The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.

Accumulating evidences have pointed that foxc1a is essential for vascular development and integrity maintenance through regulating the expression of downstream genes and interacting with signaling pathways. However, the underling cellular and molecular mechanisms of foxc1a in regulating vascular development remain undetermined. Based on two different foxc1a mutant zebrafish lines (foxc1anju18 and foxc1anju19 which generated predicted truncated foxc1a proteins with 50aa and 315aa respectively), we found that around 30 % of foxc1anju18 zebrafish exhibited severe vascular developmental defects with obvious hemorrhage in hindbrain and trunk at embryonic stages. Confocal imaging analysis revealed that the formation of middle cerebral vein (MCeV), intra-cerebral central arteries (CtAs) and dorsal longitudinal vein (DLV) of brain vessels was significantly blocked in foxc1anju18enbryos. Injection of exogenous full length and foxc1anju19 truncated foxc1a mRNA both rescued the deficiency of foxc1anju18 embryos. Transcriptome analysis revealed 186 DEGs in foxc1anju18 zebrafish among which amotl2a and ctnnb1 expression were reduced and functionally associated with adherens junctions. Dual-Luciferase assays validated amotl2a and ctnnb1 were both directly transactivated by foxc1a. Rescue experiments demonstrated that amotl2a was mainly responsible for the vascular integrity caused by foxc1a mutation and also coordinated with ctnnb1 to regulate brain vascular development. Our data point to a novel clue that foxc1a regulates vascular integrity and brain vascular development through targeting amotl2a and ctnnb1.

Chamber maturation is a significant process in cardiac development. Disorders of this crucial process lead to a range of congenital heart defects. Foxc1a is a critical transcription factor reported to regulate the specification of cardiac progenitor cells. However, little is known about the role of Foxc1a in modulating chamber maturation. Previously, we reported that foxc1a-null zebrafish embryos exhibit disrupted heart structures and functions. In this study, we observe that ventricle structure and cardiomyocyte proliferation are abolished during chamber maturation in foxc1a-null zebrafish embryos. To observe the endogenous localization of Foxc1a in the hearts of living embryos, we insert eyfp at the foxc1a genomic locus using TALEN. Analysis of the knockin zebrafish show that foxc1a is widely expressed in ventricular cardiomyocytes during chamber development. Cardiac RNA sequencing analysis reveals the downregulated expression of the Hippo signaling effector wwtr1. Dual-luciferase and chromatin immunoprecipitation assays reveal that Foxc1a can bind directly to three sites in the wwtr1 promoter region. Furthermore, wwtr1 mRNA overexpression is sufficient to reverse the ventricle defects during chamber maturation. Conditional overexpression of nkx2.5 also partially rescues the ventricular defects during chamber development. These findings demonstrate that wwtr1 and nkx2.5 are direct targets of Foxc1a during ventricular chamber maturation.

Cardiogenesis is a tightly controlled biological process required for formation of a functional heart. The transcription factor Foxc1 not only plays a crucial role in outflow tract development in mice, but is also involved in cardiac structure formation and normal function in humans. However, the molecular mechanisms by which Foxc1 controls cardiac development remain poorly understood. Previously, we reported that zebrafish embryos deficient in foxc1a, an ortholog of mammalian Foxc1, display pericardial edemas and die 9-10 days postfertilization. To further investigate Foxc1a\'s role in zebrafish cardiogenesis and identify its downstream target genes during early heart development, we comprehensively analyzed the cardiovascular phenotype of foxc1a-null zebrafish embryos. Our results confirmed that foxc1a-null mutants exhibit disrupted cardiac morphology, structure, and function. Performing transcriptome analysis on the foxc1a mutants, we found that the expression of the cardiac progenitor marker gene nkx2.5 was significantly decreased, but the expression of germ layer-patterning genes was unaffected. Dual-fluorescence in situ hybridization assays revealed that foxc1a and nkx2.5 are co-expressed in the anterior lateral plate mesoderm at the somite stage. Chromatin immunoprecipitation and promoter truncation assays disclosed that Foxc1a regulates nkx2.5 expression via direct binding to two noncanonical binding sites in the proximal nkx2.5 promoter. Moreover, functional rescue experiments revealed that developmental stage-specific nkx2.5 overexpression partially rescues the cardiac defects of the foxc1a-null embryos. Taken together, our results indicate that during zebrafish cardiogenesis, Foxc1a is active directly upstream of nkx2.5.

Foxc1a is a member of the forkhead transcription factors. It plays an essential role in zebrafish somitogenesis. However, little is known about the molecular mechanisms underlying its controlling somitogenesis. To uncover how foxc1a regulates zebrafish somitogenesis, we generated foxc1a knock-out zebrafish using TALEN (transcription activator-like effector nuclease) technology. The foxc1a null embryos exhibited defective somites at early development. Analyses on the expressions of the key genes that control processes of somitogenesis revealed that foxc1a controlled early somitogenesis by regulating the expression of myod1. In the somites of foxc1a knock-out embryos, expressions of fgf8a and deltaC were abolished, whereas the expression of aldh1a2 (responsible for providing retinoic acid signaling) was significantly increased. Once the increased retinoic acid level in the foxc1a null embryos was reduced by knocking down aldh1a2, the reduced expression of myod1 was partially rescued by resuming expressions of fgf8a and deltaC in the somites of the mutant embryos. Moreover, a chromatin immunoprecipitation assay on zebrafish embryos revealed that Foxc1a bound aldh1a2 promoter directly. On the other hand, neither knocking down fgf8a nor inhibiting Notch signaling affected the expression of aldh1a2, although knocking down fgf8a reduced expression of deltaC in the somites of zebrafish embryos at early somitogenesis and vice versa. Taken together, our results demonstrate that foxc1a plays an essential role in early somitogenesis by controlling Fgf and Notch signaling through restricting the expression of aldh1a2 in paraxial mesoderm directly.

Neural connectivity between the spinal cord and paired appendages is key to the superior locomotion of tetrapods and aquatic vertebrates. In contrast to nerves that innervate axial muscles, those innervating appendages converge at a specialized structure, the plexus, where they topographically reorganize before navigating towards their muscle targets. Despite its importance for providing appendage mobility, the genetic program that drives nerve convergence at the plexus, as well as the functional role of this convergence, are not well understood. Here, we show that in zebrafish the transcription factor foxc1a is dispensable for trunk motor nerve guidance but is required to guide spinal nerves innervating the pectoral fins, equivalent to the tetrapod forelimbs. In foxc1a null mutants, instead of converging with other nerves at the plexus, pectoral fin nerves frequently bypass the plexus. We demonstrate that foxc1a expression in muscle cells delineating the nerve path between the spinal cord and the plexus region restores convergence at the plexus. By labeling individual fin nerves, we show that mutant nerves bypassing the plexus enter the fin at ectopic positions, yet innervate their designated target areas, suggesting that motor axons can select their appropriate fin target area independently of their migration through the plexus. Although foxc1a mutants display topographically correct fin innervation, mutant fin muscles exhibit a reduction in the levels of pre- and postsynaptic structures, concomitant with reduced pectoral fin function. Combined, our results reveal foxc1a as a key player in the development of connectivity between the spinal cord and paired appendages, which is crucial for appendage mobility.

Elucidating the gene regulatory networks that control kidney development can provide information about the origins of renal birth defects and kidney disease, as well as insights relevant to the design of clinical interventions for these conditions. The kidney is composed of functional units termed nephrons. Renal malfunction often arises from damage to cells known as podocytes, which are highly specialized epithelial cells that comprise the blood filter, or glomerulus, located on each nephron. Podocytes interact with the vasculature to create an elaborate sieve that collects circulatory fluid, and this filtrate enters the nephron where it is modified to produce urine and balance water homeostasis. Podocytes are an essential cellular component of the glomerular filtration barrier, helping to protect nephrons from the entry of large proteins and circulatory cells. Podocyte loss has catastrophic consequences for renal function and overall health, as podocyte destruction leads to nephron damage and pathological conditions like chronic kidney disease. Despite their importance, there is still a rather limited understanding about the molecular pathways that control podocyte formation. In recent years, however, studies of podocyte development using the zebrafish embryonic kidney, or pronephros, have been an expanding area of nephrology research. Zebrafish form an anatomically simple pronephros comprised of two nephrons that share a single blood filter, and podocyte progenitors can be easily visualized throughout the process of glomerular development. The zebrafish is an especially useful system for studying the mechanisms that are essential for formation of nephron cell types like podocytes due to the high genetic conservation between vertebrate species, including humans. In this review, we discuss how research using the zebrafish has provided new insights into the molecular regulation of the podocyte lineage during kidney ontogeny, complementing contemporary research in other animal models.