standing challenge in biology, because up to now most efforts have been devoted to understand the genetic requirements for cell specification, and the lack of availability of user-friendly tools to be paired with imaging in order to extract biological information from large imaging datasets.
Lately, we generated the complete lineage tree of the neurosensory elements of the inner ear and of specific hindbrain progenitor populations by high spatial and temporal resolution imaging. Our approach uniquely allows to quantitatively and simultaneously studying progenitor cells in their native/modified environment such as progeny number, location and differentiation status. We provide dynamic maps of progenitor pools in the whole organ context, and correlate the progenitor potentials to the temporal and spatial gene requirements [Dyballa et al, 2017].
DECIPHERING HOW CELL DIVERSITY IS GENERATED
The generation of brain cell diversity occurs during embryogenesis at the same time that the brain undergoes a dramatic transformation from a simple tubular structure, the neural tube, to a highly convoluted structure –the brain-. This brain morphogenesis results in changes in the position of neuronal progenitors and their derivatives upon time. One of the main unsolved questions is how multiple cell types are generated and maintained in highly organized spatial patterns upon brain morphogenesis. Thus, to better comprehend how cell diversity is generated, we need to understand: i) how the neurogenic/gliogenic capacity is allocated to specific territories, ii) how progenitor and differentiation capacities are balanced, and iii) how their relative spatial distribution changes upon brain morphogenesis. For this, we combine functional analysis with high-resolution in vivo imaging.
UNDERSTANDING THE ROLE OF MORPHOMECHANICS OF TISSUE SEGMENTATION ON CELL FATE
Our aim is to study the impact of morphomechanical changes on tissue subdivision and cell organization within the hindbrain. Actomyosin cables act downstream of EphA/Ephrin signaling in the segregation of rhombomeric cell populations to avoid cell intermingling between different neuronal types [Calzolari et al 2014]. More recently, we explored the cellular machinery responsible of the assembly of this cellular mechanical barrier and demonstrated that the capacity of boundaries to act as an elastic mesh for segregating rhombomeric cells evolved by cooption of critical genes to a novel regulatory block, refining the mechanisms for hindbrain segmentation [Letelier et al, 2018]. We unveiled the role of Yap/Taz-signaling as a mechanosensor and demonstrated the pivotal role of Yap/Taz-TEAD signaling in maintaining progenitor features in the hindbrain boundary cell population [Voltes et al, 2019].
DEVELOPING THE DIGITAL Z-HINDBRAIN
Through a combination of genetic and in vivo imaging techniques paired with image-analysis tools, we are building a zebrafish 3D-hindbrain map. We register experimental whole hindbrain images and map neuronal differentiation patterns with a unified spatial representation, using a confocal microscopy imaging setup and cross-platform segmentation and registration tools.
Our aim is to share an established protocol using open-access tools and algorithms that allow standardized and accessible tissue-wide cell population atlas construction. We hereby validate it through the creation of our own atlas - the digital Z-hindbrain atlas – in an effort to reveal the remodeling of the neuronal progenitor and neuronal cluster domains upon time [Blanc et al, unpub].