Biology subjects

Pujades, C.

Publications and source records attributed to Pujades, C..

8 recordsLinked to original sources

bioRxiv2025

Multimodal cell lineage reconstruction in the hindbrain reveals a link between progenitor origin and activity patterning

Does the stem cell origin impact how daughter neurons acquire functional characteristics and assemble into circuits? Here, by multimodal cell lineage reconstruction in the zebrafish hindbrain we related a neurons embryonic origin to its future terminal differentiation features, such as neurotransmitter identity, and neuronal activity pattern. Intersectional lineage tracing, new developed computational tools, and genetic knockouts revealed that different progenitors formed functionally distinct neuron subtypes and could not compensate for the loss of adjacent progenitor pools, indicating developmental hardwiring. Dynamics of neuronal production suggest that progenitor competence changes over time. Whereas neurog1-expressing progenitors contributed to both glutamatergic and GABAergic lineages at early embryonic stages, later, other progenitor pools also assumed this role. Whole-hindbrain 3D atlases combining calcium imaging to monitor spontaneous neuronal activity, with genetic perturbations and progenitor origin information, unveiled that the emergence of neuronal activity patterns was presaged by their progenitor origins. This reveals a link between cell ontogeny and neuronal activity in the zebrafish hindbrain.

developmental biology↗

bioRxiv2024

Her9 controls the stemness properties of the hindbrain boundary cells

Different spatiotemporal distribution of progenitor/neurogenic capacities permits that brain regions engage asynchronously in neurogenesis. In the hindbrain, rhombomere progenitor cells are the main contributors to neurons during the first neurogenic phase, whereas boundary cells participate later, relying on Notch3-activity. To analyze the mechanism(s) that maintain boundary cells as proliferative progenitors not engaging in neurogenesis, we addressed the role of the zebrafish Hes1 ortholog, Her9, in this cell population. her9 expression is temporarily sustained in boundary cells in a Notch-independent manner while they behave as non-neurogenic progenitors. Functional manipulations demonstrate that Her9 inhibits the onset of Notch-signaling and the neurogenic program, thus keeping boundary cells in the progenitor state. Combining multicolor clonal analysis with functional approaches, we reveal a role of Her9 in the expansion of boundary progenitors by promoting symmetric proliferative divisions and preventing neurogenic cell divisions. Moreover, Her9 regulates the proliferation of boundary cells by inhibiting the cell cycle gene cdkn1ca and potentially interplaying with CyclinD1. Altogether, Her9 maintains the stemness and proliferation of hindbrain boundary progenitors at early embryonic stages.

developmental biology↗

bioRxiv2023

Hindbrain rhombomere centers harbor a heterogenous population of dividing progenitors which rely on Notch-signaling

Tissue growth and morphogenesis are interrelated processes, whose tight coordination is essential for the production of different cell fates and the timely precise allocation of stem cell capacities. The zebrafish embryonic brainstem, the hindbrain, exemplifies such coupling between spatiotemporal cell diversity acquisition and tissue growth, as the neurogenic commitment is differentially distributed over time. Here, we combined cell lineage and in vivo imaging approaches to reveal the emergence of different specific cell population properties within the very same rhombomeres, and focused on the hindbrain rhombomere centers. We studied the molecular identity of rhombomere centers, and showed that they harbor different progenitor capacities that change over time. By clonal analysis, we revealed that cells within the rhombomeres decrease the proliferative capacity over time to remain mainly in G1-phase. Proliferating progenitors give rise to neurons by asymmetric and symmetric neurogenic divisions, while maintaining the pool of progenitors. The proliferative capacity of these cells differs from their neighbors, and they are delayed in the onset of Notch-activity. By functional studies we demonstrated that they rely on Notch3-signaling to be maintained as non-committed progenitors. In this study we show that cells in rhombomere centers might share steps of a similar program, despite the neurogenic asynchrony from the rhombomere counterparts, to ensure proper tissue growth.

developmental biology↗

bioRxiv2021

TEMPO: A system to sequentially label and genetically manipulate vertebrate cell lineages

During development, regulatory factors appear in a precise order to determine cell fates over time. To investigate complex tissue development, one should not just label cell lineages but further visualize and manipulate cells with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labelling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation/inactivation of reporters/effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification. One-Sentence SummaryGaining sequential genetic access to vertebrate cell lineages.

developmental biology↗

bioRxiv2021

The neurogenic fate of the hindbrain boundaries: a Notch-dependent behavioral switch triggers asymmetric division of boundary stem cells

The generation of cell diversity in the central nervous system occurs during embryogenesis and requires a precise balance between cell proliferation, commitment to specific fates, and further neuronal differentiation. The cellular and molecular mechanisms regulating this balance in the embryonic brain are still poorly understood. Here we study how the neurogenic capacity in the embryonic hindbrain is spatiotemporally allocated, and how the fate and the growth of the hindbrain boundary cells are regulated. By generating a CRISPR-based knock-in zebrafish transgenic line to specifically label the hindbrain boundaries, we unveiled that boundary cells undergo a functional transition to become neurogenic during hindbrain segmentation concurrently as they maintain the progenitor cell pool. Boundary cells engaged in neurogenesis coinciding with the onset of Notch signaling, which triggered their asymmetrical cell division. Our findings reveal that distinct neurogenic phases take place during hindbrain growth and suggest that boundary cells contribute to refine the final number, identity, and proportion of neurons in the brain. SUMMARYHindbrain boundary cells undergo a functional transition to become neurogenic concurrently as they are maintained as a progenitor cell pool. This involves a behavioral switch from symmetrically dividing progenitor cells to asymmetrically dividing progenitors, which depends on Notch-activity.

developmental biology↗

bioRxiv2021

The 3D-atlas builder: a dynamic and expandable 3D-tool for monitoring the changes in the neuronal differentiation domain during hindbrain morphogenesis

Reconstruction of prototypic three-dimensional (3D) atlases at the scale of whole tissues or organs requires specific methods to be developed. We have established a digital 3D-atlas maker (DAMAKER) and built a digital 3D-temporal atlas to monitor the changes in the growth of the tissue and neuronal differentiation domain in the zebrafish hindbrain. DAMAKER integrates spatial and temporal data from cell populations, neuronal differentiation and brain morphogenesis, through in vivo imaging techniques paired with image analyses and segmentation tools. First, we generated a 3D-reference from several imaged hindbrains and segmented them using a trainable tool; these were aligned using rigid registration, revealing distribution of neuronal differentiation growth patterns along the axes. Second, we quantified the dynamic growth of the neuronal differentiation domain vs. the progenitor domain, and by in vivo neuronal birthdating experiments we generated a digital 3D-temporal map of the neuronal growth in the whole hindbrain, revealing the spatiotemporal dynamics of neuronal differentiation upon morphogenesis. Last, we applied it to glutamatergic and GABAergic neurons, as proof-of-concept that the digital 3D-temporal map could be used as a proxy to infer neuronal birthdate. As this protocol uses open-access tools and algorithms, it can be shared for standardized, accessible, tissue-wide cell population atlas construction.

developmental biology↗

bioRxiv2019

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

BACKGROUNDThe 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.\n\nRESULTSIn 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.\n\nCONCLUSIONSOur 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.

developmental biology↗

bioRxiv2018

YAP/TAZ-TEAD Activity Links Mechanical Cues To Specific Cell Fate Within The Hindbrain Boundaries

Cells perceive their microenvironment through chemical and physical cues. However, how mechanical signals are interpreted during embryonic tissue deformation resulting in specific cell behaviors is largely unexplored. The Yap/Taz family of transcriptional co-activators has emerged as an important regulator of tissue growth and regeneration, responding to physical cues from the extracellular matrix, cell shape and actomyosin cytoskeleton. In this work, we unveiled the role of Yap/Taz-TEAD activity as sensor of mechanical signals in the regulation of the progenitor behavior of boundary cells during hindbrain compartmentalization. Monitoring in vivo Yap/Taz-activity during hindbrain segmentation we discovered that boundary cells respond to mechanical cues in a cell-autonomous manner through Yap/Taz-TEAD activity. Cell-lineage analysis revealed that Yap/Taz-TEAD boundary cells decrease their proliferative activity when Yap/Taz-TEAD ceased, preceding changes of cell fate: from proliferating progenitors to differentiated neurons. Functional experiments demonstrated the pivotal role of Yap/Taz-TEAD signaling in maintaining the progenitor features in the hindbrain boundary cell population.

developmental biology↗