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.

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.

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.

Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration.

BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, whereas expression of the erythroid regulator GATA1 directs SMAD1 loss on nonerythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.

Mutations in Bcl9 and Pygo genes cause congenital heart defects by tissue-specific perturbation of Wnt/ß-catenin signaling.

Bcl9 and Pygopus (Pygo) are obligate Wnt/ß-catenin cofactors in Drosophila, yet their contribution to Wnt signaling during vertebrate development remains unresolved. Combining zebrafish and mouse genetics, we document a conserved, ß-catenin-associated function for BCL9 and Pygo proteins during vertebrate heart development. Disrupting the ß-catenin-BCL9-Pygo complex results in a broadly maintained canonical Wnt response yet perturbs heart development and proper expression of key cardiac regulators. Our work highlights BCL9 and Pygo as selective ß-catenin cofactors in a subset of canonical Wnt responses during vertebrate development. Moreover, our results implicate alterations in BCL9 and BCL9L in human congenital heart defects.

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 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.

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.

Switch and Trace: Recombinase Genetics in Zebrafish.

Transgenic approaches are instrumental for labeling and manipulating cells and cellular machineries in vivo. Transgenes have traditionally been static entities that remained unaltered following genome integration, limiting their versatility. The development of DNA recombinase-based methods to modify, excise, or rearrange transgene cassettes has introduced versatile control of transgene activity and function. In particular, recombinase-controlled transgenes enable regulation of exogenous gene expression, conditional mutagenesis, and genetic lineage tracing. In zebrafish, transgenesis-based recombinase genetics using Cre/lox, Flp/FRT, and ΦC31 are increasingly applied to study development and homeostasis, and to generate disease models. Intersected with the versatile imaging capacity of the zebrafish model and recent breakthroughs in genome editing, we review and discuss past, current, and potential future approaches and resources for recombinase-based techniques in zebrafish.

Beta-catenin hits chromatin: regulation of Wnt target gene activation.

The canonical Wnt pathway has gathered much attention in recent years owing to its fundamental contribution to metazoan development, tissue homeostasis and human malignancies. Wnt target gene transcription is regulated by nuclear beta-catenin, and genetic assays have revealed various collaborating protein cofactors. Their daunting number and diverse nature, however, make it difficult to arrange an orderly picture of the nuclear Wnt transduction events. Yet, these findings emphasize that beta-catenin-mediated transcription affects chromatin. How does beta-catenin cope with chromatin regulation to turn on Wnt target genes?

An exclusive cellular and molecular network governs intestinal smooth muscle cell differentiation in vertebrates.

Intestinal smooth muscle cells (iSMCs) are a crucial component of the adult gastrointestinal tract and support intestinal differentiation, peristalsis and epithelial homeostasis during development. Despite these crucial roles, the origin of iSMCs and the mechanisms responsible for their differentiation and function remain largely unknown in vertebrates. Here, we demonstrate that iSMCs arise from the lateral plate mesoderm (LPM) in a stepwise process. Combining pharmacological and genetic approaches, we show that TGFß/Alk5 signaling drives the LPM ventral migration and commitment to an iSMC fate. The Alk5-dependent induction of zeb1a and foxo1a is required for this morphogenetic process: zeb1a is responsible for driving LPM migration around the gut, whereas foxo1a regulates LPM predisposition to iSMC differentiation. We further show that TGFß, zeb1a and foxo1a are tightly linked together by miR-145 In iSMC-committed cells, TGFß induces the expression of miR-145, which in turn is able to downregulate zeb1a and foxo1a The absence of miR-145 results in only a slight reduction in the number of iSMCs, which still express mesenchymal genes but fail to contract. Together, our data uncover a cascade of molecular events that govern distinct morphogenetic steps during the emergence and differentiation of vertebrate iSMCs.

Anterior trunk muscle shows mix of axial and appendicular developmental patterns.

BACKGROUND: Skeletal muscle in the trunk derives from the somites, paired segments of paraxial mesoderm. Whereas axial musculature develops within the somite, appendicular muscle develops following migration of muscle precursors into lateral plate mesoderm. The development of muscles bridging axial and appendicular systems appears mixed. RESULTS: We examine development of three migratory muscle precursor-derived muscles in zebrafish: the sternohyoideus (SH), pectoral fin (PF), and posterior hypaxial (PHM) muscles. We show there is an anterior to posterior gradient to the developmental gene expression and maturation of these three muscles. SH muscle precursors exhibit a long delay between migration and differentiation, PF muscle precursors exhibit a moderate delay in differentiation, and PHM muscle precursors show virtually no delay between migration and differentiation. Using lineage tracing, we show that lateral plate contribution to the PHM muscle is minor, unlike its known extensive contribution to the PF muscle and absence in the ventral extension of axial musculature. CONCLUSIONS: We propose that PHM development is intermediate between a migratory muscle mode and an axial muscle mode of development, wherein the PHM differentiates after a very short migration of its precursors and becomes more anterior primarily by elongation of differentiated muscle fibers.

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.

Cre/lox-controlled spatiotemporal perturbation of FGF signaling in zebrafish.

BACKGROUND: Spatiotemporal perturbation of signaling pathways in vivo remains challenging and requires precise transgenic control of signaling effectors. Fibroblast growth factor (FGF) signaling guides multiple developmental processes, including body axis formation and cell fate patterning. In zebrafish, mutants and chemical perturbations affecting FGF signaling have uncovered key developmental processes; however, these approaches cause embryo-wide perturbations, rendering assessment of cell-autonomous vs. non-autonomous requirements for FGF signaling in individual processes difficult. RESULTS: Here, we created the novel transgenic line fgfr1-dn-cargo, encoding dominant-negative Fgfr1a with fluorescent tag under combined Cre/lox and heatshock control to perturb FGF signaling spatiotemporally. Validating efficient perturbation of FGF signaling by fgfr1-dn-cargo primed with ubiquitous CreERT2, we established that primed, heatshock-induced fgfr1-dn-cargo behaves similarly to pulsed treatment with the FGFR inhibitor SU5402. Priming fgfr1-dn-cargo with CreERT2 in the lateral plate mesoderm triggered selective cardiac and pectoral fin phenotypes without drastic impact on overall embryo patterning. Harnessing lateral plate mesoderm-specific FGF inhibition, we recapitulated the cell-autonomous and temporal requirement for FGF signaling in pectoral fin outgrowth, as previously inferred from pan-embryonic FGF inhibition. CONCLUSIONS: As a paradigm for rapid Cre/lox-mediated signaling perturbations, our results establish fgfr1-dn-cargo as a genetic tool to define the spatiotemporal requirements for FGF signaling in zebrafish. Developmental Dynamics 247:1146-1159, 2018. © 2018 Wiley Periodicals, Inc.

A defect in the mitochondrial protein Mpv17 underlies the transparent casper zebrafish.

The casper strain of zebrafish is widely used in studies ranging from cancer to neuroscience. casper offers the advantage of relative transparency throughout adulthood, making it particularly useful for in vivo imaging by epifluorescence, confocal, and light sheet microscopy. casper was developed by selective breeding of two previously described recessive pigment mutants: 1) nacre, which harbors an inactivating mutation of the mitfa gene, rendering the fish devoid of pigmented melanocytes; and 2) roy orbison, a mutant with a so-far unidentified genetic cause that lacks reflective iridophores. To clarify the molecular nature of the roy orbison mutation, such that it can inform studies using casper, we undertook an effort to positionally clone the roy orbison mutation. We find that roy orbison is caused by an intronic defect in the gene mpv17, encoding an inner mitochondrial membrane protein that has been implicated in the human mitochondrial DNA depletion syndrome. The roy orbison mutation is phenotypically and molecularly remarkably similar to another zebrafish iridophore mutant called transparent. Using Cas9-induced crispants and germline mutants with a disrupted mpv17 open reading frame, we show in trans-heterozygote embryos that new frameshift alleles of mpv17, roy orbison, and transparent fail to complement each other. Our work provides genetic evidence that both roy orbison and transparent affect the mpv17 locus by a similar if not identical genetic lesion. Identification of mpv17 mutants will allow for further work probing the relationship between mitochondrial function and pigmentation, which has to date received little attention.

Gata2b is a restricted early regulator of hemogenic endothelium in the zebrafish embryo.

The adult blood system is established by hematopoietic stem cells (HSCs), which arise during development from an endothelial-to-hematopoietic transition of cells comprising the floor of the dorsal aorta. Expression of aortic runx1 has served as an early marker of HSC commitment in the zebrafish embryo, but recent studies have suggested that HSC specification begins during the convergence of posterior lateral plate mesoderm (PLM), well before aorta formation and runx1 transcription. Further understanding of the earliest stages of HSC specification necessitates an earlier marker of hemogenic endothelium. Studies in mice have suggested that GATA2 might function at early stages within hemogenic endothelium. Two orthologs of Gata2 exist in zebrafish: gata2a and gata2b. Here, we report that gata2b expression initiates during the convergence of PLM, becoming restricted to emerging HSCs. We observe Notch-dependent gata2b expression within the hemogenic subcompartment of the dorsal aorta that is in turn required to initiate runx1 expression. Our results indicate that Gata2b functions within hemogenic endothelium from an early stage, whereas Gata2a functions more broadly throughout the vascular system.

Novel cardiovascular gene functions revealed via systematic phenotype prediction in zebrafish.

Comprehensive functional annotation of vertebrate genomes is fundamental to biological discovery. Reverse genetic screening has been highly useful for determination of gene function, but is untenable as a systematic approach in vertebrate model organisms given the number of surveyable genes and observable phenotypes. Unbiased prediction of gene-phenotype relationships offers a strategy to direct finite experimental resources towards likely phenotypes, thus maximizing de novo discovery of gene functions. Here we prioritized genes for phenotypic assay in zebrafish through machine learning, predicting the effect of loss of function of each of 15,106 zebrafish genes on 338 distinct embryonic anatomical processes. Focusing on cardiovascular phenotypes, the learning procedure predicted known knockdown and mutant phenotypes with high precision. In proof-of-concept studies we validated 16 high-confidence cardiac predictions using targeted morpholino knockdown and initial blinded phenotyping in embryonic zebrafish, confirming a significant enrichment for cardiac phenotypes as compared with morpholino controls. Subsequent detailed analyses of cardiac function confirmed these results, identifying novel physiological defects for 11 tested genes. Among these we identified tmem88a, a recently described attenuator of Wnt signaling, as a discrete regulator of the patterning of intercellular coupling in the zebrafish cardiac epithelium. Thus, we show that systematic prioritization in zebrafish can accelerate the pace of developmental gene function discovery.

Latent TGF-ß binding protein 3 identifies a second heart field in zebrafish.

The four-chambered mammalian heart develops from two fields of cardiac progenitor cells distinguished by their spatiotemporal patterns of differentiation and contributions to the definitive heart. The first heart field differentiates earlier in lateral plate mesoderm, generates the linear heart tube and ultimately gives rise to the left ventricle. The second heart field (SHF) differentiates later in pharyngeal mesoderm, elongates the heart tube, and gives rise to the outflow tract and much of the right ventricle. Because hearts in lower vertebrates contain a rudimentary outflow tract but not a right ventricle, the existence and function of SHF-like cells in these species has remained a topic of speculation. Here we provide direct evidence from Cre/Lox-mediated lineage tracing and loss-of-function studies in zebrafish, a lower vertebrate with a single ventricle, that latent TGF-ß binding protein 3 (ltbp3) transcripts mark a field of cardiac progenitor cells with defining characteristics of the anterior SHF in mammals. Specifically, ltbp3(+) cells differentiate in pharyngeal mesoderm after formation of the heart tube, elongate the heart tube at the outflow pole, and give rise to three cardiovascular lineages in the outflow tract and myocardium in the distal ventricle. In addition to expressing Ltbp3, a protein that regulates the bioavailability of TGF-ß ligands, zebrafish SHF cells co-express nkx2.5, an evolutionarily conserved marker of cardiac progenitor cells in both fields. Embryos devoid of ltbp3 lack the same cardiac structures derived from ltbp3(+) cells due to compromised progenitor proliferation. Furthermore, small-molecule inhibition of TGF-ß signalling phenocopies the ltbp3-morphant phenotype whereas expression of a constitutively active TGF-ß type I receptor rescues it. Taken together, our findings uncover a requirement for ltbp3-TGF-ß signalling during zebrafish SHF development, a process that serves to enlarge the single ventricular chamber in this species.

Wnt/ß-catenin signaling is required for radial glial neurogenesis following spinal cord injury.

Spinal cord injury results in permanent sensorimotor loss in mammals, in part due to a lack of injury-induced neurogenesis. The regeneration of neurons depends upon resident neural progenitors, which in zebrafish persist throughout the central nervous system as radial glia. However the molecular mechanisms regulating spinal cord progenitors remain uncharacterized. Wnt/ß-catenin signaling is necessary for the regenerative response of multiple tissues in zebrafish as well as other vertebrates, but it is not known whether the pathway has a role in spinal cord regeneration. Here we show that spinal radial glia exhibit Wnt/ß-catenin activity as they undergo neurogenesis following transection. We then use Cre-mediated lineage tracing to label the progeny of radial glia and show that Wnt/ß-catenin signaling is required for progenitors to differentiate into neurons. Finally, we show that axonal regrowth after injury also requires Wnt/ß-catenin signaling, suggesting coordinated roles for the pathway in functional recovery. Our data thus establish Wnt/ß-catenin pathway activation as a necessary step in spinal cord regeneration.