The vertebrate heart develops from several progenitor lineages. After early-differentiating first heart field (FHF) progenitors form the linear heart tube, late-differentiating second heart field (SHF) progenitors extend atrium, ventricle, and form the inflow and outflow tracts (IFT/OFT). However, the position and migration of late-differentiating progenitors during heart formation remains unclear. Here, we tracked zebrafish heart development using transgenics based on the cardiopharyngeal transcription factor gene tbx1. Live-imaging uncovered a tbx1 reporter-expressing cell sheath that from anterior lateral plate mesoderm continuously disseminates towards the forming heart tube. High-speed imaging and optogenetic lineage tracing corroborated that the zebrafish ventricle forms through continuous addition from the undifferentiated progenitor sheath followed by late-phase accrual of the bulbus arteriosus (BA). FGF inhibition during sheath migration reduced ventricle size and abolished BA formation, refining the window of FGF action during OFT formation. Our findings consolidate previous end-point analyses and establish zebrafish ventricle formation as a continuous process.

Hematopoietic stem cells (HSCs) are rare cells that arise in the embryo and sustain adult hematopoiesis. Although the functional potential of nascent HSCs is detectable by transplantation, their native contribution during development is unknown, in part due to the overlapping genesis and marker gene expression with other embryonic blood progenitors. Using single-cell transcriptomics, we define gene signatures that distinguish nascent HSCs from embryonic blood progenitors. Applying a lineage-tracing approach to selectively track HSC output in situ, we find significantly delayed lymphomyeloid contribution. An inducible HSC injury model demonstrates a negligible impact on larval lymphomyelopoiesis following HSC depletion. HSCs are not merely dormant at this developmental stage, as they showed robust regeneration after injury. Combined, our findings illuminate that nascent HSCs self-renew but display differentiation latency, while HSC-independent embryonic progenitors sustain developmental hematopoiesis. Understanding these differences could improve de novo generation and expansion of functional HSCs.

Transgenic animals are invaluable for modeling cancer genomics, but often require complex crosses of multiple germline alleles to obtain the desired combinations.Zebrafish models have advantages in that transgenes can be rapidly tested by mosaic expression, but these typically lack spatial and temporal control of tumor onset, which limits their utility for the study of tumor progression and metastasis. To overcome these limitations, we have developed a method called Transgene Electroporation in Adult Zebrafish (TEAZ). TEAZ can deliver DNA constructs with promoter elements of interest 2 to drive fluorophores, oncogenes, or CRISPR-Cas9-based mutagenic cassettes in specific cell types. Using TEAZ, we created a highly aggressive melanoma model by expression of BRAF V600E in spatially constrained melanocytes in the context of p53 deficiency and Cas9-mediated inactivation of Rb1. Unlike prior models that take ~4 months to develop, we found that TEAZ leads to tumor onset in ~7 weeks and these develop in fully immunocompetent animals. As the resulting tumors initiated at highly defined locations, we could track their progression via fluorescence and documented deep invasion into tissues and metastatic deposits. TEAZ can be deployed to other tissues and cell types such as the heart with the use of suitable transgenic promoters.The versatility of TEAZ makes it widely accessible for rapid modeling of somatic gene alterations and cancer progression at a scale not achievable in other in vivo systems.

The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration, and their dysregulation can result in congenital anomalies of the viscera, ventral wall defects, and mesothelioma tumors. Nonetheless, the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here, we combine genetic lineage tracing, in toto live imaging, and single-cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm (LPM). Our single-cell analysis uncovers a post-gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos, we chart the origin of mesothelial progenitors to the lateral-most, hand2-expressing LPM and confirm evolutionary conservation in mouse. Our time-lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation, documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally, hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium, we find de novo expression of LPM-associated transcription factors, and in particular of Hand2, indicating the re-initiation of a developmental transcriptional program in mesothelioma. Taken together, our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2, contributing to our understanding of mesothelial pathologies and mesothelioma.

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