Zebrafish casr affects swim bladder inflation by regulating heart development

Zebrafish casr affects swim bladder inflation by regulating heart development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Zebrafish casr affects swim bladder inflation by regulating heart development Ling Liu, Yuyao Hu, Binling Xie, Junwei Zhu, Ting Zeng, Wen Huang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4498455/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Calcium is fundamental to biological processes, and the Calcium sensing receptor (CaSR) plays a key role in maintaining calcium homeostasis. This process is intimately related to numerous cardiovascular diseases and various types of cancers. However, the role of CaSR in heart development is yet to be thoroughly understood. To delve into this, we conducted a casr gene knockout experiment, analyzed cardiac physiological functions, and performed transcriptomics to investigate the mechanism of the casr gene in zebrafish heart development. Results: We successfully established casr gene knockout lines in zebrafish with Tuebingen (TU) backgrounds. Compared to the control, casr mutant embryos exhibited a smaller heart size, reduced heart rate, and diminished cardiac output. Additionally, these mutants exhibited a curved body structure and a mal-developed swim bladder. Zebrafish larvae began to die at 11 days post-fertilization (dpf). Subsequent transcriptome sequencing andbioinformatics analysis revealed that the loss of casr disrupts cardiac muscle contraction, leading to defective swim bladder inflation and ultimately death. Furthermore, we crossbred casr mutant lines with Tupfel long-fin (TL) background nkx2.5: ZsYellow transgenic lines, and subsequently obtained a casr -/- line where the swim bladder developed normally. Furthermore, qPCR results indicated that the expression of genes linked to cardiac muscle contraction turned to normal. Further experimental results demonstrated that the survival rate of casr mutants was influenced by the TL background. Conclusions: Taken together, casr is vital for zebrafish swim bladder inflation and heart development, exerting its regulatory role through the Wnt signaling pathway and the cardiac muscle contraction. Importantly, the TL background significantly impacts the development of casr zebrafish mutant embryos. casr heart development zebrafish gene knockout Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Calcium (Ca 2+ ) serves as an essential and universal intracellular messenger and is implicated in a multitude of cellular and biological functions [ 1 ], ranging from bone formation and neurotransmission to hormone secretion [ 2 ], muscle contraction [ 3 ], gene expression [ 4 ] and cell proliferation [ 3 , 5 ]. Cells regulate the extracellular calcium ion concentration through various pathways, such as through calcium ion channels or calcium-binding proteins. Calcium ions are the primary physiological ligands of G protein-coupled receptors (GPCRs), which exhibit strong activity in the parathyroid gland and kidney and regulate systemic calcium homeostasis [ 6 – 8 ]. Central to extracellular calcium signaling is the calcium-sensing receptor (CaSR), which is located on the plasma membrane. CaSR, a class C G protein-coupled receptor discovered by Brown and colleagues in 1993 [ 9 ], is the first membrane protein observed to have ion-sensing ability; it contains an extracellular domain consisting of a flytrap (VFT) domain of 613 amino acids and a cysteine-rich domain (CRD), a 7-transmembrane helix (TMD) domain consisting of 250 amino acids and a carboxyl terminus of approximately 200 amino acids [ 10 , 11 ]. The phosphorylation sites of protein kinase A (PKA) and protein kinase C (PKC) are located within their respective C-terminal structural domains. Upon binding with protein kinases, they initiate downstream signaling pathways [ 12 , 13 ]. The human CaSR gene is located on chromosome 3 and is composed of eight exons [ 14 ]. It is expressed in all crucial tissues involved in extracellular calcium homeostasis, such as parathyroid cells, thyrocalcitonin-secreting C cells[ 15 ], bone[ 16 ] and the intestine[ 17 ]. This finding implies its significant role in calcium-related processes. The CaSR protein primarily maintains systemic calcium homeostasis by regulating the biosynthesis and secretion of parathyroid hormone (PTH) [ 18 ]. Upon detecting a decrease in the extracellular calcium concentration, the calcium-sensing receptor (CaSR) stimulates the parathyroid to secrete PTH. Secreted PTH functions to reduce renal calcium excretion, augment intestinal calcium absorption, encourage bone resumption, and release skeletal calcium. On the other hand, elevated physiological calcium levels result in receptor activation, consequently suppressing the synthesis and secretion of parathyroid hormone [ 19 ]. Additionally, numerous studies have demonstrated the expression of CaSR in various other in vivo tissues, including blood vessels, the liver and different regions of the brain[ 20 ]. In conclusion, CaSR is instrumental for maintaining intracellular calcium homeostasis; cell secretion, proliferation, differentiation, chemotaxis and apoptosis[ 21 ]. These intracellular signaling pathways are mutually dependent and can interact with each other. The CaSR also plays an important role in various diseases, including calciotropic disorders caused by its mutations, such as familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, autosomal dominant hypocalcemia, and pseudo-Bartter's syndrome [ 22 , 23 ]. Its abnormal function also causes the development of certain non-calciotropic diseases, such as cardiovascular diseases and cancer [ 24 ]. Lu et al. demonstrated that disturbance of extracellular calcium homeostasis disrupts the calcium balance within the endoplasmic reticulum (ER) and mitochondria, ultimately leading to apoptosis and the release of stress hormones, resulting in heart failure [ 25 ]. Additionally, alterations in serum calcium levels can instigate cardiovascular diseases, including vascular calcification [ 26 ], coronary artery disease and myocardial infarction [ 27 ], and may exacerbate blood pressure and lipid levels, all of which are known risk factors for cardiovascular disease (CVD). In conclusion, it can be speculated that CaSR can act as a protective barrier against hypertension and CVD by regulating the concentration of calcium ions in systemic and local tissues. Zebrafish exhibit significant physiological, pharmacological, and genetic similarities with humans, as they possess organs and systems that resemble those of mammals [ 28 ]. Zebrafish exhibit rapid development, with organogenesis occurring just three days postfertilization. The genome of these viruses comprises approximately 70% of the genes found in humans and, notably, approximately 87% of the known human drug target genes [ 29 , 30 ]. The swim bladder, an organ unique to fish, provides buoyancy and allows fish to maintain balance with minimal energy expenditure within a certain depth range in the water[ 31 ]. Despite humans lacking a swim bladder, this organ bears resemblance to the human lung in aspects of function, structure, development, and transcription. The composition of surfactant in the swim bladder closely parallels that found in the lung [ 32 ]. Furthermore, the maturation of the swim bladder strongly correlates with cardiac function. Research indicates that cardiac impairment can result in the inability to inflate the bladder [ 33 ]. Zebrafish are especially conducive to cardiovascular development studies due to their ability to receive sufficient oxygen through passive diffusion, even when they exhibit the most severe cardiovascular defects. Additionally, the unique attributes of zebrafish, such as optical transparency, in vitro fertilization, and a high number of offspring, greatly facilitate forward genetic screening[ 34 ]. These advantages render zebrafish an appealing vertebrate model organism for congenital heart disease and heart development, as they supplement human and other mammalian studies on this disease. In the present study, we generated three independent casr knockout lines to investigate the role of casr in zebrafish heart development. Our findings revealed that at 5 days post fertilization (dpf), the swim bladder of casr −/− zebrafish fails to form properly, and the homozygotes ultimately died between 11–14 dpf. Subsequent experiments indicated a decrease in heart rate and cardiac output in homozygotes. Notably, in the homozygous with TL background, swim bladder development and heart function defects were rectified, suggesting the significant influence of the TL genetic background on the development of casr mutant zebrafish embryos. Furthermore, we provided evidence that the observed abnormal heart development in casr mutants is closely linked to Wnt signaling. Results Generation of Zebrafish casr knockout lines To investigate the function of casr , we generated casr mutant lines using CRISPR/Cas9 gene editor tools [ 35 ]. Two target sites with a distance of 117 bp were designed. We screened three independent strains, each of which was crossed with a control strain for two generations to mitigate any off-target effects induced by the gene knockout. The mutant lines were obtained from different F0 founders: an 80 bp deletion (named casr hunnu01 ), a 76 bp deletion (named casr hunnu02 ) and an 83 bp deletion (named casr hunnu03 ). Each of these mutations resulted in a disrupted open reading frame and premature stop codons, potentially abrogating all casr functions (Fig. 1 A- 1 D). All the heterozygous zebrafish showed no discernable phenotype and could develop into adults. The genotyping results of F0 and F1 for casr hunnu01 are presented in Fig. 1 E and Fig. 1 F, respectively. The Sanger sequencing and genotyping results for casr hunnu02 and casr hunnu03 could be seen in S1 Fig. We performed real-time quantitative PCR (RT–qPCR) and found that the expression level of casr was significantly decreased in the mutant lines (Fig. 1 G). Loss of casr leads to defective swim bladder inflation and spinal curvature To explore whether casr deletion leads to developmental defects, heterozygous adult fish were crossed, and the resulting embryos were collected for observation at 12-hour intervals. No significant differences were observed between control and casr mutant embryos before 4 dpf. However, at 5 dpf, defective swim bladder inflation was observed in 24% (52/214) of the embryos (Fig. 2 A and 2 B) (S2D Fig). Concurrently, these larvae were found at the bottom of the petri dish and displayed impaired swimming ability. Fifteen larvae were selected for genotyping, and the results showed that all the larvae with defective swim bladder inflation were homozygous (S2A Fig). Then, the embryos with normal swim bladders were genotyped, and the results indicated that these embryos were either heterozygous or wild type (S2B Fig). Furthermore, spinal curvature was observed in larvae with abnormal swim bladders at 7 dpf (Fig. 2 C and 2 D), and in later developmental stages, the symptoms became more pronounced. These larvae were subsequently genotyped, and as expected, all the larvae were homozygous (S2C Fig). Notably, few homozygous mutants died at 11 days, and all the homozygous mutants died at 14 days (S2E Fig). Taken together, these results demonstrated that casr is essential for zebrafish development. Given that the casr hunnu01 , casr hunnu02 and casr hunnu03 larvae exhibited the same defective phenotypes, casr hunnu03 mutants ( casr +/− mutants and casr −/− mutants henceforth) were selected for further analysis. Similar to mammalian lungs, zebrafish swim bladders originate from the foregut endoderm [ 36 , 37 ]. In zebrafish, two Sonic Hedgehog (Shh)-related proteins and two Indian Hedgehog (Ihh)-related proteins have been identified [ 38 – 40 ], along with two homologs of their receptors, Patched 1 (Ptc1) and Patched 2 (Ptc2)[ 41 , 42 ]. The Hedgehog (Hh) pathway is involved in the development of endodermal organs in zebrafish and could also be involved in swim bladder development [ 43 – 45 ]. It has been previously established that the growth and development of the swim bladder are regulated by the Wnt signaling pathway [ 46 ] and that defective swim bladder development caused by blockade of Wnt signaling is partially attributed to reduced cell proliferation and increased cell apoptosis [ 47 ]. To investigate the mechanisms that affect casr -mediated swim bladder development, we employed qPCR to measure the mRNA expression of genes related to the Hh pathway and Wnt signaling pathway during embryogenesis. The results showed that the mRNA expression levels of ihhb , shha , shhb , ptc1 and ptc2 in the Hh pathway (Fig. 2 E) and the mRNA expression levels of fzd1 , fzd5 , fzd7b , tcf3b , tcf3b , wnt1 and wnt9b in the Wnt signaling pathway (Fig. 2 F) were down-regulated in casr −/− . Next, we performed a global transcriptome analysis of casr KO zebrafish vs. WT zebrafish using RNA-seq and visualized the Hh and Wnt signaling pathways through GSEA. The results further substantiated the down-regulation of the Hh (Fig. 2 G) and Wnt (Fig. 2 H) signaling pathways. Our findings indicated that casr regulated swim bladder development via the Hh and Wnt signaling pathways. Loss of casr resulted in reduced ventricular diastole and cardiac output Abnormal swim bladder development, as previously reported, may be a secondary effect of abnormal cardiac function [ 33 ]. To determine whether cardiac function is affected in casr −/− , we examined physiological function. Microphotography was employed to examine the structure and functional performance of the hearts from casr KO zebrafish at 52 hpf. After the high-speed movies were acquired, semi-automated optical analysis was used for further analysis [ 48 ]. M-modes were generated from the movie and included the heart period (HP), diastolic diameter (DD) and systolic diameter (SD) (Fig. 3 A and 3 B). Figure 3 C and 3 D show the control and casr mutant ventricular morphology, respectively. Compared with those in the control group, the heart rate and cardiac output were notably lower in the casr −/− group (P < 0.01), suggesting that cardiac function was significantly impaired in the casr KO zebrafish (Fig. 3 F and 3 G). Moreover, by analyzing the long diameter, short diameter and area of the control and mutant ventricles, we found that these three parameters were significantly lower during diastole than in the control ( p < 0.01). However, no significant difference was observed during the systolic stage (Fig. 3 H- 3 J). Collectively, these findings suggest that the casr gene influences ventricular diastole and cardiac output. casr was required for heart development in zebrafish To elucidate the impact of casr KO on heart development, we conducted an RNA-seq experiment and bioinformatics analysis. A total of 702 genes were differentially expressed between the control and casr −/− zebrafish, including 337 up-regulated genes and 365 down-regulated genes (Fig. 4 A). Subsequent signaling pathway analysis, as annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG), revealed that casr deficiency influences cardiac muscle contraction (Fig. 4 B). In addition, MeV software was used to generate heatmaps for the DEGs to facilitate further selection of genes with similar functionalities (Fig. 4 C). To discern the biological processes altered by casr disruption, a Gene Ontology (GO) analysis was performed using the DAVID bioinformatics resource [ 49 ]. The analysis showed that the major biological processes associated with the significant changes were associated primarily with molecular function (Fig. 4 D). Extracellular calcium ions play an essential role in the nonclassical Wnt signaling pathway [ 50 ]. We next carried out a GSEA-based KEGG signal enrichment analysis and visualized four signaling pathways: calcium signaling, cardiac muscle contraction, adrenergic signaling in cardiomyocytes, and the actin cytoskeleton. The results demonstrated that the mRNA expression levels of the genes within these four signaling pathways were down-regulated (Fig. 4 E). Since the primary function of CaSR is to regulate intracellular and extracellular calcium levels to maintain systemic calcium homeostasis [ 14 ], we then performed RT‒qPCR to evaluate the mRNA expression of genes related to the calcium signaling pathway to further explain the molecular regulatory mechanism of casr in zebrafish development. Our results showed that the mRNA expression levels of genes, including ncx1b , pmca2 , pth2 , actc1 , vdra , vdrb and runx2a , were significantly lower in casr −/− embryos than in control embryos ( p < 0.05). However, the mRNA expression level of pth1 was significantly increased in casr −/− embryos than in WT embryos ( p < 0.01) (Fig. 4 F). According to the results, we assumed that casr influences Ca 2+ levels together with sodium-calcium channels and hormones, thereby regulating calcium homeostasis. To further validate the transcriptome sequencing results, six down-regulated genes in the cardiac muscle contraction pathway were selected, including fosab , tnnt2a , tnni2b.1 , cnn1a , myhc4 and myh7l . The RT‒qPCR results showed that the mRNA expression of these genes was down-regulated in the casr −/− zebrafish compared to the control zebrafish (Fig. 4 G), demonstrating that loss of casr affects the contractile function of cardiomyocytes. The functions of the abovementioned genes are shown in S1 Table. Taken together, these data suggested that casr KO affects heart development by affecting the calcium signaling pathway and cardiac muscle contraction. casr deletion-induced phenotypic defects and impairments in cardiac physiological function can be partially mitigated in zebrafish with a Tupfel long-fin background As the loss of casr resulted in impaired cardiac physiology, the casr +/− fish were subsequently crossed with nkx2.5: ZsYellow transgenic fish on a TL background to better understand the role of casr in heart development. Embryos expressing yellow fluorescence were selected at 24 hpf, and genotyping was carried out two months later. After that, the embryos generated from heterozygous crosses were raised and genotyped. Contrary to our expectations, the homozygous individuals survived, and their swim bladders developed normally (Fig. 5 A- 5 C). This finding was unexpected, as all three lines could not survive on a TU background for approximately five generations but could mature to adulthood on a TL background. Therefore, we hypothesized that the TL background of nkx2.5: ZsYellow transgenic fish could compensate for the defect caused by the deletion of casr . Additionally, we observed that some homozygotes did not exhibit a nkx2.5: ZsYellow fluorescence signal, but all homozygotes had long fins, a characteristic feature of the TL background (S3A and S3B Fig). Therefore, we propose that the TL background could have mitigated the defective phenotype. To further confirm the genetic background of these homozygotes, we conducted a gene mapping cloning experiment. We discovered that the product amplified by the primer of linkage group 13 (LG13) could be used to distinguish between the TU and TL backgrounds by agarose gel electrophoresis. The genomic DNA was extracted from the tail fin of the adult homozygotes for background identification. The results indicated that all the homozygotes had either a pure TL background or a mixture of TU and TL backgrounds, with no fish having a pure TU background (S3C Fig). This finding supported our hypothesis that zebrafish with a TL background could compensate for swim bladder defects or that other genes that could rescue the casr mutant presented during the casr +/− cross-occurrence with nkx2.5: ZsYellow transgenic fish. To investigate whether the surviving casr mutant could ameliorate cardiac function defects, we compared the physiological functions of wild type and homozygous from the two backgrounds. The results showed no significant difference in heart rate between the TL background casr −/− and TU background casr −/− zebrafish (Fig. 5 L), but there was a significant increase in cardiac output (Fig. 5 M). Figure 5 D- 5 F and 5 G- 5 I show M-Mode images and ventricle morphology of the control, TU background casr −/− and TL background casr −/− zebrafish, respectively (Movies S1-3). Subsequently, RT‒qPCR was performed with a control and two background homozygotes. The results showed that most genes in the calcium signaling pathway and cardiac muscle contraction pathway were up-regulated in the TL homozygotes compared with the TU homozygotes (Fig. 5 J and 5 K), which further confirmed our hypothesis that the TL background can rescue defects in cardiac physiological function. casr is required for zebrafish cardiomyocyte differentiation To determine the role of the casr gene in zebrafish cardiomyocyte differentiation, we analyzed the expression levels of the cardiac differentiation markers cmlc1 and cmlc2 using in situ hybridization experiments. The results revealed that the heart of mutant was smaller than that of the wild type (TU), which is consistent with previous results (Fig. 6 A- 6 F). To further determine whether the mRNA expression levels of these two genes were changed, we performed RT‒qPCR. The results showed that the mRNA expression levels of cmlc1 and cmlc2 were significantly lower in the casr −/− of TU background than in the WT (TU) (p < 0.001), and compared to casr −/− of TU background, the mRNA expression level of cmlc2 was significantly higher in the casr −/− of TL background ( p < 0.001); however, cmlc1 expression was not significantly different. These findings demonstrated that casr affected zebrafish cardiomyocyte differentiation in the TU background but not in the TL background (Fig. 6 G). Discussion CaSR is implicated in a variety of cardiac pathological processes. It triggers numerous intracellular signaling pathways associated with diseases such as atherosclerosis, vascular calcification, cardiomyopathy, cardiac fibrosis, and myocardial infarction [ 22 , 25 , 51 ]. Our study elucidated the crucial roles of zebrafish casr during early development. Utilizing CRISPR/Cas9 technology, we generated three casr -mutant zebrafish lines and discovered that casr deficiency led to morphogenetic abnormalities in the swim bladder and spine. All the homozygous larvae in the TU genetic background died before 14 dpf. It has been reported that bladder inflation is complete when zebrafish develop to 120 hpf. At this developmental stage, zebrafish larvae start to feed and need to take up exogenous nutrients to maintain their normal growth and development. The swim bladder plays a crucial role in enabling larvae to float in water and engage in predation. Should the swim bladder fail to develop or inflate at this point, the larvae experience a loss in motility and predation capacity, ultimately resulting in death due to nutritional deficiency [ 52 ]. Furthermore, studies have reported a correlation between the failure of swim bladder inflation and cardiac dysfunction. Subsequently, we analyzed cardiac physiological function in both control and casr mutant larvae. Our findings indicate that, compared to those in the control group, the heart rate and cardiac output in the mutant zebrafish were significantly lower. Additionally, both the long and short diameters of the heart decreased at diastole. It has been suggested that an increased concentration of extracellular Ca 2+ or other CaSR agonists might lead to increased sarcoplasmic reticulum Ca 2+ release through the G protein-phospholipase C-inositol triphosphate signaling pathway, thereby triggering cardiac contraction [ 30 ]. Conversely, a decrease in CaSR expression could result in a reduced Ca 2+ concentration in the sarcoplasmic reticulum, impairing myocardial cell excitation–contraction coupling and subsequently leading to compromised myocardial contractile function [ 53 , 54 ]. These findings suggest that the absence of casr could lead to heart failure, which is often characterized by impaired cardiac contractility or arrhythmias. The Wnt signaling pathway, implicated in the induction of cardiac fibrosis and in cardiac fibroblasts (CFs), represents a potential therapeutic target for cardiac fibrosis [ 55 ]. It exerts significant influence on embryonic development, adult tissue homeostasis, and regeneration. The canonical Wnt/β-catenin signaling pathway differentially regulates cardiac development at different developmental stages, with early promotion followed by later inhibition [ 56 ]. However, the role of the Wnt/β-catenin signaling pathway in postnatal cardiomyocyte (CM) growth and development has been scarcely explored in previous studies. The development of the swim bladder has been primarily linked to the Wnt signaling pathway. The inhibition of Wnt signaling interferes with the formation of epithelial, mesenchymal, and mesothelial tissue layers in the swim bladder of zebrafish, consequently hindering their growth and differentiation[ 47 ]. The relevance of Hh signaling pathways in the development of lungs in mice and chickens has been underscored [ 57 , 58 ]. This study investigated the impact of casr knockout on the Wnt and Hh signaling pathways in zebrafish through quantitative analysis. The results showed that the mRNA expression of genes in both signaling pathways was down-regulated, which suggested that loss of casr affects the Wnt and Hh signaling pathways, leading to defective swim bladder inflation, thus affecting the normal growth and development of zebrafish. Given that the primary function of casr is to maintain calcium homeostasis by regulating the calcium ion concentration, a quantitative analysis of the ncx1b , pmca2 , pth2 , vdra , vdrb , actc1 , and runx2a genes in the calcium signaling pathway was also conducted. The results demonstrated a general down-regulation of these genes, whereas pth1 exhibited an upward trend. As the calcium signaling pathway is a part of the nonclassical Wnt signaling pathway, it is suggested that casr regulates calcium homeostasis via the Wnt signaling pathway. Following casr knockdown, the parathyroid hormone pth1 undergoes a compensatory expression increase to maintain calcium homeostasis. However, this study examined only the RNA level. The impacts of casr knockout on the translation and posttranslational modification of these proteins warrant further investigation. The main function of the heart is to receive and pump blood. This is achieved through the coordinated contraction of the ventricles, which relies on the contractile function and electrophysiological activity of cardiomyocytes. Cardiac systolic function is primarily regulated by the sliding of actin filament units, known as myofibrils, which enable cardiac muscle cells to contract and eject blood from the heart[ 59 ]. Wang et al.[ 60 ] initially reported the expression of Casr in neonatal rat cardiomyocytes, highlighting that an increase in calcium levels within in vitro cell cultures correspondingly escalated intracellular calcium levels and cardiac activity. To further investigate the impact of casr genes on zebrafish heart development, we carried out transcriptome sequencing and bioinformatics analysis to identify potential genes influencing phenotype and function. Subsequently, 702 significantly varied candidate genes were identified and subjected to further GO enrichment and KEGG signaling pathway analysis. The analysis indicated that the up-regulated genes were predominantly enriched in cellular processes such as cell matrix adhesion, protein folding, cell differentiation and redox, with no discernible pathways related to cardiac developmental signaling. Conversely, the down-regulated genes were chiefly enriched in protein hydrolysis, the immune response, lipid metabolism, energy metabolism, and other processes intimately associated with calcium ions. KEGG signaling pathway analysis revealed that these genes were enriched primarily in the cardiac muscle contraction pathway. Moreover, the MAPK, FoxO, and mToR signaling pathways potentially interact with the Wnt signaling pathway, thereby influencing zebrafish heart development. Subsequent RT‒qPCR analysis of the mRNA expression of several down-regulated genes revealed a significant decrease ( p < 0.05) in the mRNA expression of genes associated with cardiovascular tissue, myocardial tissue regeneration, calmodulin, cardiac contraction, myosin, and troponin. This finding substantiates that casr knockout indeed impacts zebrafish heart function. The core genes of the relevant pathways were analyzed using KEGG, the results of which are shown in a chord diagram (Fig. 7 ). However, the exact regulatory mechanism involved remains unclear and warrants further scrutiny. Considering that homozygotes within the TU background are nonviable, casr +/− zebrafish were crossed with nkx2.5: ZsYellow transgenic fish on a TL background to further elucidate the role of casr in zebrafish cardiac function. We selected embryos demonstrating heart-specific yellow fluorescence expression for subsequent experiments. Intriguingly, none of the embryos from these casr +/− incrosse exhibited swim bladder deficiency or a curved spine phenotype. All the zebrafish survived and reproduced normally, and all the homozygotes displayed an elongated fin phenotype. Prior studies have indicated that diverse zebrafish strains possess unique morphological and behavioral traits, such as AB, Tubingen (TU), Wild India Kolkata (WIK), and Tupfel long fin (TL) strains. These traits, including swimming ability, are influenced by factors such as water temperature, caudal fin length, and genetic background [ 61 ]. Furthermore, variations in fin size, inherited traits, and physiological and behavioral characteristics in the juvenile and adult stages have been observed [ 62 – 64 ]. We thus postulated that the nkx2.5: ZsYellow transgenic fish with a TL background could effectively counteract defects induced by casr deletion. In a subsequent screening, some nonfluorescent homozygotes were observed to grow normally into adulthood and reproduce, suggesting that the nkx2.5 transcription factor does not contribute to defect rectification. Physiological functional tests and RT‒qPCR experiments were subsequently conducted, which revealed partial restoration of cardiac function in the TL homozygotes compared to their TU counterparts. Real-time fluorescence further revealed significant upregulation of the majority of genes within the Wnt signaling pathway, calcium signaling pathway, and cardiac muscle contraction pathway. These results collectively suggest that the TL background can markedly ameliorate the developmental defects of casr zebrafish mutants. However, it remains unclear whether the TL background directly influences cardiac development or alters the expression levels of associated genes, ultimately affecting cardiac development (Fig. 8 ). Muscle myosin is a hexamer that consists of two myosin heavy chains (MHCs), two regulatory light chains (RLCs) and two essential light chains (ELCs)[ 65 ]. Since ELCs and RLCs contain two calcium-binding EF-hand motifs, they may regulate the calcium sensitivity of force generation and crossbridge kinetics, thereby fine-tuning cardiac muscle contraction [ 66 ]. Mutations in the genes encoding ELCs and RLCs have been shown to be causally associated with 1% of human cardiomyopathies[ 67 , 68 ]. In zebrafish, the cardiomyocyte differentiation marker genes cmlc1 and cmlc2 are the primary homologs of ELC and RLC, respectively. Therefore, we performed in situ hybridization and RT‒qPCR experiments on these two marker genes. The RT‒qPCR results showed that the expression of both genes was significantly lower in the TU background homozygote than in the control ( p < 0.05) and that the expression level of the cmlc2 gene was significantly higher in the TL background homozygote than in the TU background homozygote, whereas the mRNA expression of cmlc1 remained unchanged. Previous research has indicated that deletion of cmlc1 in zebrafish results in an increase in both the size and number of cardiomyocytes, leading to enlarged ventricular chamber volumes. Conversely, deletion of cmlc2 causes a reduction in the size and number of cardiomyocytes [ 69 ]. These findings further substantiate that the loss of casr can result in a smaller heart and that casr plays a pivotal role in heart development. However, the relationships between these two genes and casr , as well as their interactions with one another, need to be further explored. In summary, the data in this study revealed a critical role of casr in heart development, and different genetic backgrounds had different effects on heart development in casr -mutant zebrafish. Furthermore, we found that loss of casr resulted in down-regulated expression of genes involved in the Wnt and cardiac muscle contraction pathways. The TL genetic background could significantly affect the development of casr mutant embryos. Conclusion Herein, we demonstrate the importance of the casr gene in regulating zebrafish development and its close relationship with swim bladder inflation and heart function. Our RT‒qPCR and transcriptome sequencing experiments revealed that the Wnt signaling pathway and cardiac muscle contraction pathway were down-regulated after casr knockout. Additionally, we observed that different genetic backgrounds have varying regulatory functions. In summary, our work provides new insights into the role of the casr gene in heart development. Materials and Methods Zebrafish husbandry and ethics approval Zebrafish TU and TL lines were used and maintained under standard conditions in this study. Embryos were collected from natural mating, incubated at 28.5°C and staged according to standard protocols[ 70 ]. All the experiments were performed following animal use protocols approved by the Animal Care and Use Committee of Hunan Normal University. CRISPR/Cas9-mediated gene knockout casr mutant lines were generated with the CRISPR/Cas9 system following previous methods [ 71 ]. Briefly, the casr target site sequences are located in the second exon, and the two target sites are 117 bp apart from each other. The sgRNAs were synthesized in vitro with T7 RNA polymerase. A total of 400 pg of Cas9-mRNA and 50 pg of casr -gRNA were co-injected into zebrafish embryos at the one-cell stage. For genotyping, polymerase chain reaction (PCR) was carried out using the following primer sets: 5′- GGCTGTTGAAACTAGAGGAAA-3’ and 3′- GAGAAGCTGACCATGTTTTGT-5’. Quantitative real-time PCR (RT–qPCR) Total RNA was prepared from 50 embryos at 5 dpf using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. First-strand cDNA was generated using a reverse transcription kit (TaKaRa) with random primers. Quantitative real-time PCR (qPCR) was performed using a SYBR green kit (Takara, Dalian, China) on a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific). Relative expression levels of the tested mRNAs were determined using β-actin as an internal reference and the comparative Ct (2 −△△Ct ) method ( p < 0.05). The sequences of primers used in this study are listed in S2 Table. Whole-mount in situ hybridization (WISH) Zebrafish embryos at the desired stages were fixed in 4% paraformaldehyde (PFA) overnight before processing for WISH analysis as previously described [ 72 ]. Digoxigenin-UTP-labeled antisense RNA probes for cmlc1 and cmlc2 were generated via an in vitro transcription method using T7 RNA polymerase (Thermo Fisher, Waltham, MA, USA). RNA sequencing A total of 6 cDNA libraries were prepared from three biological replicates of casr −/− and wild type zebrafish at 5 dpf. RNA quality was confirmed by a Nano200 nucleic acid analyzer (allsheng, China), and the libraries were sequenced with an Illumina Nova seq6000. The RNAseq results were validated by qPCR. The results represent the average of 3 repeat experiments, and each experiment was repeated 3 times. The RNA sequencing data from this study were deposited in the NCBI Sequence Read Archive under accession SRA: PRJNA967502, PRJNA967502, PRJNA967502, PRJNA967502, PRJNA967502, PRJNA967502. Cardiac function analysis At least 10 juvenile zebrafish were selected and anesthetized with triclocaine. Movies of beating hearts from embryos at 52 h postfertilization (hpf) were recorded using an IX71-Olympus inverted microscope equipped with a Hamamatsu C9300 digital camera. Heart rate was counted for 15 s and then multiplied by 4 to calculate the heart rate in beats per minute (bpm). The long-diameter (a), short-diameter (b) and M-mode heart segments at diastole and systole were obtained by semi-automated optical analysis[ 48 , 73 ]. All images and data were obtained from zebrafish ventricles. Each experiment was repeated at least three times to ensure reliability. The ventricular volume was measured by the following ellipsoidal formula: \(\text{V}\text{=}\frac{\text{4}}{\text{3}}\text{π×(}\frac{\text{a}}{\text{2}}\text{)×}{\text{(}\frac{\text{b}}{\text{2}}\text{)}}^{\text{2}}\) \(\text{Stroke volume=}{\text{V}}_{\text{diastole}}\text{-}{\text{V}}_{\text{systole}}\) \(\text{Cardiac output=Stroke volume}\text{ }\text{×}\text{ }\text{heart rate}\) Bioinformatics analysis Gene set enrichment analysis (GSEA)[ 74 ] was performed on the list of genes sorted by fold change in the experiment, and the enrichment of the up- or down-regulated gene sets in the KEGG pathway database was calculated. The gene sets with fewer than 15 genes or more than 500 genes were excluded, and the t-statistic mean of the genes was computed for each KEGG pathway using a permutation test with 1000 replications. The up-regulated pathways were defined by a normalized enrichment score (NES) > 0, and the down-regulated pathways were defined by an NES < 0[ 75 ]. R v3.2.2 was used to perform the data preprocessing. The mapping process was performed through packages such as ClusterProfiler and Bioconductor. A chord diagram was drawn on Sangerbox [ 76 ]. Gene mapping cloning experiment The genetic background of the zebrafish was distinguished based on simple sequence length polymorphisms (SSLP) on different chromosomes[ 77 , 78 ]. First, primers for identifying 25 pairs of sequence tagging sites (STSs) on zebrafish chromosomes were found in the NCBI database, after which the wild-type (WT) zebrafish genome with a TU and TL background was used as a template for in vitro amplification. The sequences of primers used for LG13 (linkage group 13) in this study were as follows: Z6007-F, AGCCCTAAACAAACCGGC; and Z6007-R, GAGCATGATCTGCTCGTTAGG. Statistical analysis All the values are expressed as the means ± SEMs. The statistical analysis was performed using Student’s t test. P values < 0.05 were considered to indicate statistical significance and are indicated with asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001). Data availability The source data behind the figures can be found in S1 Raw Data. Declarations Acknowledgments We would like to express our appreciation to all the members of the Laboratory of Animal Nutrition and Human Health at Hunan Normal University for their assistance and encouragement. Author contributions Conceptualization: Ling Liu, Chengbo Yang, Huaping Xie. Data curation: Ling Liu, Jiaxin Liang. Funding acquisition: Wen Huang, Huaping Xie. Investigation: Junwei Zhu, Jian Huang, Xiangding Chen. Methodology: Ling Liu, Yuyao Hu, Binling Xie, Ting Zeng, Huaping Xie. Project administration: Jianzhong Li, Huaping Xie, Xiangding Chen. Resources: Wen Huang, Xiaochun Lu, Huaping Xie. Software: Ling Liu, Yuyao Hu, Binling Xie, Ting Zeng. Supervision: Jianzhong Li, Huaping Xie, Xiangding Chen. Validation: Ling Liu, Yuyao Hu. 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Supplementary Files Supportinginformation.docx S1Movie.mp4 S2Movie.mp4 S3Movie.mp4 floatimage1.jpeg Graphical Abstract casr affects heart development through the cardiac muscle contraction pathway Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4498455","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":311872770,"identity":"11a907c8-30b0-44b5-96c4-68d8a86b6cb0","order_by":0,"name":"Ling Liu","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Liu","suffix":""},{"id":311872771,"identity":"94652f62-25e1-4a86-b3e3-264a4d74662c","order_by":1,"name":"Yuyao Hu","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yuyao","middleName":"","lastName":"Hu","suffix":""},{"id":311872772,"identity":"c4519c57-c81d-4edc-98bc-5141826b78ff","order_by":2,"name":"Binling Xie","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Binling","middleName":"","lastName":"Xie","suffix":""},{"id":311872773,"identity":"9a41e420-179f-469d-b838-87aee2a2b71f","order_by":3,"name":"Junwei Zhu","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Junwei","middleName":"","lastName":"Zhu","suffix":""},{"id":311872774,"identity":"864cf8b4-034f-4906-acdb-d038edf6e9a9","order_by":4,"name":"Ting Zeng","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Zeng","suffix":""},{"id":311872775,"identity":"2853d1da-6dc1-498f-8f2f-b67240fa9a44","order_by":5,"name":"Wen Huang","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Huang","suffix":""},{"id":311872776,"identity":"cccc228e-bb3d-4f29-b66c-3f93fef0b7f2","order_by":6,"name":"Jian Huang","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Huang","suffix":""},{"id":311872777,"identity":"aec5f05b-06a5-45f7-a8f3-5b64846a25d3","order_by":7,"name":"Xiaochun Lu","email":"","orcid":"","institution":"Chinese PLA General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaochun","middleName":"","lastName":"Lu","suffix":""},{"id":311872778,"identity":"4cb8d6da-7271-4c41-90dc-c8b68d0e1206","order_by":8,"name":"Chengbo Yang","email":"","orcid":"","institution":"University of Manitoba","correspondingAuthor":false,"prefix":"","firstName":"Chengbo","middleName":"","lastName":"Yang","suffix":""},{"id":311872779,"identity":"c81b3c16-ed94-4dcb-a7a9-fcc98ab3ee91","order_by":9,"name":"Jianzhong Li","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jianzhong","middleName":"","lastName":"Li","suffix":""},{"id":311872780,"identity":"cdbbf45b-c042-4090-a879-88d4e4e6cb0e","order_by":10,"name":"Xiangding Chen","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiangding","middleName":"","lastName":"Chen","suffix":""},{"id":311872781,"identity":"05434f2a-f21f-4e21-90d4-46ebee295dfa","order_by":11,"name":"Huaping Xie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACAyA+wFBxAMplI1rLGVK1MDC2kaLFXLrH8MDHeXcSt0v3GDB8KDvMwD+7Ab8WyznHEg7O3PYsceecMwaMM84dZpC4cwC/FoMbyQcO8247nLjhRo4BM2/bYQYDiQRCWhIbDv+dA9XylzgtQFsYG6BaGInScgfol55jh4033EgrONhzLp1H4gYhLbd7jD/8qDksu+FG8sYHP8qs5fhnENDCIIHEPgDEPATUo2kZBaNgFIyCUYAVAAC9Wk8amWVmwgAAAABJRU5ErkJggg==","orcid":"","institution":"Hunan Normal University","correspondingAuthor":true,"prefix":"","firstName":"Huaping","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2024-05-29 16:36:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4498455/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4498455/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58235793,"identity":"7f807d91-2d57-4590-b40c-bd5ced3a2135","added_by":"auto","created_at":"2024-06-12 21:41:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5100313,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ecasr \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ewas knocked out in zebrafish.\u003c/strong\u003e (A) Schematic representation of the CRSIPR/Cas9 target region in the \u003cem\u003ecasr\u003c/em\u003e gene. The green box represents the exon, the red box indicates the targeted exon, and the solid lines signify introns. (B-D) Comparative analysis of the genomic DNA and amino acid sequences of the WT (TU) and three independently mutated alleles. The \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu01\u003c/sup\u003e mutant had 80 bp deleted, the \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu02\u003c/sup\u003e mutant had a 76 bp deletion, and the \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu03\u003c/sup\u003e mutant had an 83 bp deletion. The target sequences are highlighted in red. The rectangular box indicates deletion sites in the mutants, while the blue box signifies the termination of protein translation. (E, F) Screening results for the F0 (E) and F1 (F) generation adult fish in the \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu01\u003c/sup\u003e. M: DNA marker; Open and closed arrowheads denote wild type and knockout alleles, respectively. (G) RT‒qPCR analysis of the expression level of \u003cem\u003ecasr\u003c/em\u003e in the mutant line at 5 dpf. n = 3 independent experiments. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. WT: wild type of TU.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/626215786b0c3ac50a8463a2.png"},{"id":58235792,"identity":"863c38e0-6218-4c22-8026-4b97c2192c6a","added_by":"auto","created_at":"2024-06-12 21:41:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":679807,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ecasr\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deficiency leads to abnormalities in the swim bladder and spine.\u003c/strong\u003e (A-D) Compared to control larvae, \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e larvae exhibited abnormal swim bladder development at 5 dpf (A, B), and spinal curvature occurred at 7 dpf (C, D). The red and blue arrowheads indicate the positions of the swim bladder and spine, respectively. (E, F) The relative expression of mRNAs associated with the Hh signaling pathway (E) and the Wnt signaling pathway (F) in the control and \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e (F). (G, H) Line chart of GSEA results for the Hh signaling pathway (G) and Wnt signaling pathway (H). The ordinate represents the enrichment score (ES), and the ES at the highest peak is the ES value of the gene set. All the genes were tested with 3 replicates (N = 3), each containing 50 embryos (n = 50). The data are presented as the means ± SEMs. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/dde07450f99150e07768d96a.png"},{"id":58235947,"identity":"ee146488-43f3-4a4d-8a02-a148d6b9891c","added_by":"auto","created_at":"2024-06-12 21:49:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":923532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysiological Functions Analysis.\u003c/strong\u003e (A, B) Five-second M-Modes obtained from video footage of both wild type and \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e heart ventricles are presented. HP: heart period; DD: diastolic diameter; SD: systolic diameter. (C, D) The ventricular morphology at the end of diastole in both the wild type and \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e zebrafish is shown. (E) A diagram demonstrating the heart position of the long and short diameters is provided. (F-J) A comparative analysis of heart rate (F), cardiac output (G), short diameter (H), long diameter (I), and cross-sectional area (J) between control and \u003cem\u003ecasr\u003c/em\u003e mutant zebrafish was conducted. The data are presented as the means ± SEMs (n\u0026gt;10). \u003csup\u003ens\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/526b147fa0051f9f70664887.png"},{"id":58236297,"identity":"91062cf0-5c1e-4150-bffc-9c26afe2431e","added_by":"auto","created_at":"2024-06-12 21:57:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2840674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDepicts the regulation of cardiac muscle contraction via the calcium signaling pathway in the absence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecasr\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) A volcano plot of the differentially expressed genes is presented, with up-regulated genes denoted by red dots and down-regulated genes denoted by blue dots. (B) A KEGG enrichment bubble diagram showing the different genes. The bubble size corresponds to the number of genes, and the bubble color represents the p-correction value. The y-axis shows the pathway name, and the x-axis displays the enrichment index. (C) A cluster diagram of the differentially expressed genes is shown. Red indicates relatively highly expressed protein-coding genes, blue indicates relatively low protein-coding genes, and gray color signifies genes with nonsignificant differences. (D) A diagram of the GO enrichment analysis of biological processes in \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e zebrafish is displayed, with the y-axis representing the number of genes and the x-axis showing the name of the subclass. (E) A line chart of the GSEA results is shown, where the right ordinate represents the enrichment score. (F, G) The relative mRNA expression of genes in the calcium signaling pathway (F) and the cardiac muscle contraction pathway (G) in the wild type (TU) and \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e zebrafish at 5 dpf is shown. All the genes were tested with 3 replicates (N = 3), each containing 50 embryos (n = 50). The data are presented as the means ± SEMs. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/626af6ea7542c79e01109516.png"},{"id":58235949,"identity":"87320f75-86d7-4f13-9c09-89d400704a1a","added_by":"auto","created_at":"2024-06-12 21:49:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1005739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTL gene background can rescue the defects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecasr\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant zebrafish embryos.\u003c/strong\u003e (A-I) Diagrams of the phenotype (A-C), M-Mode (D-F) and ventricular morphology (G-I) of TU, TU \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e and TL \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e zebrafish. The red arrow indicates the swim bladder. HP: heart period; DD: diastolic diameter; SD: systolic diameter. (J, K) mRNA expression levels of genes involved in the calcium signaling pathway (J) and cardiac muscle contraction pathway (K) in wild-type of TU, TU \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e and TL \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e zebrafish. (L, M) Comparison of heart rate (L) and cardiac output (M) between the wild type and mutant lines. The plotted data are presented as the means ± SEMs (n\u0026gt;10). \u003csup\u003ens\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/066aeb98a1c262fe1096eefe.png"},{"id":58235800,"identity":"6c2b7014-b194-4e0e-9cf4-651f8bd28979","added_by":"auto","created_at":"2024-06-12 21:41:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":911839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of myocardial differentiation genes.\u003c/strong\u003e (A-F) In situ hybridization results of \u003cem\u003ecmlc1\u003c/em\u003e (A-C) and \u003cem\u003ecmlc2\u003c/em\u003e (D-F) in the wild type of Tuebingen (TU), TU background \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e and TL background \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e at 48 hpf. n\u0026gt;30. (G) The mRNA expression of the \u003cem\u003ecmlc1\u003c/em\u003e and \u003cem\u003ecmlc2\u003c/em\u003e genes in the WT (TU) and \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e lines with different genetic backgrounds. The plotted data are presented as the means ± SEMs (n\u0026gt;10). \u003csup\u003ens\u003c/sup\u003e p \u0026gt; 0.05; * p \u0026lt; 0.05; ** p \u0026lt; 0.01; *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/2a079f0c745eddca4b37f8f0.png"},{"id":58235796,"identity":"682f09c8-f100-4669-b1e5-f279b6971080","added_by":"auto","created_at":"2024-06-12 21:41:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2620024,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChord diagram illustrating the key genes within the significant pathways.\u003c/strong\u003e The fold change in the expression of these core genes is depicted on the left, while connections from left to right represent the gene’s membership within a pathway’s leading-edge subset.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/6c64b7fe4bfac567d7414486.png"},{"id":58235797,"identity":"dcce1b79-563c-4722-a01d-8c46640eaf75","added_by":"auto","created_at":"2024-06-12 21:41:42","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":491860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression changes of cardiac muscle contraction pathway genes after \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecasr\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene knockout.\u003c/strong\u003e The green shading and red indicate down-regulated and up-regulated expression in the \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003ewith a TU background, while the orange shading and gray indicate up-regulated and insignificant differences expression in the \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e with a TL background.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/a127d293c38180002d1b870d.png"},{"id":59674709,"identity":"14c8d0ca-bb50-4ebf-a2e5-535761e3a1b8","added_by":"auto","created_at":"2024-07-04 15:17:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15503045,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/3fd7c32b-e962-4c37-a692-a2ea635b116a.pdf"},{"id":58235804,"identity":"0fb4e9f6-da6e-4551-950e-8fbe64a8628d","added_by":"auto","created_at":"2024-06-12 21:41:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":41380340,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/4f60f9c1dd0557d8628c0f65.docx"},{"id":58236298,"identity":"70979bf8-2301-4fc4-9418-0b30e5ee65b1","added_by":"auto","created_at":"2024-06-12 21:57:42","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14042742,"visible":true,"origin":"","legend":"","description":"","filename":"S1Movie.mp4","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/d33b9c2310c2825f03148af8.mp4"},{"id":58235951,"identity":"da86d663-7344-45cc-b248-01731bcfa752","added_by":"auto","created_at":"2024-06-12 21:49:42","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14059573,"visible":true,"origin":"","legend":"","description":"","filename":"S2Movie.mp4","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/efbdada3a491b3757a6ff4ee.mp4"},{"id":58235803,"identity":"b2989c00-56c4-470f-a93c-e27832e1f10a","added_by":"auto","created_at":"2024-06-12 21:41:42","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14111722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"S3Movie.mp4","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/b97933a752ad692f5658d248.mp4"},{"id":58235802,"identity":"ada4b231-06e2-4706-a998-8e1405545278","added_by":"auto","created_at":"2024-06-12 21:41:42","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":399362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ecasr\u003c/em\u003eaffects heart development through the cardiac muscle contraction pathway\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4498455/v1/253c6d611ec697ed1ce09804.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Zebrafish casr affects swim bladder inflation by regulating heart development","fulltext":[{"header":"Background","content":"\u003cp\u003eCalcium (Ca\u003csup\u003e2+\u003c/sup\u003e) serves as an essential and universal intracellular messenger and is implicated in a multitude of cellular and biological functions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], ranging from bone formation and neurotransmission to hormone secretion [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], muscle contraction [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], gene expression [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and cell proliferation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Cells regulate the extracellular calcium ion concentration through various pathways, such as through calcium ion channels or calcium-binding proteins. Calcium ions are the primary physiological ligands of G protein-coupled receptors (GPCRs), which exhibit strong activity in the parathyroid gland and kidney and regulate systemic calcium homeostasis [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCentral to extracellular calcium signaling is the calcium-sensing receptor (CaSR), which is located on the plasma membrane. CaSR, a class C G protein-coupled receptor discovered by Brown and colleagues in 1993 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], is the first membrane protein observed to have ion-sensing ability; it contains an extracellular domain consisting of a flytrap (VFT) domain of 613 amino acids and a cysteine-rich domain (CRD), a 7-transmembrane helix (TMD) domain consisting of 250 amino acids and a carboxyl terminus of approximately 200 amino acids [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The phosphorylation sites of protein kinase A (PKA) and protein kinase C (PKC) are located within their respective C-terminal structural domains. Upon binding with protein kinases, they initiate downstream signaling pathways [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe human \u003cem\u003eCaSR\u003c/em\u003e gene is located on chromosome 3 and is composed of eight exons [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. It is expressed in all crucial tissues involved in extracellular calcium homeostasis, such as parathyroid cells, thyrocalcitonin-secreting C cells[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], bone[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and the intestine[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This finding implies its significant role in calcium-related processes. The CaSR protein primarily maintains systemic calcium homeostasis by regulating the biosynthesis and secretion of parathyroid hormone (PTH) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Upon detecting a decrease in the extracellular calcium concentration, the calcium-sensing receptor (CaSR) stimulates the parathyroid to secrete PTH. Secreted PTH functions to reduce renal calcium excretion, augment intestinal calcium absorption, encourage bone resumption, and release skeletal calcium. On the other hand, elevated physiological calcium levels result in receptor activation, consequently suppressing the synthesis and secretion of parathyroid hormone [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, numerous studies have demonstrated the expression of CaSR in various other in vivo tissues, including blood vessels, the liver and different regions of the brain[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In conclusion, CaSR is instrumental for maintaining intracellular calcium homeostasis; cell secretion, proliferation, differentiation, chemotaxis and apoptosis[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These intracellular signaling pathways are mutually dependent and can interact with each other.\u003c/p\u003e \u003cp\u003eThe CaSR also plays an important role in various diseases, including calciotropic disorders caused by its mutations, such as familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, autosomal dominant hypocalcemia, and pseudo-Bartter's syndrome [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Its abnormal function also causes the development of certain non-calciotropic diseases, such as cardiovascular diseases and cancer [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Lu et al. demonstrated that disturbance of extracellular calcium homeostasis disrupts the calcium balance within the endoplasmic reticulum (ER) and mitochondria, ultimately leading to apoptosis and the release of stress hormones, resulting in heart failure [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Additionally, alterations in serum calcium levels can instigate cardiovascular diseases, including vascular calcification [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], coronary artery disease and myocardial infarction [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and may exacerbate blood pressure and lipid levels, all of which are known risk factors for cardiovascular disease (CVD). In conclusion, it can be speculated that CaSR can act as a protective barrier against hypertension and CVD by regulating the concentration of calcium ions in systemic and local tissues.\u003c/p\u003e \u003cp\u003eZebrafish exhibit significant physiological, pharmacological, and genetic similarities with humans, as they possess organs and systems that resemble those of mammals [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Zebrafish exhibit rapid development, with organogenesis occurring just three days postfertilization. The genome of these viruses comprises approximately 70% of the genes found in humans and, notably, approximately 87% of the known human drug target genes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The swim bladder, an organ unique to fish, provides buoyancy and allows fish to maintain balance with minimal energy expenditure within a certain depth range in the water[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Despite humans lacking a swim bladder, this organ bears resemblance to the human lung in aspects of function, structure, development, and transcription. The composition of surfactant in the swim bladder closely parallels that found in the lung [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Furthermore, the maturation of the swim bladder strongly correlates with cardiac function. Research indicates that cardiac impairment can result in the inability to inflate the bladder [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Zebrafish are especially conducive to cardiovascular development studies due to their ability to receive sufficient oxygen through passive diffusion, even when they exhibit the most severe cardiovascular defects. Additionally, the unique attributes of zebrafish, such as optical transparency, in vitro fertilization, and a high number of offspring, greatly facilitate forward genetic screening[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These advantages render zebrafish an appealing vertebrate model organism for congenital heart disease and heart development, as they supplement human and other mammalian studies on this disease.\u003c/p\u003e \u003cp\u003eIn the present study, we generated three independent \u003cem\u003ecasr\u003c/em\u003e knockout lines to investigate the role of \u003cem\u003ecasr\u003c/em\u003e in zebrafish heart development. Our findings revealed that at 5 days post fertilization (dpf), the swim bladder of \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e zebrafish fails to form properly, and the homozygotes ultimately died between 11\u0026ndash;14 dpf. Subsequent experiments indicated a decrease in heart rate and cardiac output in homozygotes. Notably, in the homozygous with TL background, swim bladder development and heart function defects were rectified, suggesting the significant influence of the TL genetic background on the development of \u003cem\u003ecasr\u003c/em\u003e mutant zebrafish embryos. Furthermore, we provided evidence that the observed abnormal heart development in \u003cem\u003ecasr\u003c/em\u003e mutants is closely linked to Wnt signaling.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGeneration of Zebrafish\u003c/b\u003e \u003cb\u003ecasr\u003c/b\u003e \u003cb\u003eknockout lines\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the function of \u003cem\u003ecasr\u003c/em\u003e, we generated \u003cem\u003ecasr\u003c/em\u003e mutant lines using CRISPR/Cas9 gene editor tools [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Two target sites with a distance of 117 bp were designed. We screened three independent strains, each of which was crossed with a control strain for two generations to mitigate any off-target effects induced by the gene knockout. The mutant lines were obtained from different F0 founders: an 80 bp deletion (named \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu01\u003c/sup\u003e), a 76 bp deletion (named \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu02\u003c/sup\u003e) and an 83 bp deletion (named \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu03\u003c/sup\u003e). Each of these mutations resulted in a disrupted open reading frame and premature stop codons, potentially abrogating all \u003cem\u003ecasr\u003c/em\u003e functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). All the heterozygous zebrafish showed no discernable phenotype and could develop into adults. The genotyping results of F0 and F1 for \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu01\u003c/sup\u003e are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, respectively. The Sanger sequencing and genotyping results for \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu02\u003c/sup\u003e and \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu03\u003c/sup\u003e could be seen in S1 Fig. We performed real-time quantitative PCR (RT\u0026ndash;qPCR) and found that the expression level of \u003cem\u003ecasr\u003c/em\u003e was significantly decreased in the mutant lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLoss of\u003c/b\u003e \u003cb\u003ecasr\u003c/b\u003e \u003cb\u003eleads to defective swim bladder inflation and spinal curvature\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore whether \u003cem\u003ecasr\u003c/em\u003e deletion leads to developmental defects, heterozygous adult fish were crossed, and the resulting embryos were collected for observation at 12-hour intervals. No significant differences were observed between control and \u003cem\u003ecasr\u003c/em\u003e mutant embryos before 4 dpf. However, at 5 dpf, defective swim bladder inflation was observed in 24% (52/214) of the embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) (S2D Fig). Concurrently, these larvae were found at the bottom of the petri dish and displayed impaired swimming ability. Fifteen larvae were selected for genotyping, and the results showed that all the larvae with defective swim bladder inflation were homozygous (S2A Fig). Then, the embryos with normal swim bladders were genotyped, and the results indicated that these embryos were either heterozygous or wild type (S2B Fig). Furthermore, spinal curvature was observed in larvae with abnormal swim bladders at 7 dpf (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), and in later developmental stages, the symptoms became more pronounced. These larvae were subsequently genotyped, and as expected, all the larvae were homozygous (S2C Fig). Notably, few homozygous mutants died at 11 days, and all the homozygous mutants died at 14 days (S2E Fig). Taken together, these results demonstrated that \u003cem\u003ecasr\u003c/em\u003e is essential for zebrafish development. Given that the \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu01\u003c/sup\u003e, \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu02\u003c/sup\u003e and \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu03\u003c/sup\u003e larvae exhibited the same defective phenotypes, \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003ehunnu03\u003c/sup\u003e mutants (\u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003e mutants and \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mutants henceforth) were selected for further analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilar to mammalian lungs, zebrafish swim bladders originate from the foregut endoderm [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In zebrafish, two Sonic Hedgehog (Shh)-related proteins and two Indian Hedgehog (Ihh)-related proteins have been identified [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], along with two homologs of their receptors, Patched 1 (Ptc1) and Patched 2 (Ptc2)[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The Hedgehog (Hh) pathway is involved in the development of endodermal organs in zebrafish and could also be involved in swim bladder development [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. It has been previously established that the growth and development of the swim bladder are regulated by the Wnt signaling pathway [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and that defective swim bladder development caused by blockade of Wnt signaling is partially attributed to reduced cell proliferation and increased cell apoptosis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. To investigate the mechanisms that affect \u003cem\u003ecasr\u003c/em\u003e-mediated swim bladder development, we employed qPCR to measure the mRNA expression of genes related to the Hh pathway and Wnt signaling pathway during embryogenesis. The results showed that the mRNA expression levels of \u003cem\u003eihhb\u003c/em\u003e, \u003cem\u003eshha\u003c/em\u003e, \u003cem\u003eshhb\u003c/em\u003e, \u003cem\u003eptc1\u003c/em\u003e and \u003cem\u003eptc2\u003c/em\u003e in the Hh pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) and the mRNA expression levels of \u003cem\u003efzd1\u003c/em\u003e, \u003cem\u003efzd5\u003c/em\u003e, \u003cem\u003efzd7b\u003c/em\u003e, \u003cem\u003etcf3b\u003c/em\u003e, \u003cem\u003etcf3b\u003c/em\u003e, \u003cem\u003ewnt1\u003c/em\u003e and \u003cem\u003ewnt9b\u003c/em\u003e in the Wnt signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) were down-regulated in \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e. Next, we performed a global transcriptome analysis of \u003cem\u003ecasr\u003c/em\u003e KO zebrafish vs. WT zebrafish using RNA-seq and visualized the Hh and Wnt signaling pathways through GSEA. The results further substantiated the down-regulation of the Hh (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) and Wnt (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) signaling pathways. Our findings indicated that \u003cem\u003ecasr\u003c/em\u003e regulated swim bladder development via the Hh and Wnt signaling pathways.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLoss of\u003c/b\u003e \u003cb\u003ecasr\u003c/b\u003e \u003cb\u003eresulted in reduced ventricular diastole and cardiac output\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAbnormal swim bladder development, as previously reported, may be a secondary effect of abnormal cardiac function [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. To determine whether cardiac function is affected in \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, we examined physiological function. Microphotography was employed to examine the structure and functional performance of the hearts from \u003cem\u003ecasr\u003c/em\u003e KO zebrafish at 52 hpf. After the high-speed movies were acquired, semi-automated optical analysis was used for further analysis [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. M-modes were generated from the movie and included the heart period (HP), diastolic diameter (DD) and systolic diameter (SD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD show the control and \u003cem\u003ecasr\u003c/em\u003e mutant ventricular morphology, respectively. Compared with those in the control group, the heart rate and cardiac output were notably lower in the \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting that cardiac function was significantly impaired in the \u003cem\u003ecasr\u003c/em\u003e KO zebrafish (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Moreover, by analyzing the long diameter, short diameter and area of the control and mutant ventricles, we found that these three parameters were significantly lower during diastole than in the control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, no significant difference was observed during the systolic stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Collectively, these findings suggest that the \u003cem\u003ecasr\u003c/em\u003e gene influences ventricular diastole and cardiac output.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ecasr\u003c/b\u003e \u003cb\u003ewas required for heart development in zebrafish\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the impact of \u003cem\u003ecasr\u003c/em\u003e KO on heart development, we conducted an RNA-seq experiment and bioinformatics analysis. A total of 702 genes were differentially expressed between the control and \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e zebrafish, including 337 up-regulated genes and 365 down-regulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Subsequent signaling pathway analysis, as annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG), revealed that \u003cem\u003ecasr\u003c/em\u003e deficiency influences cardiac muscle contraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In addition, MeV software was used to generate heatmaps for the DEGs to facilitate further selection of genes with similar functionalities (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). To discern the biological processes altered by \u003cem\u003ecasr\u003c/em\u003e disruption, a Gene Ontology (GO) analysis was performed using the DAVID bioinformatics resource [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The analysis showed that the major biological processes associated with the significant changes were associated primarily with molecular function (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Extracellular calcium ions play an essential role in the nonclassical Wnt signaling pathway [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. We next carried out a GSEA-based KEGG signal enrichment analysis and visualized four signaling pathways: calcium signaling, cardiac muscle contraction, adrenergic signaling in cardiomyocytes, and the actin cytoskeleton. The results demonstrated that the mRNA expression levels of the genes within these four signaling pathways were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince the primary function of CaSR is to regulate intracellular and extracellular calcium levels to maintain systemic calcium homeostasis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], we then performed RT‒qPCR to evaluate the mRNA expression of genes related to the calcium signaling pathway to further explain the molecular regulatory mechanism of \u003cem\u003ecasr\u003c/em\u003e in zebrafish development. Our results showed that the mRNA expression levels of genes, including \u003cem\u003encx1b\u003c/em\u003e, \u003cem\u003epmca2\u003c/em\u003e, \u003cem\u003epth2\u003c/em\u003e, \u003cem\u003eactc1\u003c/em\u003e, \u003cem\u003evdra\u003c/em\u003e, \u003cem\u003evdrb\u003c/em\u003e and \u003cem\u003erunx2a\u003c/em\u003e, were significantly lower in \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e embryos than in control embryos (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the mRNA expression level of \u003cem\u003epth1\u003c/em\u003e was significantly increased in \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e embryos than in WT embryos (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). According to the results, we assumed that \u003cem\u003ecasr\u003c/em\u003e influences Ca\u003csup\u003e2+\u003c/sup\u003e levels together with sodium-calcium channels and hormones, thereby regulating calcium homeostasis.\u003c/p\u003e \u003cp\u003eTo further validate the transcriptome sequencing results, six down-regulated genes in the cardiac muscle contraction pathway were selected, including \u003cem\u003efosab\u003c/em\u003e, \u003cem\u003etnnt2a\u003c/em\u003e, \u003cem\u003etnni2b.1\u003c/em\u003e, \u003cem\u003ecnn1a\u003c/em\u003e, \u003cem\u003emyhc4\u003c/em\u003e and \u003cem\u003emyh7l\u003c/em\u003e. The RT‒qPCR results showed that the mRNA expression of these genes was down-regulated in the \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e zebrafish compared to the control zebrafish (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), demonstrating that loss of \u003cem\u003ecasr\u003c/em\u003e affects the contractile function of cardiomyocytes. The functions of the abovementioned genes are shown in S1 Table.\u003c/p\u003e \u003cp\u003eTaken together, these data suggested that \u003cem\u003ecasr\u003c/em\u003e KO affects heart development by affecting the calcium signaling pathway and cardiac muscle contraction.\u003c/p\u003e \u003cp\u003e \u003cb\u003ecasr\u003c/b\u003e \u003cb\u003edeletion-induced phenotypic defects and impairments in cardiac physiological function can be partially mitigated in zebrafish with a Tupfel long-fin background\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs the loss of \u003cem\u003ecasr\u003c/em\u003e resulted in impaired cardiac physiology, the \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003e fish were subsequently crossed with nkx2.5: ZsYellow transgenic fish on a TL background to better understand the role of \u003cem\u003ecasr\u003c/em\u003e in heart development. Embryos expressing yellow fluorescence were selected at 24 hpf, and genotyping was carried out two months later. After that, the embryos generated from heterozygous crosses were raised and genotyped. Contrary to our expectations, the homozygous individuals survived, and their swim bladders developed normally (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This finding was unexpected, as all three lines could not survive on a TU background for approximately five generations but could mature to adulthood on a TL background. Therefore, we hypothesized that the TL background of nkx2.5: ZsYellow transgenic fish could compensate for the defect caused by the deletion of \u003cem\u003ecasr\u003c/em\u003e. Additionally, we observed that some homozygotes did not exhibit a nkx2.5: ZsYellow fluorescence signal, but all homozygotes had long fins, a characteristic feature of the TL background (S3A and S3B Fig). Therefore, we propose that the TL background could have mitigated the defective phenotype. To further confirm the genetic background of these homozygotes, we conducted a gene mapping cloning experiment. We discovered that the product amplified by the primer of linkage group 13 (LG13) could be used to distinguish between the TU and TL backgrounds by agarose gel electrophoresis. The genomic DNA was extracted from the tail fin of the adult homozygotes for background identification. The results indicated that all the homozygotes had either a pure TL background or a mixture of TU and TL backgrounds, with no fish having a pure TU background (S3C Fig). This finding supported our hypothesis that zebrafish with a TL background could compensate for swim bladder defects or that other genes that could rescue the \u003cem\u003ecasr\u003c/em\u003e mutant presented during the \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003e cross-occurrence with nkx2.5: ZsYellow transgenic fish.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether the surviving \u003cem\u003ecasr\u003c/em\u003e mutant could ameliorate cardiac function defects, we compared the physiological functions of wild type and homozygous from the two backgrounds. The results showed no significant difference in heart rate between the TL background \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and TU background \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e zebrafish (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL), but there was a significant increase in cardiac output (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI show M-Mode images and ventricle morphology of the control, TU background \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and TL background \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e zebrafish, respectively (Movies S1-3). Subsequently, RT‒qPCR was performed with a control and two background homozygotes. The results showed that most genes in the calcium signaling pathway and cardiac muscle contraction pathway were up-regulated in the TL homozygotes compared with the TU homozygotes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK), which further confirmed our hypothesis that the TL background can rescue defects in cardiac physiological function.\u003c/p\u003e \u003cp\u003e \u003cb\u003ecasr\u003c/b\u003e \u003cb\u003eis required for zebrafish cardiomyocyte differentiation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the role of the \u003cem\u003ecasr\u003c/em\u003e gene in zebrafish cardiomyocyte differentiation, we analyzed the expression levels of the cardiac differentiation markers \u003cem\u003ecmlc1\u003c/em\u003e and \u003cem\u003ecmlc2\u003c/em\u003e using in situ hybridization experiments. The results revealed that the heart of mutant was smaller than that of the wild type (TU), which is consistent with previous results (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). To further determine whether the mRNA expression levels of these two genes were changed, we performed RT‒qPCR. The results showed that the mRNA expression levels of \u003cem\u003ecmlc1\u003c/em\u003e and \u003cem\u003ecmlc2\u003c/em\u003e were significantly lower in the \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e of TU background than in the WT (TU) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and compared to \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e of TU background, the mRNA expression level of \u003cem\u003ecmlc2\u003c/em\u003e was significantly higher in the \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e of TL background (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001); however, \u003cem\u003ecmlc1\u003c/em\u003e expression was not significantly different. These findings demonstrated that \u003cem\u003ecasr\u003c/em\u003e affected zebrafish cardiomyocyte differentiation in the TU background but not in the TL background (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCaSR is implicated in a variety of cardiac pathological processes. It triggers numerous intracellular signaling pathways associated with diseases such as atherosclerosis, vascular calcification, cardiomyopathy, cardiac fibrosis, and myocardial infarction [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Our study elucidated the crucial roles of zebrafish \u003cem\u003ecasr\u003c/em\u003e during early development. Utilizing CRISPR/Cas9 technology, we generated three \u003cem\u003ecasr\u003c/em\u003e-mutant zebrafish lines and discovered that \u003cem\u003ecasr\u003c/em\u003e deficiency led to morphogenetic abnormalities in the swim bladder and spine. All the homozygous larvae in the TU genetic background died before 14 dpf. It has been reported that bladder inflation is complete when zebrafish develop to 120 hpf. At this developmental stage, zebrafish larvae start to feed and need to take up exogenous nutrients to maintain their normal growth and development. The swim bladder plays a crucial role in enabling larvae to float in water and engage in predation. Should the swim bladder fail to develop or inflate at this point, the larvae experience a loss in motility and predation capacity, ultimately resulting in death due to nutritional deficiency [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Furthermore, studies have reported a correlation between the failure of swim bladder inflation and cardiac dysfunction. Subsequently, we analyzed cardiac physiological function in both control and \u003cem\u003ecasr\u003c/em\u003e mutant larvae. Our findings indicate that, compared to those in the control group, the heart rate and cardiac output in the mutant zebrafish were significantly lower. Additionally, both the long and short diameters of the heart decreased at diastole. It has been suggested that an increased concentration of extracellular Ca\u003csup\u003e2+\u003c/sup\u003e or other CaSR agonists might lead to increased sarcoplasmic reticulum Ca\u003csup\u003e2+\u003c/sup\u003e release through the G protein-phospholipase C-inositol triphosphate signaling pathway, thereby triggering cardiac contraction [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Conversely, a decrease in CaSR expression could result in a reduced Ca\u003csup\u003e2+\u003c/sup\u003e concentration in the sarcoplasmic reticulum, impairing myocardial cell excitation\u0026ndash;contraction coupling and subsequently leading to compromised myocardial contractile function [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. These findings suggest that the absence of \u003cem\u003ecasr\u003c/em\u003e could lead to heart failure, which is often characterized by impaired cardiac contractility or arrhythmias.\u003c/p\u003e \u003cp\u003eThe Wnt signaling pathway, implicated in the induction of cardiac fibrosis and in cardiac fibroblasts (CFs), represents a potential therapeutic target for cardiac fibrosis [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. It exerts significant influence on embryonic development, adult tissue homeostasis, and regeneration. The canonical Wnt/β-catenin signaling pathway differentially regulates cardiac development at different developmental stages, with early promotion followed by later inhibition [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. However, the role of the Wnt/β-catenin signaling pathway in postnatal cardiomyocyte (CM) growth and development has been scarcely explored in previous studies. The development of the swim bladder has been primarily linked to the Wnt signaling pathway. The inhibition of Wnt signaling interferes with the formation of epithelial, mesenchymal, and mesothelial tissue layers in the swim bladder of zebrafish, consequently hindering their growth and differentiation[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The relevance of Hh signaling pathways in the development of lungs in mice and chickens has been underscored [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. This study investigated the impact of \u003cem\u003ecasr\u003c/em\u003e knockout on the Wnt and Hh signaling pathways in zebrafish through quantitative analysis. The results showed that the mRNA expression of genes in both signaling pathways was down-regulated, which suggested that loss of \u003cem\u003ecasr\u003c/em\u003e affects the Wnt and Hh signaling pathways, leading to defective swim bladder inflation, thus affecting the normal growth and development of zebrafish. Given that the primary function of \u003cem\u003ecasr\u003c/em\u003e is to maintain calcium homeostasis by regulating the calcium ion concentration, a quantitative analysis of the \u003cem\u003encx1b\u003c/em\u003e, \u003cem\u003epmca2\u003c/em\u003e, \u003cem\u003epth2\u003c/em\u003e, \u003cem\u003evdra\u003c/em\u003e, \u003cem\u003evdrb\u003c/em\u003e, \u003cem\u003eactc1\u003c/em\u003e, and \u003cem\u003erunx2a\u003c/em\u003e genes in the calcium signaling pathway was also conducted. The results demonstrated a general down-regulation of these genes, whereas pth1 exhibited an upward trend. As the calcium signaling pathway is a part of the nonclassical Wnt signaling pathway, it is suggested that \u003cem\u003ecasr\u003c/em\u003e regulates calcium homeostasis via the Wnt signaling pathway. Following \u003cem\u003ecasr\u003c/em\u003e knockdown, the parathyroid hormone pth1 undergoes a compensatory expression increase to maintain calcium homeostasis. However, this study examined only the RNA level. The impacts of \u003cem\u003ecasr\u003c/em\u003e knockout on the translation and posttranslational modification of these proteins warrant further investigation.\u003c/p\u003e \u003cp\u003eThe main function of the heart is to receive and pump blood. This is achieved through the coordinated contraction of the ventricles, which relies on the contractile function and electrophysiological activity of cardiomyocytes. Cardiac systolic function is primarily regulated by the sliding of actin filament units, known as myofibrils, which enable cardiac muscle cells to contract and eject blood from the heart[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWang et al.[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] initially reported the expression of \u003cem\u003eCasr\u003c/em\u003e in neonatal rat cardiomyocytes, highlighting that an increase in calcium levels within in vitro cell cultures correspondingly escalated intracellular calcium levels and cardiac activity. To further investigate the impact of \u003cem\u003ecasr\u003c/em\u003e genes on zebrafish heart development, we carried out transcriptome sequencing and bioinformatics analysis to identify potential genes influencing phenotype and function. Subsequently, 702 significantly varied candidate genes were identified and subjected to further GO enrichment and KEGG signaling pathway analysis. The analysis indicated that the up-regulated genes were predominantly enriched in cellular processes such as cell matrix adhesion, protein folding, cell differentiation and redox, with no discernible pathways related to cardiac developmental signaling. Conversely, the down-regulated genes were chiefly enriched in protein hydrolysis, the immune response, lipid metabolism, energy metabolism, and other processes intimately associated with calcium ions. KEGG signaling pathway analysis revealed that these genes were enriched primarily in the cardiac muscle contraction pathway. Moreover, the MAPK, FoxO, and mToR signaling pathways potentially interact with the Wnt signaling pathway, thereby influencing zebrafish heart development. Subsequent RT‒qPCR analysis of the mRNA expression of several down-regulated genes revealed a significant decrease (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the mRNA expression of genes associated with cardiovascular tissue, myocardial tissue regeneration, calmodulin, cardiac contraction, myosin, and troponin. This finding substantiates that \u003cem\u003ecasr\u003c/em\u003e knockout indeed impacts zebrafish heart function. The core genes of the relevant pathways were analyzed using KEGG, the results of which are shown in a chord diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, the exact regulatory mechanism involved remains unclear and warrants further scrutiny.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering that homozygotes within the TU background are nonviable, \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003e zebrafish were crossed with nkx2.5: ZsYellow transgenic fish on a TL background to further elucidate the role of \u003cem\u003ecasr\u003c/em\u003e in zebrafish cardiac function. We selected embryos demonstrating heart-specific yellow fluorescence expression for subsequent experiments. Intriguingly, none of the embryos from these \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003e incrosse exhibited swim bladder deficiency or a curved spine phenotype. All the zebrafish survived and reproduced normally, and all the homozygotes displayed an elongated fin phenotype. Prior studies have indicated that diverse zebrafish strains possess unique morphological and behavioral traits, such as AB, Tubingen (TU), Wild India Kolkata (WIK), and Tupfel long fin (TL) strains. These traits, including swimming ability, are influenced by factors such as water temperature, caudal fin length, and genetic background [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Furthermore, variations in fin size, inherited traits, and physiological and behavioral characteristics in the juvenile and adult stages have been observed [\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. We thus postulated that the nkx2.5: ZsYellow transgenic fish with a TL background could effectively counteract defects induced by \u003cem\u003ecasr\u003c/em\u003e deletion. In a subsequent screening, some nonfluorescent homozygotes were observed to grow normally into adulthood and reproduce, suggesting that the nkx2.5 transcription factor does not contribute to defect rectification. Physiological functional tests and RT‒qPCR experiments were subsequently conducted, which revealed partial restoration of cardiac function in the TL homozygotes compared to their TU counterparts. Real-time fluorescence further revealed significant upregulation of the majority of genes within the Wnt signaling pathway, calcium signaling pathway, and cardiac muscle contraction pathway. These results collectively suggest that the TL background can markedly ameliorate the developmental defects of \u003cem\u003ecasr\u003c/em\u003e zebrafish mutants. However, it remains unclear whether the TL background directly influences cardiac development or alters the expression levels of associated genes, ultimately affecting cardiac development (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMuscle myosin is a hexamer that consists of two myosin heavy chains (MHCs), two regulatory light chains (RLCs) and two essential light chains (ELCs)[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Since ELCs and RLCs contain two calcium-binding EF-hand motifs, they may regulate the calcium sensitivity of force generation and crossbridge kinetics, thereby fine-tuning cardiac muscle contraction [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Mutations in the genes encoding ELCs and RLCs have been shown to be causally associated with 1% of human cardiomyopathies[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. In zebrafish, the cardiomyocyte differentiation marker genes \u003cem\u003ecmlc1\u003c/em\u003e and \u003cem\u003ecmlc2\u003c/em\u003e are the primary homologs of ELC and RLC, respectively. Therefore, we performed in situ hybridization and RT‒qPCR experiments on these two marker genes. The RT‒qPCR results showed that the expression of both genes was significantly lower in the TU background homozygote than in the control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and that the expression level of the \u003cem\u003ecmlc2\u003c/em\u003e gene was significantly higher in the TL background homozygote than in the TU background homozygote, whereas the mRNA expression of \u003cem\u003ecmlc1\u003c/em\u003e remained unchanged. Previous research has indicated that deletion of \u003cem\u003ecmlc1\u003c/em\u003e in zebrafish results in an increase in both the size and number of cardiomyocytes, leading to enlarged ventricular chamber volumes. Conversely, deletion of \u003cem\u003ecmlc2\u003c/em\u003e causes a reduction in the size and number of cardiomyocytes [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. These findings further substantiate that the loss of \u003cem\u003ecasr\u003c/em\u003e can result in a smaller heart and that \u003cem\u003ecasr\u003c/em\u003e plays a pivotal role in heart development. However, the relationships between these two genes and \u003cem\u003ecasr\u003c/em\u003e, as well as their interactions with one another, need to be further explored.\u003c/p\u003e \u003cp\u003eIn summary, the data in this study revealed a critical role of \u003cem\u003ecasr\u003c/em\u003e in heart development, and different genetic backgrounds had different effects on heart development in \u003cem\u003ecasr\u003c/em\u003e-mutant zebrafish. Furthermore, we found that loss of \u003cem\u003ecasr\u003c/em\u003e resulted in down-regulated expression of genes involved in the Wnt and cardiac muscle contraction pathways. The TL genetic background could significantly affect the development of \u003cem\u003ecasr\u003c/em\u003e mutant embryos.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eHerein, we demonstrate the importance of the \u003cem\u003ecasr\u003c/em\u003e gene in regulating zebrafish development and its close relationship with swim bladder inflation and heart function. Our RT‒qPCR and transcriptome sequencing experiments revealed that the Wnt signaling pathway and cardiac muscle contraction pathway were down-regulated after \u003cem\u003ecasr\u003c/em\u003e knockout. Additionally, we observed that different genetic backgrounds have varying regulatory functions. In summary, our work provides new insights into the role of the \u003cem\u003ecasr\u003c/em\u003e gene in heart development.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eZebrafish husbandry and ethics approval\u003c/h2\u003e \u003cp\u003eZebrafish TU and TL lines were used and maintained under standard conditions in this study. Embryos were collected from natural mating, incubated at 28.5\u0026deg;C and staged according to standard protocols[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. All the experiments were performed following animal use protocols approved by the Animal Care and Use Committee of Hunan Normal University.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCRISPR/Cas9-mediated gene knockout\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003ecasr\u003c/em\u003e mutant lines were generated with the CRISPR/Cas9 system following previous methods [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Briefly, the \u003cem\u003ecasr\u003c/em\u003e target site sequences are located in the second exon, and the two target sites are 117 bp apart from each other. The sgRNAs were synthesized in vitro with T7 RNA polymerase. A total of 400 pg of Cas9-mRNA and 50 pg \u003cem\u003eof casr\u003c/em\u003e-gRNA were co-injected into zebrafish embryos at the one-cell stage. For genotyping, polymerase chain reaction (PCR) was carried out using the following primer sets: 5\u0026prime;- GGCTGTTGAAACTAGAGGAAA-3\u0026rsquo; and 3\u0026prime;- GAGAAGCTGACCATGTTTTGT-5\u0026rsquo;.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time PCR (RT–qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was prepared from 50 embryos at 5 dpf using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s protocol. First-strand cDNA was generated using a reverse transcription kit (TaKaRa) with random primers. Quantitative real-time PCR (qPCR) was performed using a SYBR green kit (Takara, Dalian, China) on a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific). Relative expression levels of the tested mRNAs were determined using β-actin as an internal reference and the comparative Ct (2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e) method (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The sequences of primers used in this study are listed in S2 Table.\u003c/p\u003e\n\u003ch3\u003eWhole-mount in situ hybridization (WISH)\u003c/h3\u003e\n\u003cp\u003eZebrafish embryos at the desired stages were fixed in 4% paraformaldehyde (PFA) overnight before processing for WISH analysis as previously described [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Digoxigenin-UTP-labeled antisense RNA probes for \u003cem\u003ecmlc1\u003c/em\u003e and \u003cem\u003ecmlc2\u003c/em\u003e were generated via an in vitro transcription method using T7 RNA polymerase (Thermo Fisher, Waltham, MA, USA).\u003c/p\u003e\n\u003ch3\u003eRNA sequencing\u003c/h3\u003e\n\u003cp\u003eA total of 6 cDNA libraries were prepared from three biological replicates of \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and wild type zebrafish at 5 dpf. RNA quality was confirmed by a Nano200 nucleic acid analyzer (allsheng, China), and the libraries were sequenced with an Illumina Nova seq6000. The RNAseq results were validated by qPCR. The results represent the average of 3 repeat experiments, and each experiment was repeated 3 times. The RNA sequencing data from this study were deposited in the NCBI Sequence Read Archive under accession SRA: PRJNA967502, PRJNA967502, PRJNA967502, PRJNA967502, PRJNA967502, PRJNA967502.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCardiac function analysis\u003c/h2\u003e \u003cp\u003eAt least 10 juvenile zebrafish were selected and anesthetized with triclocaine. Movies of beating hearts from embryos at 52 h postfertilization (hpf) were recorded using an IX71-Olympus inverted microscope equipped with a Hamamatsu C9300 digital camera. Heart rate was counted for 15 s and then multiplied by 4 to calculate the heart rate in beats per minute (bpm). The long-diameter (a), short-diameter (b) and M-mode heart segments at diastole and systole were obtained by semi-automated optical analysis[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. All images and data were obtained from zebrafish ventricles. Each experiment was repeated at least three times to ensure reliability. The ventricular volume was measured by the following ellipsoidal formula:\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{V}\\text{=}\\frac{\\text{4}}{\\text{3}}\\text{\u0026pi;\u0026times;(}\\frac{\\text{a}}{\\text{2}}\\text{)\u0026times;}{\\text{(}\\frac{\\text{b}}{\\text{2}}\\text{)}}^{\\text{2}}\\)\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{Stroke volume=}{\\text{V}}_{\\text{diastole}}\\text{-}{\\text{V}}_{\\text{systole}}\\)\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{Cardiac output=Stroke volume}\\text{ }\\text{\u0026times;}\\text{ }\\text{heart rate}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis\u003c/h2\u003e \u003cp\u003eGene set enrichment analysis (GSEA)[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e] was performed on the list of genes sorted by fold change in the experiment, and the enrichment of the up- or down-regulated gene sets in the KEGG pathway database was calculated. The gene sets with fewer than 15 genes or more than 500 genes were excluded, and the t-statistic mean of the genes was computed for each KEGG pathway using a permutation test with 1000 replications. The up-regulated pathways were defined by a normalized enrichment score (NES)\u0026thinsp;\u0026gt;\u0026thinsp;0, and the down-regulated pathways were defined by an NES\u0026thinsp;\u0026lt;\u0026thinsp;0[\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. R v3.2.2 was used to perform the data preprocessing. The mapping process was performed through packages such as ClusterProfiler and Bioconductor. A chord diagram was drawn on Sangerbox [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGene mapping cloning experiment\u003c/h2\u003e \u003cp\u003eThe genetic background of the zebrafish was distinguished based on simple sequence length polymorphisms (SSLP) on different chromosomes[\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. First, primers for identifying 25 pairs of sequence tagging sites (STSs) on zebrafish chromosomes were found in the NCBI database, after which the wild-type (WT) zebrafish genome with a TU and TL background was used as a template for in vitro amplification. The sequences of primers used for LG13 (linkage group 13) in this study were as follows: Z6007-F, AGCCCTAAACAAACCGGC; and Z6007-R, GAGCATGATCTGCTCGTTAGG.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the values are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs. The statistical analysis was performed using Student\u0026rsquo;s t test. P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered to indicate statistical significance and are indicated with asterisks (*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe source data behind the figures can be found in S1 Raw Data.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe would like to express our appreciation to all the members of the Laboratory of Animal Nutrition and Human Health at Hunan Normal University for their assistance and encouragement.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u003c/strong\u003e Ling Liu, Chengbo Yang, Huaping Xie.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData curation:\u003c/strong\u003e Ling Liu, Jiaxin Liang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding acquisition:\u003c/strong\u003e Wen Huang, Huaping Xie.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation:\u003c/strong\u003e Junwei Zhu, Jian Huang, Xiangding Chen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology:\u003c/strong\u003e Ling Liu, Yuyao Hu, Binling Xie, Ting Zeng, Huaping Xie.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProject administration:\u003c/strong\u003e Jianzhong Li, Huaping Xie, Xiangding Chen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResources:\u003c/strong\u003e Wen Huang, Xiaochun Lu, Huaping Xie.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoftware:\u003c/strong\u003e Ling Liu, Yuyao Hu, Binling Xie, Ting Zeng.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupervision:\u003c/strong\u003e Jianzhong Li, Huaping Xie, Xiangding Chen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation:\u003c/strong\u003e Ling Liu, Yuyao Hu.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisualization:\u003c/strong\u003e Ling Liu, Yuyao Hu, Binling Xie, Jiaxin Liang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting \u0026ndash; original draft:\u003c/strong\u003e Ling Liu, Yuyao Hu.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting \u0026ndash; review \u0026amp; editing:\u003c/strong\u003e Ling Liu, Chengbo Yang, Huaping Xie.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBerridge MJ, Lipp P, Bootman MD. 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Genomics. 2002; 79(6):756-9. https://doi.org/10.1006/geno.2002.6769.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"casr, heart development, zebrafish, gene knockout","lastPublishedDoi":"10.21203/rs.3.rs-4498455/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4498455/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Calcium is fundamental to biological processes, and the Calcium sensing receptor (CaSR) plays a key role in maintaining calcium homeostasis. This process is intimately related to numerous cardiovascular diseases and various types of cancers. However, the role of CaSR in heart development is yet to be thoroughly understood. To delve into this, we conducted a \u003cem\u003ecasr\u003c/em\u003e gene knockout experiment, analyzed cardiac physiological functions, and performed transcriptomics to investigate the mechanism of the \u003cem\u003ecasr\u003c/em\u003e gene in zebrafish heart development.\u003c/p\u003e\n\u003cp\u003eResults: We successfully established \u003cem\u003ecasr\u003c/em\u003e gene knockout lines in zebrafish with Tuebingen (TU) backgrounds. Compared to the control, \u003cem\u003ecasr\u003c/em\u003e mutant embryos exhibited a smaller heart size, reduced heart rate, and diminished cardiac output. Additionally, these mutants exhibited a curved body structure and a mal-developed swim bladder. Zebrafish larvae began to die at 11 days post-fertilization (dpf). Subsequent transcriptome sequencing andbioinformatics analysis revealed that the loss of casr disrupts cardiac muscle contraction, leading to defective swim bladder inflation and ultimately death. Furthermore, we crossbred \u003cem\u003ecasr\u003c/em\u003e mutant lines with Tupfel long-fin (TL) background nkx2.5: ZsYellow transgenic lines, and subsequently obtained a \u003cem\u003ecasr\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e line where the swim bladder developed normally. Furthermore, qPCR results indicated that the expression of genes linked to cardiac muscle contraction turned to normal. Further experimental results demonstrated that the survival rate of \u003cem\u003ecasr\u003c/em\u003e mutants was influenced by the TL background.\u003c/p\u003e\n\u003cp\u003eConclusions: Taken together, \u003cem\u003ecasr\u003c/em\u003e is vital for zebrafish swim bladder inflation and heart development, exerting its regulatory role through the Wnt signaling pathway and the cardiac muscle contraction. Importantly, the TL background significantly impacts the development of casr zebrafish mutant embryos.\u003c/p\u003e","manuscriptTitle":"Zebrafish casr affects swim bladder inflation by regulating heart development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-12 21:41:37","doi":"10.21203/rs.3.rs-4498455/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"170dc860-32b6-485b-b3f6-fdee7ae91be3","owner":[],"postedDate":"June 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-04T15:09:41+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-12 21:41:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4498455","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4498455","identity":"rs-4498455","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}