Biology subjects

Garcia-Castro, H.

Publications and source records attributed to Garcia-Castro, H..

7 recordsLinked to original sources

bioRxiv2026

A sex-specific regulator expands the embryonic ocular field to generate a novel visual system

Morphological novelties often emerge through the redeployment of conserved developmental programs, but how such programs become activated in new developmental contexts remains poorly understood. We investigated the evolutionary origin of the turbanate eyes (TurbEyes) of mayflies, a male-specific additional visual system. Using single-cell transcriptomics, chromatin accessibility profiling and functional genetics, we show that this novelty develops through deployment of the ancestral retinal determination gene network (RDGN). We identify a novel, male-specific paralog of the sex determination transcription factor Doublesex, DsxM, that regulates the eye-specification gene eyeless/Pax6 and promotes the expansion of the embryonic ocular field. Functional assays impairing dsxM activity reduce this male-specific expansion and causes defects in TurbEye development. Our findings reveal a developmental mechanism by which a sex-specific regulator generates a new developmental territory, enabling the spatial redeployment of a conserved developmental program and to generate a novel male-specific visual system.

evolutionary biology↗

bioRxiv2024

The Regulatory Logic of Planarian Stem Cell Differentiation

Cell type identity is determined by gene regulatory networks (GRNs), comprising the expression of specific transcription factors (TFs) regulating target genes (TGs) via binding to open chromatin regions (OCRs). The regulatory logic of differentiation includes factors specific to one or multiple cell types, functioning in a combinatorial fashion. Classic approaches of GRN discovery used perturbational data to elucidate TF-TG links, but are laborious and not scalable across the tree of life. Single cell transcriptomics has emerged as a revolutionary approach to study gene expression with cell type resolution, but incorporating perturbational data is challenging. Planarians, with their pluripotent neoblast stem cells continuously giving rise to all cell types, offer an ideal model to attempt this integration. Despite extensive single cell transcriptomic studies, the transcriptional and chromatin regulation at the cell type level remains unexplored. Here, we investigate the regulatory logic of planarian stem cell differentiation by obtaining an organism-level integration of single cell transcriptomics and single cell accessibility data. We identify specific open chromatin profiles for major differentiated cell types and analyse their transcriptomic landscape, revealing distinct gene modules expressed in individual types and combinations of them. Integrated analysis unveils gene networks reflecting known TF interactions in each type and identifies TFs potentially driving differentiation across multiple cell types. To validate our predictions, we combined TF knockdown RNAi experiments with single cell transcriptomics. We focus on hnf4, a TF known to be expressed in gut phagocytes, and confirm its influence on other types, including parenchymal cells. Our results demonstrate high overlap between predicted targets and experimentally-validated differentially-regulated genes. Overall, our study integrates TFs, TGs and OCRs to reveal the regulatory logic of planarian stem cell differentiation, showcasing that the combination of single cell methods and perturbational studies will be key for characterising GRNs widely.

systems biology↗

bioRxiv2024

The Hydractinia cell atlas reveals cellular and molecular principles of cnidarian coloniality

Coloniality is a widespread growth form in cnidarians, tunicates, and bryozoans, among others. Despite being modular, composed of multiple zooids and supporting tissues, colonies function as a single physiological unit. A major question in the biology of colonies is the cellular mechanism of generating structurally and functionally distinct colony parts. The cnidarian Hydractinia establishes colonies with different types of zooids (polyps), interconnected by a gastrovascular system that is attached to the substrate and known as stolons. We obtained single cell transcriptomic profiles of [~]200K Hydractinia cells, including isolated stolons and two polyp types. We characterised the major Hydractinia cell types and quantified their abundance across colony parts. Overall, we find that distinct colony parts are characterised primarily by distinct combinations of shared cell types and to a lesser extent by part-specific cell types. Therefore, we propose that both cell type combinations, as well as rarer cell type innovations, have been the main mechanism in the evolution of coloniality in cnidarians. We identified cell type-specific transcription factors (TFs) and gene networks expressed within these cell types. Notably, we discovered a previously unidentified, stolon-specific cell type, which expresses enzymes related to biomineralization and chitin synthesis, reminiscent of molluscan shell matrix proteins that may represent a crucial adaptation to the animals habitat. In summary, the Hydractinia cell atlas elucidates the fundamental cellular and molecular mechanisms underlying coloniality.

systems biology↗

bioRxiv2024

Multiplex single-cell analysis of serotonergic neuron function in planarians reveals widespread effects in diverse cell types

Neurons function by interacting with each other and with other cell types, often exerting organism-wide regulation. Serotonergic neurons play a systemic role in processes such as appetite, sleep and motor control. Functional studies in the planarian Schmidtea mediterranea have shown that impairment of serotonergic neurons results in systemic effects. Studying neurons and the tissues they interact with is challenging using either bulk or single-cell analysis techniques. While bulk methods merge the information from all cell types, single-cell methods show promise in overcoming this limitation. However, current single-cell approaches encounter other challenges including stress of cell dissociation, high cost, multiplexing capacity, batch effects, replication and statistical analysis. Here we used ACME and SPLiT-seq to generate a multiplex single-cell analysis of serotonergic neuron function in planarians by inhibiting pitx and lhx1/5-1, two transcription factors expressed in them. We recovered single-cell transcriptomic profiles of 47,292 cells from knockdown and control animals, including biological and technical replicates. Our results show that epidermal, muscular and the recently described parenchymal cell types are affected the most by serotonergic neuron impairment. By computationally dissecting each cell type, we elucidated gene expression changes in each, including changes in epidermis cilia genes and myofiber genes in muscle. Interestingly, parenchymal cells downregulate genes involved in neurotransmitter recycling, suggesting a glial-like function of these recently described enigmatic cell types. Our results will allow disentangling the complexity of serotonergic neuron inhibition by studying the downstream effectors and the affected tissues, and offer new data on the function of parenchymal cells in planarians. Ultimately, our results pave the way for dissecting complex phenotypes through multiplex single-cell transcriptomics.

systems biology↗

bioRxiv2023

Annelid adult cell type diversity and their pluripotent cellular origins

Annelids are a broadly distributed, highly diverse, economically and environmentally important group of animals. Most species can regenerate missing body parts, and many are able to reproduce asexually. Therefore, many annelids can generate all adult cell types in adult stages. However, the putative adult stem cell populations involved in these processes, as well as the diversity of adult cell types generated by them, are still unknown. Here, we recover 75,218 single cell transcriptomes of Pristina leidyi, a highly regenerative and asexually-reproducing freshwater annelid. We characterise all major annelid adult cell types, and validate many of our observations by HCR in situ hybridisation. Our results uncover complex patterns of regionally expressed genes in the annelid gut, as well as neuronal, muscle and epidermal specific genes. We also characterise annelid-specific cell types such as the chaetal sacs and globin+ cells, and novel cell types of enigmatic affinity, including a vigilin+ cell type, a lumbrokinase+ cell type, and a diverse set of metabolic cells. Moreover, we characterise transcription factors and gene networks that are expressed specifically in these populations. Finally, we uncover a broadly abundant cluster of putative stem cells with a pluripotent signature. This population expresses well-known stem cell markers such as vasa, piwi and nanos homologues, but also shows heterogeneous expression of differentiated cell markers and their transcription factors. In these piwi+ cells, we also find conserved expression of pluripotency regulators, including multiple chromatin remodelling and epigenetic factors. Finally, lineage reconstruction analyses reveal the existence of differentiation trajectories from piwi+ cells to diverse adult types. Our data reveal the cell type diversity of adult annelids for the first time and serve as a resource for studying annelid cell types and their evolution. On the other hand, our characterisation of a piwi+ cell population with a pluripotent stem cell signature will serve as a platform for the study of annelid stem cells and their role in regeneration.

genomics↗

bioRxiv2022

An atlas of spider development at single-cell resolution provides new insights into arthropod embryogenesis

Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis, particular studies using the common house spider Parasteatoda tepidariorum, has made important contributions to understanding the evolution of animal development, including axis formation, segmentation, and patterning. However, we lack knowledge about the cells that build spider embryos, their gene expression profiles and fate. Single-cell transcriptomic analyses have been revolutionary in describing these complex landscapes of cellular genetics in a range of animals. Therefore, we carried out single-cell RNA sequencing of P. tepidariorum embryos at stages 7, 8 and 9, which encompass the establishment and patterning of the body plan, and initial differentiation of many tissues and organs. We identified 20 cell clusters, from 18.5k cells, which were marked by many developmental toolkit genes, as well as a plethora of genes not previously investigated. There were differences in the cell cycle transcriptional signatures, suggestive of different proliferation dynamics, which related to distinctions between endodermal and some mesodermal clusters, compared with ectodermal clusters. We found many Hox genes were markers of cell clusters, and Hox gene ohnologs often were present in different clusters. This provided additional evidence of sub- and/or neo-functionalisation of these important developmental genes after the whole genome duplication in the arachnopulmonate ancestor (spiders, scorpions, and allies). We also examined the spatial expression of marker genes for each cluster to generate a comprehensive cell atlas of these embryonic stages. This revealed new insights into the cellular basis and genetic regulation of head patterning, hematopoiesis, limb development, gut development, and posterior segmentation. This atlas will serve as a platform for future analysis of spider cell specification and fate, and studying the evolution of these processes among animals at cellular resolution.

developmental biology↗

bioRxiv2020

ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics

Single-cell sequencing technologies are revolutionizing biology, but are limited by the need to dissociate fresh samples that can only be fixed at later stages. We present ACME (ACetic-MEthanol) dissociation, a cell dissociation approach that fixes cells as they are being dissociated. ACME-dissociated cells have high RNA integrity, can be cryopreserved multiple times, can be sorted by Fluorescence-Activated Cell Sorting (FACS) and are permeable, enabling combinatorial single-cell transcriptomic approaches. As a proof of principle, we have performed SPLiT-seq with ACME cells to obtain around ∼34K single cell transcriptomes from two planarian species and identified all previously described cell types in similar proportions. ACME is based on affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of cell types across the tree of life.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗