Lineage labeling with zebrafish Cre and CreERT2 recombinase CRISPR knock-ins.

Background: The ability to generate endogenous Cre recombinase drivers using CRISPR-Cas9 knock-in technology allows lineage tracing, cell type specific gene studies, and validation of inferred developmental trajectories from phenotypic and gene expression analyses. This report describes endogenous zebrafish Cre and CreERT2 drivers generated with GeneWeld CRISPR-Cas9 precision targeted integration.

Results: and knock-ins crossed with ubiquitous -based Switch reporters led to broad labeling in expected mesodermal and neural crest-derived lineages in cardiac, pectoral fins, pharyngeal arch, liver, intestine, and mesothelial tissues, as well as enteric neurons.

Quantification of subcellular RNA localization through direct detection of RNA oxidation.

Across cell types and organisms, thousands of RNAs display asymmetric subcellular distributions. The study of this process often requires quantifying abundances of specific RNAs at precise subcellular locations. To analyze subcellular transcriptomes, multiple proximity-based techniques have been developed in which RNAs near a localized bait protein are specifically labeled, facilitating their biotinylation and purification.

The pericardium forms as a distinct structure during heart formation.

The heart integrates diverse cell lineages into a functional unit, including the pericardium, a mesothelial sac that supports heart movement, homeostasis, and immune responses. However, despite its critical roles, the developmental origins of the pericardium remain uncertain due to disparate models. Here, using live imaging, lineage tracking, and single-cell transcriptomics in zebrafish, we find the pericardium forms within the lateral plate mesoderm from dedicated anterior mesothelial progenitors and distinct from the classic heart field.

: Safe harbor landing sites for reproducible and efficient transgenesis in zebrafish.

Standard zebrafish transgenesis involves random transgene integration with resource-intensive screening. While phiC31 integrase-based / recombination has streamlined transgenesis in mice and , validated -based landing sites for universal applications are lacking in zebrafish. Here, we developed () as transgenesis approach, with two landing sites and from well-validated Tol2 transgenes.

: Safe harbor landing sites for reproducible and efficient transgenesis in zebrafish.

Standard methods for transgenesis in zebrafish depend on random transgene integration into the genome followed by resource-intensive screening and validation. Targeted vector integration into validated genomic loci using phiC31 integrase-based / recombination has transformed mouse and transgenesis. However, while the phiC31 system functions in zebrafish, validated loci carrying -based landing or safe harbor sites suitable for universal transgenesis applications in zebrafish have not been established.

Conserved enhancers control notochord expression of vertebrate Brachyury.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene.

Rosettes guide the way for mesodermal MET.

Mesenchymal-epithelial transition (MET) is a critical process in development, guiding tissue morphogenesis. In this issue of Developmental Cell, two studies, one by Gredler et al. and one by Abboud Asleh et al.

A median fin derived from the lateral plate mesoderm and the origin of paired fins.

The development of paired appendages was a key innovation during evolution and facilitated the aquatic to terrestrial transition of vertebrates. Largely derived from the lateral plate mesoderm (LPM), one hypothesis for the evolution of paired fins invokes derivation from unpaired median fins via a pair of lateral fin folds located between pectoral and pelvic fin territories. Whilst unpaired and paired fins exhibit similar structural and molecular characteristics, no definitive evidence exists for paired lateral fin folds in larvae or adults of any extant or extinct species.

Conserved enhancer logic controls the notochord expression of vertebrate .

The cell type-specific expression of key transcription factors is central to development. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian gene.

Lateral thinking in syndromic congenital cardiovascular disease.

Syndromic birth defects are rare diseases that can present with seemingly pleiotropic comorbidities. Prime examples are rare congenital heart and cardiovascular anomalies that can be accompanied by forelimb defects, kidney disorders and more. Whether such multi-organ defects share a developmental link remains a key question with relevance to the diagnosis, therapeutic intervention and long-term care of affected patients.

Rbm8a deficiency causes hematopoietic defects by modulating Wnt/PCP signaling.

Defects in blood development frequently occur among syndromic congenital anomalies. Thrombocytopenia-Absent Radius (TAR) syndrome is a rare congenital condition with reduced platelets (hypomegakaryocytic thrombocytopenia) and forelimb anomalies, concurrent with more variable heart and kidney defects. TAR syndrome associates with hypomorphic gene function for that encodes a component of the exon junction complex involved in mRNA splicing, transport, and nonsense-mediated decay.

Urp1 and Urp2 act redundantly to maintain spine shape in zebrafish larvae.

Urp1 and Urp2 are two neuropeptides, members of the Urotensin 2 family, that have been recently involved in the control of body axis morphogenesis in zebrafish. They are produced by a population of sensory spinal neurons, called cerebrospinal fluid contacting neurons (CSF-cNs), under the control of signals relying on the Reissner fiber, an extracellular thread bathing in the CSF. Here, we have investigated further the function of Urp1 and Urp2 (Urp1/2) in body axis formation and maintenance.

The centrosomal protein 83 (CEP83) regulates human pluripotent stem cell differentiation toward the kidney lineage.

During embryonic development, the mesoderm undergoes patterning into diverse lineages including axial, paraxial, and lateral plate mesoderm (LPM). Within the LPM, the so-called intermediate mesoderm (IM) forms kidney and urogenital tract progenitor cells, while the remaining LPM forms cardiovascular, hematopoietic, mesothelial, and additional progenitor cells. The signals that regulate these early lineage decisions are incompletely understood.

Endothelial versus pronephron fate decision is modulated by the transcription factors Cloche/Npas4l, Tal1, and Lmo2.

Endothelial specification is a key event during embryogenesis; however, when, and how, endothelial cells separate from other lineages is poorly understood. In zebrafish, Npas4l is indispensable for endothelial specification by inducing the expression of the transcription factor genes , , and . We generated a knock-in reporter in zebrafish to visualize endothelial progenitors and their derivatives in wild-type and mutant embryos.

Heterogeneity and genomic loci of ubiquitous transgenic Cre reporter lines in zebrafish.

Background: The most-common strategy for zebrafish Cre/lox-mediated lineage labeling experiments combines ubiquitously expressed, lox-based Switch reporter transgenes with tissue-specific Cre or 4-OH-Tamoxifen-inducible CreERT2 driver lines. Although numerous Cre driver lines have been produced, only a few broadly expressed Switch reporters exist in zebrafish and their generation by random transgene integration has been challenging due to position-effect sensitivity of the lox-flanked recombination cassettes. Here, we compare commonly used Switch reporter lines for their recombination efficiency and reporter expression pattern during zebrafish development.

Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma.

The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood.

Definitive hematopoietic stem cells minimally contribute to embryonic hematopoiesis.

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.

Persistent Ventricle Partitioning in the Adult Zebrafish Heart.

The vertebrate heart integrates cells from the early-differentiating first heart field (FHF) and the later-differentiating second heart field (SHF), both emerging from the lateral plate mesoderm. In mammals, this process forms the basis for the development of the left and right ventricle chambers and subsequent chamber septation. The single ventricle-forming zebrafish heart also integrates FHF and SHF lineages during embryogenesis, yet the contributions of these two myocardial lineages to the adult zebrafish heart remain incompletely understood.

From Stripes to a Beating Heart: Early Cardiac Development in Zebrafish.

The heart is the first functional organ to form during vertebrate development. Congenital heart defects are the most common type of human birth defect, many originating as anomalies in early heart development. The zebrafish model provides an accessible vertebrate system to study early heart morphogenesis and to gain new insights into the mechanisms of congenital disease.

The lateral plate mesoderm.

The lateral plate mesoderm (LPM) forms the progenitor cells that constitute the heart and cardiovascular system, blood, kidneys, smooth muscle lineage and limb skeleton in the developing vertebrate embryo. Despite this central role in development and evolution, the LPM remains challenging to study and to delineate, owing to its lineage complexity and lack of a concise genetic definition. Here, we outline the processes that govern LPM specification, organization, its cell fates and the inferred evolutionary trajectories of LPM-derived tissues.