pIGLET: 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 attP/attB recombination has streamlined transgenesis in mice and Drosophila, validated attP-based landing sites for universal applications are lacking in zebrafish. Here, we developed phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET) as transgenesis approach, with two attP landing sites pIGLET14a and pIGLET24b from well-validated Tol2 transgenes. Both sites facilitate diverse transgenesis applications including reporters and Cre/loxP transgenes. The pIGLET14a and pIGLET24b landing sites consistently yield 25 to 50% germline transmission, substantially reducing the resources needed for transgenic line generation. Transgenesis into these sites enables reproducible expression patterns in F0 zebrafish embryos for enhancer discovery and testing of gene regulatory variants. Together, our new landing sites streamline targeted, reproducible zebrafish transgenesis as a robust platform for various applications while minimizing the workload for generating transgenic lines.

Pluripotency of a founding field: rebranding developmental biology.

The field of developmental biology has declined in prominence in recent decades, with off-shoots from the field becoming more fashionable and highly funded. This has created inequity in discovery and opportunity, partly due to the perception that the field is antiquated or not cutting edge. A 'think tank' of scientists from multiple developmental biology-related disciplines came together to define specific challenges in the field that may have inhibited innovation, and to provide tangible solutions to some of the issues facing developmental biology. The community suggestions include a call to the community to help 'rebrand' the field, alongside proposals for additional funding apparatuses, frameworks for interdisciplinary innovative collaborations, pedagogical access, improved science communication, increased diversity and inclusion, and equity of resources to provide maximal impact to the community.

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 RBM8A/Y14 that encodes a component of the exon junction complex involved in mRNA splicing, transport, and nonsense-mediated decay. How perturbing a general mRNA-processing factor causes the selective TAR Syndrome phenotypes remains unknown. Here, we connect zebrafish rbm8a perturbation to early hematopoietic defects via attenuated non-canonical Wnt/Planar Cell Polarity (PCP) signaling that controls developmental cell re-arrangements. In hypomorphic rbm8a zebrafish, we observe a significant reduction of cd41-positive thrombocytes. rbm8a-mutant zebrafish embryos accumulate mRNAs with individual retained introns, a hallmark of defective nonsense-mediated decay; affected mRNAs include transcripts for non-canonical Wnt/PCP pathway components. We establish that rbm8a-mutant embryos show convergent extension defects and that reduced rbm8a function interacts with perturbations in non-canonical Wnt/PCP pathway genes wnt5b, wnt11f2, fzd7a, and vangl2. Using live-imaging, we found reduced rbm8a function impairs the architecture of the lateral plate mesoderm (LPM) that forms hematopoietic, cardiovascular, kidney, and forelimb skeleton progenitors as affected in TAR Syndrome. Both mutants for rbm8a and for the PCP gene vangl2 feature impaired expression of early hematopoietic/endothelial genes including runx1 and the megakaryocyte regulator gfi1aa. Together, our data propose aberrant LPM patterning and hematopoietic defects as consequence of attenuated non-canonical Wnt/PCP signaling upon reduced rbm8a function. These results also link TAR Syndrome to a potential LPM origin and a developmental mechanism.

pIGLET: 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 attP/attB recombination has transformed mouse and Drosophila transgenesis. However, while the phiC31 system functions in zebrafish, validated loci carrying attP-based landing or safe harbor sites suitable for universal transgenesis applications in zebrafish have not been established. Here, using CRISPR-Cas9, we converted two well-validated single insertion Tol2-based zebrafish transgenes with long-standing genetic stability into two attP landing sites, called phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET). Generating fluorescent reporters, loxP-based Switch lines, CreERT2 drivers, and gene-regulatory variant reporters in the pIGLET14a and pIGLET24b landing site alleles, we document their suitability for transgenesis applications across cell types and developmental stages. For both landing sites, we routinely achieve 25-50% germline transmission of targeted transgene integrations, drastically reducing the number of required animals and necessary resources to generate individual transgenic lines. We document that phiC31 integrase-based transgenesis into pIGLET14a and pIGLET24b reproducibly results in representative reporter expression patterns in injected F0 zebrafish embryos suitable for enhancer discovery and qualitative and quantitative comparison of gene-regulatory element variants. Taken together, our new phiC31 integrase-based transgene landing sites establish reproducible, targeted zebrafish transgenesis for numerous applications while greatly reducing the workload of generating new transgenic zebrafish lines.

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. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

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., reveal how multicellular rosettes critically contribute to MET in earliest notochord and lateral plate mesoderm formation, respectively.

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. The heart, endothelial and blood lineages develop together from the lateral plate mesoderm (LPM), which also harbors the progenitor cells for limb connective tissue, kidneys, mesothelia and smooth muscle. This developmental plasticity of the LPM, which founds on multi-lineage progenitor cells and shared transcription factor expression across different descendant lineages, has the potential to explain the seemingly disparate syndromic defects in rare congenital diseases. Combining patient genome-sequencing data with model organism studies has already provided a wealth of insights into complex LPM-associated birth defects, such as heart-hand syndromes. Here, we summarize developmental and known disease-causing mechanisms in early LPM patterning, address how defects in these processes drive multi-organ comorbidities, and outline how several cardiovascular and hematopoietic birth defects with complex comorbidities may be LPM-associated diseases. We also discuss strategies to integrate patient sequencing, data-aggregating resources and model organism studies to mechanistically decode congenital defects, including potentially LPM-associated orphan diseases. Eventually, linking complex congenital phenotypes to a common LPM origin provides a framework to discover developmental mechanisms and to anticipate comorbidities in congenital diseases affecting the cardiovascular system and beyond.

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 territories1. 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. As unpaired fin core components are regarded as exclusively derived from paraxial mesoderm, any transition presumes both co-option of a fin developmental programme to the LPM and bilateral duplication2. Here, we identify that the larval zebrafish unpaired pre-anal fin fold (PAFF) is derived from the LPM and thus may represent a developmental intermediate between median and paired fins. We trace the contribution of LPM to the PAFF in both cyclostomes and gnathostomes, supporting the notion that this is an ancient trait of vertebrates. Finally, we observe that the PAFF can be bifurcated by increasing bone morphogenetic protein signalling, generating LPM-derived paired fin folds. Our work provides evidence that lateral fin folds may have existed as embryonic anlage for elaboration to paired fins.

Conserved enhancer logic controls the notochord expression of vertebrate Brachyury.

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 Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three Brachyury-controlling notochord enhancers T3, C, and I in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, deletion of all three enhancers in mouse abolishes Brachyury/T expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. Sequence and functional conservation of Brachyury-driving notochord enhancers with the brachyury/tbxtb loci from diverse lineages of fishes dates their origin to the last common ancestor of jawed vertebrates. Our data define the enhancers for Brachyury/T/TBXTB notochord expression as ancient mechanism in axis development.

Next-generation plasmids for transgenesis in zebrafish and beyond.

Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible modular system. Here, we establish several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2 and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Finally, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker that is active before hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish and other models.

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. We showed that urp1;urp2 double mutants develop strong body axis defects during larval growth, revealing the redundancy between the two neuropeptides. These defects were similar to those previously reported in uts2r3 mutants. We observed that this phenotype is not associated with congenital defects in vertebrae formation, but by using specific inhibitors, we found that, at least in the embryo, the action of Urp1/2 signaling depends on myosin II contraction. Finally, we provide evidence that while the Urp1/2 signaling is functioning during larval growth, it is dispensable for embryonic development. Taken together, our results show that Urp1/2 signaling is required in larvae to promote correct vertebral body axis, most likely by regulating muscle tone.

Molecular Function and Contribution of TBX4 in Development and Disease.

Over the past decade, recognition of the profound impact of the TBX4 (T-box 4) gene, which encodes a member of the evolutionarily conserved family of T-box-containing transcription factors, on respiratory diseases has emerged. The developmental importance of TBX4 is emphasized by the association of TBX4 variants with congenital disorders involving respiratory and skeletal structures; however, the exact role of TBX4 in human development remains incompletely understood. Here, we discuss the developmental, tissue-specific, and pathological TBX4 functions identified through human and animal studies and review the published TBX4 variants resulting in variable disease phenotypes. We also outline future research directions to fill the gaps in our understanding of TBX4 function and of how TBX4 disruption affects development.

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. Here, we found that the centrosomal protein 83 (CEP83), a centriolar component necessary for primary cilia formation and mutated in pediatric kidney disease, influences the differentiation of human-induced pluripotent stem cells (hiPSCs) toward IM. We induced inactivating deletions of CEP83 in hiPSCs and applied a 7-day in vitro protocol of IM kidney progenitor differentiation, based on timed application of WNT and FGF agonists. We characterized induced mesodermal cell populations using single-cell and bulk transcriptomics and tested their ability to form kidney structures in subsequent organoid culture. While hiPSCs with homozygous CEP83 inactivation were normal regarding morphology and transcriptome, their induced differentiation into IM progenitor cells was perturbed. Mesodermal cells induced after 7 days of monolayer culture of CEP83-deficient hiPCS exhibited absent or elongated primary cilia, displayed decreased expression of critical IM genes (PAX8, EYA1, HOXB7), and an aberrant induction of LPM markers (e.g. FOXF1, FOXF2, FENDRR, HAND1, HAND2). Upon subsequent organoid culture, wildtype cells differentiated to form kidney tubules and glomerular-like structures, whereas CEP83-deficient cells failed to generate kidney cell types, instead upregulating cardiomyocyte, vascular, and more general LPM progenitor markers. Our data suggest that CEP83 regulates the balance of IM and LPM formation from human pluripotent stem cells, identifying a potential link between centriolar or ciliary function and mesodermal lineage induction.

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 etsrp, tal1, and lmo2. We generated a knock-in reporter in zebrafish npas4l to visualize endothelial progenitors and their derivatives in wild-type and mutant embryos. Unexpectedly, we find that in npas4l mutants, npas4l reporter-expressing cells contribute to the pronephron tubules. Single-cell transcriptomics and live imaging of the early lateral plate mesoderm in wild-type embryos indeed reveals coexpression of endothelial and pronephron markers, a finding confirmed by creERT2-based lineage tracing. Increased contribution of npas4l reporter-expressing cells to pronephron tubules is also observed in tal1 and lmo2 mutants and is reversed in npas4l mutants injected with tal1 mRNA. Together, these data reveal that Npas4l/Tal1/Lmo2 regulate the fate decision between the endothelial and pronephron lineages.

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. RESULTS: Using different experimental setups, we show that ubi:Switch and hsp70l:Switch outperform current generations of the two additional Switch reporters actb2:BFP-DsRed and actb2:Stop-DsRed. Our comparisons also document preferential Cre-dependent recombination of ubi:Switch and hsp70l:Switch in distinct zebrafish tissues at early developmental stages. To investigate what genomic features may influence Cre accessibility and lox recombination efficiency in highly functional Switch lines, we mapped these transgenes and charted chromatin dynamics at their integration sites. CONCLUSIONS: Our data documents the heterogeneity among lox-based Switch transgenes toward informing suitable transgene selection for lineage labeling experiments. Our work further proposes that ubi:Switch and hsp70l:Switch define genomic integration sites suitable for universal transgene or switch reporter knock-in in zebrafish.

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. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies.

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. 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.

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. Here, we characterize the myocardial labeling of FHF descendants in both the developing and adult zebrafish ventricle. Expanding previous findings, late gastrulation-stage labeling using drl-driven CreERT2 recombinase with a myocardium-specific, myl7-controlled, loxP reporter results in the predominant labeling of FHF-derived outer curvature and the right side of the embryonic ventricle. Raised to adulthood, such lineage-labeled hearts retain broad areas of FHF cardiomyocytes in a region of the ventricle that is positioned at the opposite side to the atrium and encompasses the apex. Our data add to the increasing evidence for a persisting cell-based compartmentalization of the adult zebrafish ventricle even in the absence of any physical boundary.

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. Although composed of only two chambers compared with the four-chambered mammalian heart, the zebrafish heart integrates the core processes and cellular lineages central to cardiac development across vertebrates. The rapid, translucent development of zebrafish is amenable to in vivo imaging and genetic lineage tracing techniques, providing versatile tools to study heart field migration and myocardial progenitor addition and differentiation. Combining transgenic reporters with rapid genome engineering via CRISPR-Cas9 allows for functional testing of candidate genes associated with congenital heart defects and the discovery of molecular causes leading to observed phenotypes. Here, we summarize key insights gained through zebrafish studies into the early patterning of uncommitted lateral plate mesoderm into cardiac progenitors and their regulation. We review the central genetic mechanisms, available tools, and approaches for modeling congenital heart anomalies in the zebrafish as a representative vertebrate model.

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. Finally, we discuss the development of seemingly disparate organ systems that share a common LPM origin.