Cibelli, J. | Search bioRxiv

bioRxiv2026

Uncovering zebrafish embryonic proteome dynamics across 16 time points during the first 24 hours of development

Defining how proteins change over developmental time is amenable to studies deciphering regulatory genetic networks in vertebrate development, biology, and pharmacology. In an approach toward such quantitative studies of dynamic network behavior, we produced an atlas using the mass spectrometry-based method to investigate protein expression changes across 16 time points from the zygote to the early pharyngula stage zebrafish embryos. We systematically summarize 8 clusters for interrogating changes in protein expression associated with the development of zebrafish embryos. Specifically, we identified a class of zinc finger-related transcription factors primarily located on the long arm of chromosome 4, which are highly expressed during zygotic genome activation. Furthermore, we highlight the power of this analysis to assign developmental stage-specific expression information to chromosomes and tissues. Time-resolved analyses reveal significant discordance between differential transcript and protein expression, whereas no time lag is observed for proteins involved in stable and fundamental biological processes, such as metabolism (e.g., Ppt2a and Gatm), cytoskeletal organization (e.g., Col18a1), and the translation machinery (e.g., Eif4enif1). This atlas offers high-resolution and in-depth molecular insights into zebrafish development, providing a resource for developmental biologists to generate hypotheses for functional analysis of proteins during early vertebrate embryogenesis. HighlightsO_LIA global protein expression database with high time resolution is created for zebrafish embryos. C_LIO_LIDistinct patterns of protein expression correlate with biological processes. C_LIO_LITranscription factors have a burst of expression from the gastrulation stage. C_LIO_LIDevelopmental stage-specific protein expressions were assigned to chromosomes and tissues. C_LIO_LIHigh-resolution embryonic transcriptome and proteome datasets were compared and connected. C_LI

developmental biology↗

bioRxiv2020

Neuromesodermal Progenitors Advance Network Formation of Spinal Neurons and Support Cells in Neural Ribbons In Vitro and Unprotected Survival in a Rat Subacute Contusion Model

Improved human stem cell interventions to treat CNS trauma requires continued expansion of in vitro models and delivery platforms to fill gaps in analysis and treatment. Transplanted neural stem cells (NSCs) face unique, multi-faceted challenges beyond survival that include differentiation, maturation, and integration into a complex cytokine-releasing microenvironment that impinges on a multipotent cell type. Alternate strategies to transplant neurons and neuronal networks deserve reevaluation, particularly since novel differentiation protocols mimicking region-specific developmental and positional cues have recently emerged. To investigate transplantation of neurons and their early networks, we generate in vitro neural ribbons containing spinal neurons and support cells anatomically matched for cervical spinal cord injury (SCI). These glutamate-responsive, electrically-active neural ribbons apply a new hiPSC differentiation strategy transiting through neuromesodermal progenitors (NMps) to derive developmentally relevant spinal motor neurons (SMNs), interneurons (INs), and oligodendrocyte progenitor cells (OPCs). Bioinformatic profiling validates region-specific identities. Neurons and neuronal networks are functionally evaluated for action potential firing, calcium signaling, population activity, and synaptogenesis. NMp-derived neurons survive in vivo within the subacute phase hemi-contusion injury cavity when delivered either as free suspension or as encapsulated networks of pre-formed CNS cytoarchitectures. Delivery as encapsulated networks further supports survival of lower cell numbers and rapid graft penetration into host tissue. Neural network ribbons therefore provide a novel intermediary approach between cell suspensions and complex organoids for investigating network formation and early transplantation events with hiPSC-derived neurons, providing flexibility to rapidly tune cell type(s), cell ratios, and traceable biomarkers. Significance StatementIn the two decades since human stem cell technologies have emerged, the challenge has remained to improve the developmentally relevant derivation of therapeutic cells. The ability to now generate anatomically matched neurons for SCI necessitates a re-evaluation of these cells and their networks in vitro and in vivo. In this study, we apply developmental cues via neuromesodermal progenitors to generate spinal neurons from hiPSCs. Genetic and functional evaluation of these cells as in vitro neuronal networks, due to their capacity to survive and graft effectively within the rat subacute contusion cavity, offer novel approaches for customizing SCI transplantation. This work demonstrates a strategy to develop transplantable, chemically-responsive networks linking in vitro models with injury customization towards improved in vivo outcomes.

neuroscience↗