Xu, Xiaolei

Cilia are essential for normal development. The composition and assembly of cilia has been well characterized, but the signaling and transcriptional pathways that govern ciliogenesis remain poorly studied. Here, we report that Wnt/β-catenin signaling directly regulates ciliogenic transcription factor foxj1a expression and ciliogenesis in zebrafish Kupffer’s vesicle (KV). We show that Wnt signaling acts temporally and KV cell-autonomously to control left-right (LR) axis determination and ciliogenesis. Specifically, reduction of Wnt signaling leads to a disruption of LR patterning, shorter and fewer cilia, a loss of cilia motility and a downregulation of foxj1a expression. However, these phenotypes can be rescued by KV-targeted overexpression of foxj1a. In comparison to the FGF pathway that has been previously implicated in the control of ciliogenesis, our epistatic studies suggest a more downstream function of Wnt signaling in the regulation of foxj1a expression and ciliogenesis in KV. Importantly, enhancer analysis reveals that KV-specific expression of foxj1a requires the presence of putative Lef1/Tcf binding sites, indicating that Wnt signaling activates foxj1a transcription directly. We also find that impaired Wnt signaling leads to kidney cysts and otolith disorganization, which can be attributed to a loss of foxj1 expression and disrupted ciliogenesis in the developing pronephric ducts and otic vesicles. Together, our data reveal a novel role of Wnt/β-catenin signaling upstream of ciliogenesis, which might be a general developmental mechanism beyond KV. Moreover, our results also prompt a hypothesis that certain developmental effects of the Wnt/β-catenin pathway are due to the activation of Foxj1 and cilia formation.

Rationale: Although a cardioprotective function of target of rapamycin (TOR) signaling inhibition has been suggested by pharmacological studies using rapamycin, genetic evidences are still lacking. We explored adult zebrafish as a novel vertebrate model for dissecting signaling pathways in cardiomyopathy. Objective: We generated the second adult zebrafish cardiomyopathy model induced by doxorubicin. By genetically analyzing both the doxorubicin and our previous established anemia-induced cardiomyopathy models, we decipher the functions of TOR signaling in cardiomyopathies of different etiology. Methods and Results: Along the progression of both cardiomyopathy models, we detected dynamic TOR activity at different stages of pathogenesis as well as distinct effects of TOR signaling inhibition. Nevertheless, cardiac enlargement in both models can be effectively attenuated by inhibition of TOR signaling through short-term rapamycin treatment. To assess the long-term effects of TOR reduction, we used a zebrafish target of rapamycin ( ztor ) mutant identified from an insertional mutagenesis screen. We show that TOR haploinsufficiency in the ztor heterozygous fish improved cardiac function, prevented pathological remodeling events, and ultimately reduced mortality in both adult fish models of cardiomyopathy. Mechanistically, these cardioprotective effects are conveyed by the antihypertrophy, antiapoptosis, and proautophagy function of TOR signaling inhibition. Conclusions: Our results prove adult zebrafish as a conserved novel vertebrate model for human cardiomyopathies. Moreover, we provide the first genetic evidence to demonstrate a long-term cardioprotective effect of TOR signaling inhibition on at least 2 cardiomyopathies of distinct etiology, despite dynamic TOR activities during their pathogenesis.

The Wnt/β-catenin pathway exhibits distinct and developmental stage-specific roles during cardiogenesis. However, little is known about the molecular mechanisms of Wnt/β-catenin signaling in the establishment of cardiac left-right (LR) asymmetry. Using zebrafish as an animal model, we show here that Wnt/β-catenin signaling is differentially required in cardiac LR patterning. At an early stage, during asymmetric signal generation,Wnt/β-catenin signaling is necessary for Kupffer's vesicle development and for the regulation of both heart and visceral laterality. At a later stage, during asymmetric signal propagation, excessive Wnt/β-catenin signaling inhibits the transmission of asymmetric cues from the lateral plate mesoderm (LPM) to the cardiac field but not to the developing gut; as such, it only regulates heart laterality. Molecular analysis identifies Gata4 as the downstream target of Wnt/β-catenin signaling in the cardiac field that responds to the Wnt/β-catenin signaling and regulates the competence of the heart field to express left-sided genes. In summary, our results reveal a previously unexpected role of Wnt-Gata4 signaling in the control of asymmetric signal propagation from the LPM to the cardiac field.

The cardiac neural crest is a subpopulation of neural crest cells (NCCs) that originates from the dorsal neural tube and later migrates to the heart and pharyngeal arch (PA), where they contribute to distinct aspects of cardiogenesis and PA patterning. Wnt family members have been implicated in general neural crest induction; however, the molecular mechanisms underlying the specification of distinct cell types such as cardiac NCCs remain unclear. Here, we have identified cysteine-rich intestinal protein 2 ( crip2 ), a gene encoding for a LIM domain–containing protein, as a novel marker for cardiac NCCs in zebrafish. We observed a dynamic expression pattern for crip2 , with expression being detected in the premigratory NCCs in rhombomere 6 (r6), migrating NCCs, ventricular cardiomyocytes, and aortic vessels in PAs 3 to 6. Interestingly, expression of wnt3a was detected in regions of the dorsal neural tube encompassing r6, and reduction of wnt3a led to disrupted expression of crip2 in r6, abnormal migration of NCCs, and defective cardiac function, as well as PA development. Furthermore, these phenotypes could be recapitulated by reduction of crip2 and rescued by overexpression of crip2 . In summary, our data suggest a specific role for Wnt3a in the development of cardiac NCCs. We propose that this function of wnt3a in r6 is partially mediated by crip2 expression in the premigratory cardiac NCCs, which subsequently affects cardiac function and PA patterning.

The genetic study of titin has been notoriously difficult because of its size and complicated alternative splicing routes. Here, we have used zebrafish as an animal model to investigate the functions of individual titin isoforms. We identified 2 titin orthologs in zebrafish, ttna and ttnb , and annotated the full-length genomic sequences for both genes. We found that ttna , but not ttnb , is required for sarcomere assembly in the heart as well as the subsequent establishment of cardiac contractility. In fact, ttna is the earliest sarcomeric mRNA that is expressed in the heart, which makes it an early molecular marker for cardiomyocyte differentiation. Surprisingly, ttna is required for later steps of sarcomere assembly, including the assembly of Z-discs and A-bands, but not for early steps such as the assembly of Z-bodies and nonstriated myosin filaments. Reduction of individual titin isoforms in vivo using morpholino-modified antisense oligonucleotides indicated that (1) both N2B exon–containing and N2A exon–containing isoforms of ttna are required for sarcomere assembly in the heart; (2) N2A exon–containing isoforms of both ttna and ttnb are required for sarcomere assembly in the somites; and (3) the N2B exon–containing isoforms of ttnb are expressed later than other titin isoforms and are probably involved in modulating their expression; however, these isoforms of ttnb are not required for sarcomere assembly. Collectively, our results reveal distinct functions of different titin isoforms and suggest that various phenotypes in “titinopathies” may be attributable to the disruption of different titin isoforms.

ABSTRACT Initially, it was thought that there was no intracellular signaling mediated by NRP‐1 alone in response to its ligands. However, the emerging data from our group as well as others suggest that the signaling through NRP‐1 actually promotes angiogenesis and is mediated through its C‐terminal domain and downstream molecules such as phosphoinositide 3‐kinase. Hence, understanding the signal transduction pathways mediated by NRP‐1 and identification of its downstream molecules are of importance. By using both in vivo zebrafish model and in vitro tissue culture system, we have shown that the C‐terminal three amino acids of NRP‐1 (SEA‐COOH) are required for NRP‐1‐mediated angiogenesis. Furthermore, knocking down of RGS‐GAIP‐interacting protein C terminus (GIPC) in zebrafish, which is associated with C‐terminal domain of NRP‐1, exhibits similar vasculature phenotypes to those from NRP‐1 null. Specific and effective silencing of GIPC in vascular endothelium results in inhibition of NRP‐1‐mediated migration. In both cases as described, PDZ domain of GIPC is responsible for its function. Taken together, our data suggest a novel role of GIPC in angiogenesis and vessel formation and also support our hypothesis that NRP‐1 can facilitate downstream signaling to promote angiogenesis through GIPC.—Wang, L., Mukhopadhyay, D., Xu, X. C terminus of RGS‐GAIP‐interacting protein conveys neuropilin‐1‐mediated signaling during angiogenesis. FASEB J. 20, E732–E741 (2006)