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The morphological transformation of the pectoral/shoulder girdle is fundamental to the water-to-land transition in vertebrate evolution. Although previous studies have resolved the embryonic origins of tetrapod shoulder girdles, those of fish pectoral girdles remain uncharacterized, creating a gap in the understanding of girdle transformation mechanisms from fish to tetrapods. Here, we identify the embryonic origins of the zebrafish pectoral girdle, including the cleithrum as an ancestral girdle element lost in extant tetrapods. Our combinatorial approach of photoconversion and genetic lineage tracing demonstrates that cleithrum development combines four adjoining embryonic populations. A comparison of these pectoral girdle progenitors with extinct and extant vertebrates highlights that cleithrum loss, indispensable for neck evolution, is associated with the disappearance of its unique developmental environment at the head/trunk interface. Overall, our study establishes an embryological framework for pectoral/shoulder girdle formation and provides evolutionary trajectories from their origin in water to diversification on land. 

ABSTRACT 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. Imaging of transgenic reporters in zebrafish documents lateral plate mesoderm cells that emerge lateral of the classic heart field and among a continuous mesothelial progenitor field. Single-cell transcriptomics and trajectories ofhand2-expressing lateral plate mesoderm reveal distinct populations of mesothelial and cardiac precursors, including pericardial precursors that are distinct from the cardiomyocyte lineage. The mesothelial gene expression signature is conserved in mammals and carries over to postnatal development. Light sheet-based live-imaging and machine learning-supported cell tracking documents that during heart tube formation, pericardial precursors that reside at the anterior edge of the heart field migrate anteriorly and medially before fusing, enclosing the embryonic heart to form a single pericardial cavity. Pericardium formation proceeds even upon genetic disruption of heart tube formation, uncoupling the two structures. Canonical Wnt/β-catenin signaling modulates pericardial cell number, resulting in a stretched pericardial epithelium with reduced cell number upon canonical Wnt inhibition. We connect the pathological expression of secreted Wnt antagonists of the SFRP family found in pediatric dilated cardiomyopathy to increased pericardial stiffness: sFRP1 in the presence of increased catecholamines causes cardiomyocyte stiffness in neonatal rats as measured by atomic force microscopy. Altogether, our data integrate pericardium formation as an independent process into heart morphogenesis and connect disrupted pericardial tissue properties such as pericardial stiffness to pediatric cardiomyopathies. 

Standard zebrafish transgenesis involves random transgene integration with resource-intensive screening. While phiC31 integrase–basedattP/attBrecombination has streamlined transgenesis in mice andDrosophila, validatedattP-based landing sites for universal applications are lacking in zebrafish. Here, we developedphiC31 Integrase Genomic Loci Engineered for Transgenesis(pIGLET) as transgenesis approach, with twoattPlanding sitespIGLET14aandpIGLET24bfrom well-validated Tol2 transgenes. Both sites facilitate diverse transgenesis applications including reporters and Cre/loxPtransgenes. ThepIGLET14aandpIGLET24blanding 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. 

ABSTRACT 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. 

Abstract The cell type-specific expression of key transcription factors is central to development and disease.Brachyury/T/TBXTis 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 mammalianBrachyury/T/TBXTgene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conservedBrachyury-controlling notochord enhancers,T3,C, andI, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers,in cisdeletion 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 threeBrachyury-driving notochord enhancers are conserved beyond mammals in thebrachyury/tbxtbloci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers forBrachyury/T/TBXTBnotochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development. 

ABSTRACT 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. 

Abstract 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 1 . 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 duplication 2 . 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. 

ABSTRACT 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. 

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.