T lymphocytes, which are essential for cell-mediated immunity in vertebrates, rely on thymic seeding of lymphoid progenitors for commitment, differentiation and maturation. However, the epigenetic programming of lymphoid-primed progenitor migration and differentiation is incompletely understood. Here, we show that zebrafish embryos lacking the epigenetic modulator Atf7ip or Setdb1 methyltransferase exhibit decreased thymic homing and differentiation of lymphoid progenitor cells. We show that Atf7ip regulates T cell progenitor homing and differentiation via Setdb1-triggered H3K9 trimethylation. Atf7ip interacts with Setdb1 to catalyze H3K9me3 modification of the key immune regulator bach2b to derepress the expression of ccr9a and irf4a, thereby promoting lymphoid progenitor homing and intrathymic differentiation. In the absence of Atf7ip or Setdb1, replenishing irf4a or diminishing bach2b restores the thymic trafficking and differentiation of lymphoid progenitor cells. Notably, depletion of ATF7IP by two complementary cre-recombinase alleles in mice (CAG-CreERT2 and Mx1-iCre) impedes the migration of hematopoietic progenitors to the thymus, resulting in declined T lymphopoiesis. These findings establish the role of ATF7IP/SETDB1-mediated epigenetic programming in governing T lymphoid progenitor trafficking and differentiation, with implications for understanding the pathogenesis of human T lymphoid diseases.

Primary cilia are crucial organelles involved in various cellular signaling pathways. Prostaglandin E receptor 4 (EP4), a G protein-coupled receptor (GPCR), localizes to both cilia and cellular membranes, playing pivotal roles in inflammation, immunomodulation, and cancer progression. However, the mechanisms underlying EP4 trafficking to cilia remain poorly understood, hindering further exploration of its ciliary pathophysiological functions. In this study, we unveil EP4 expression in primary cilia across zebrafish embryos, mouse tissues, and human cells. We identify the third intracellular (IC3) loop and C-terminal (CT) region in EP4 as essential for its ciliary translocation, with the LPG motif serving as a key ciliary localization sequence. A single amino acid substitution in the LPG motif, introduced by CRISPR C-to-G base editing (CGBE1), blocks EP4 trafficking to cilia and reduces cAMP-PKA signaling. High-content siRNA screening identifies the small GTPase Rab23 and intraflagellar transport protein 43 (IFT43) as crucial regulators of EP4 ciliary trafficking in zebrafish and mammalian cells. Furthermore, EP4 physically interacts with IFT43 and Rab23 through its specific domains in the IC3 loop and CT region. These findings provide important insights into the conserved Rab23/IFT43-mediated ciliary trafficking mechanism, with significant implications for understanding the pathophysiological roles mediated by EP4 signaling in cilia.

Energy deprivation and metabolic rewiring of cardiomyocytes are widely recognized hallmarks of heart failure. Here, we report that HEY2 (a Hairy/Enhancer-of-split-related transcriptional repressor) is upregulated in hearts of patients with dilated cardiomyopathy. Induced Hey2 expression in zebrafish hearts or mammalian cardiomyocytes impairs mitochondrial respiration, accompanied by elevated ROS, resulting in cardiomyocyte apoptosis and heart failure. Conversely, Hey2 depletion in adult mouse hearts and zebrafish enhances the expression of mitochondrial oxidation genes and cardiac function. Multifaceted genome-wide analyses reveal that HEY2 enriches at the promoters of genes known to regulate metabolism (including Ppargc1, Esrra and Cpt1) and colocalizes with HDAC1 to effectuate histone deacetylation and transcriptional repression. Consequently, restoration of PPARGC1A/ESRRA in Hey2- overexpressing zebrafish hearts or human cardiomyocyte-like cells rescues deficits in mitochondrial bioenergetics. Knockdown of Hey2 in adult mouse hearts protects against doxorubicin-induced cardiac dysfunction. These studies reveal an evolutionarily conserved HEY2/HDAC1-Ppargc1/Cpt transcriptional module that controls energy metabolism to preserve cardiac function.

In organ regeneration, progenitor and stem cells reside in their native microenvironment, which provides dynamic physical and chemical cues essential to their survival, proliferation, and differentiation. However, the types of cells that form the native microenvironment for renal progenitor cells (RPCs) have not been clarified. Here, single-cell sequencing of zebrafish kidney reveals fabp10a as a principal marker of renal interstitial cells (RICs), which can be specifically labeled by GFP under the control of fabp10a promoter in the fabp10a:GFP transgenic zebrafish. During nephron regeneration, the formation of nephrons is supported by RICs that form a network to wrap the RPC aggregates. RICs that are in close contact with RPC aggregates express cyclooxygenase 2 (Cox2) and secrete prostaglandin E2 (PGE2). Inhibiting PGE2 production prevents nephrogenesis by reducing the proliferation of RPCs. PGE2 cooperates with Wnt4a to promote nephron maturation by regulating β-catenin stability of RPC aggregates. Overall, these findings indicate that RICs provide a necessary microenvironment for rapid nephrogenesis during nephron regeneration.

Hematopoietic stem and progenitor cells (HSPCs) are a heterogeneous group of cells with expansion, differentiation, and repopulation capacities. How HSPCs orchestrate the stemness state with diverse lineage differentiation at steady condition or acute stress remains largely unknown. Here, we show that zebrafish mutants that are deficient in an epigenetic regulator Atf7ip or Setdb1 methyltransferase undergo excessive myeloid differentiation with impaired HSPC expansion, manifesting a decline in T cells and erythroid lineage. We find that Atf7ip regulates hematopoiesis through Setdb1-mediated H3K9me3 modification and chromatin remodeling. During hematopoiesis, the interaction of Atf7ip and Setdb1 triggers H3K9me3 depositions in hematopoietic regulatory genes including cebpβ and cdkn1a, preventing HSPCs from loss of expansion and premature differentiation into myeloid lineage. Concomitantly, loss of Atf7ip or Setdb1 derepresses retrotransposons that instigate the viral sensor Mda5/Rig-I like receptor (RLR) signaling, leading to stress-driven myelopoiesis and inflammation. We find that ATF7IP or SETDB1 depletion represses human leukemic cell growth and induces myeloid differentiation with retrotransposon-triggered inflammation. These findings establish that Atf7ip/Setdb1-mediated H3K9me3 deposition constitutes a genome-wide checkpoint that impedes the myeloid potential and maintains HSPC stemness for diverse blood cell production, providing unique insights into potential intervention in hematological malignancy.

cAMP-PKA signaling plays a pivotal role in melanin synthesis and melanosome transport by responding to the binding of the α-melanocyte-stimulating hormone (α-MSH) to melanocortin-1 receptor (MC1R). Adenylate cyclases (ADCYs) are the enzymes responsible for the synthesis of cAMP from ATP, which comprises nine transmembrane isoforms (ADCYs 1-9) and one soluble adenylate cyclase (ADCY 10) in mammals. However, little is known about which and how ADCY isoforms regulate melanocyte generation, melanin biosynthesis, and melanosome transport in vivo. In this study, we have generated a series of single and double mutants of Adcy isoforms in zebrafish. Among them, adcy3a-/- and adcy5-/- double mutants cause defects in melanosome dispersion but do not impair melanoblast differentiation and melanocyte regeneration during the embryonic or larval stages. Activation of PKA, the main effector of cAMP signaling, significantly ameliorates the defects in melanosome dispersion in adcy3a-/- and adcy5-/- double mutants. Mechanistically, Adcy3a and Adcy5 regulate melanosome dispersion by activating kinesin-1 while inhibiting cytoplasmic dynein-1. In adult zebrafish, Adcy3a and Adcy5 participate in the regulation of the expression of microphthalmia transcription factor (Mitfa) and melanin synthesis enzymes Tyr, Dct, and Trp1b. The deletion of Adcy3a and Adcy5 inhibits melanin production and reduces pigmented melanocyte numbers, causing a defect in establishing adult melanocyte stripes. Hence, our studies demonstrate that Adcy3a and Adcy5 play essential but redundant functions in mediating α-MSH-MC1R/cAMP-PKA signaling for regulating melanin synthesis and melanosome dispersion.

Heart regeneration occurs by dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). However, the signaling mechanisms by which injury induces CM renewal remain incompletely understood. Here, we find that cardiac injury in zebrafish induces expression of the secreted Wnt inhibitors, including Dickkopf 1 (Dkk1), Dkk3, secreted Frizzled-related protein 1 (sFrp1), and sFrp2, in cardiac tissue adjacent to injury sites. Experimental blocking of Wnt activity via Dkk1 overexpression enhances CM proliferation and heart regeneration, whereas ectopic activation of Wnt8 signaling blunts injury-induced CM dedifferentiation and proliferation. Although Wnt signaling is dampened upon injury, the cytoplasmic β-catenin is unexpectedly increased at disarrayed CM sarcomeres in myocardial wound edges. Our analyses indicated that p21-activated kinase 2 (Pak2) is induced at regenerating CMs, where it phosphorylates cytoplasmic β-catenin at Ser 675 and increases its stability at disassembled sarcomeres. Myocardial-specific induction of the phospho-mimetic β-catenin (S675E) enhances CM dedifferentiation and sarcomere disassembly in response to injury. Conversely, inactivation of Pak2 kinase activity reduces the Ser 675-phosphorylated β-catenin (pS675-β-catenin) and attenuates CM sarcomere disorganization and dedifferentiation. Taken together, these findings demonstrate that coordination of Wnt signaling inhibition and Pak2/pS675-β-catenin signaling enhances zebrafish heart regeneration by supporting CM dedifferentiation and proliferation.

Prostaglandin (PG) signaling regulates a wide variety of physiological and pathological processes, including body temperature, cardiovascular homeostasis, reproduction, and inflammation. Recent studies have revealed that PGs play pivotal roles in embryo development, ciliogenesis, and organ formation. Prostaglandin E2 (PGE2) and its receptor EP4 modulate ciliogenesis by increasing the anterograde intraflagellar transport. Many G-protein-coupled receptors (GPCRs) including EP4 are localized in cilia for modulating cAMP signaling under various conditions. During development, PGE2 signaling regulates embryogenesis, hepatocyte differentiation, hematopoiesis, and kidney formation. Prostaglandins are also essential for skeletal muscle repair. This review outlines recent advances in understanding the functions and mechanisms of prostaglandin signaling in ciliogenesis, embryo development, and organ formation.

1 Shanghai Key Laboratory of Regulatory Biology, Institute of Molecular Medicine, East China Normal University School of Life Sciences, Shanghai 200241, China 2 State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhong Shan Hospital, Fudan University, Shanghai 200438, China 3 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA 4 Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA 5 Department of Pathology and Laboratory Medicine, McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA 6 Department of Developmental and Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA † These authors share first authorship. * Correspondence to: Tao P. Zhong, E-mail: tzhong@bio.ecnu.edu.cn

Gene alterations are recognized as important events in acute myeloid leukemia (AML) progression. Studies on hematopoiesis of altered genes contribute to a better understanding on their roles in AML progression. Our previous work reported a DEAH box helicase 15 (DHX15) R222G mutation in AML patients, and we showed DHX15 overexpression is associated with poor prognosis in AML patients. In this work, we further study the role of dhx15 in zebrafish developmental hematopoiesis by generating dhx15-/- zebrafish using transcription activator-like effector nuclease technology. Whole-mount in situ hybridization (WISH) analysis showed hematopoietic stem/progenitor cells were dramatically perturbed when dhx15 was deleted. Immunofluorescence staining indicated inhibited hematopoietic stem/progenitor cell (HSPC) proliferation instead of accelerated apoptosis were detected in dhx15-/- zebrafish. Furthermore, our data showed that HSPC defect is mediated through the unfolded protein response (UPR) pathway. DHX15 R222G mutation, a recurrent mutation identified in AML patients, displayed a compromised function in restoring HSPC failure in dhx15-/- ; Tg (hsp: DHX15 R222G) zebrafish. Collectively, this work revealed a vital role of dhx15 in the maintenance of definitive hematopoiesis in zebrafish through the unfolded protein respone pathway. The study of DHX15 and DHX15 R222G mutation could hold clinical significance for evaluating prognosis of AML patients with aberrant DHX15 expression.

Photoreceptor cells are highly specialized sensory neurons capable of visual phototransduction. The connecting cilia in the retinal photoreceptors link the inner segment to the outer segment and mediate the transport of opsins in vision. Although our previous study demonstrates that Prostaglandin E2 (PGE2) signaling is required for ciliogenesis in zebrafish, its roles in retinal ciliogenesis and photoreceptor cell development remain unknown. Here, we investigated the function of prostaglandin signaling in retina and photoreceptor cell development. We generated zebrafish mutations in prostaglandin endoperoxide synthase 1 (PTGS1) and prostaglandin endoperoxide synthase 2 (PTGS2), two rate-limiting enzymes responsible for prostaglandin production. We found that ptgs2b knockdown in ptgs1-/- ptgs2a-/- double mutants significantly reduced the length of connecting cilia and resulted in severe defects in photoreceptor cell differentiation. Furthermore, mutation in PGE2 transporter Leakytail (Lkt)/ATP-binding cassette transporter 4 (ABCC4) exhibited loss of connecting cilia and outer segment in photoreceptor cells, leading to mislocalization of opsins in the cell bodies of photoreceptors. Together, our findings suggest that PGE2 production and transport are crucial for connecting cilia formation and photoreceptor cell development.

The centrosomal protein γ-tubulin complex protein 3 (Tubgcp3/GCP3) is required for the assembly of γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs), which play critical roles in mitotic spindle formation during mitosis. However, its function in vertebrate embryonic development is unknown. Here, we generated the zebrafish tubgcp3 mutants using the CRISPR/Cas9 system and found that the tubgcp3 mutants exhibited the small eye phenotype. Tubgcp3 is required for the cell cycle progression of retinal progenitor cells (RPCs), and its depletion caused cell cycle arrest in the mitotic (M) phase. The M-phase arrested RPCs exhibited aberrant monopolar spindles and abnormal distributed centrioles and γ-tubulin. Moreover, these RPCs underwent apoptosis finally. Our study provides the in vivo model for the functional study of Tubgcp3 and sheds light on the roles of centrosomal γ-tubulin complexes in vertebrate development.

BACKGROUND: Intracranial aneurysm (IA) is usually a late-onset disease, affecting 1% to 3% of the general population and leading to lifethreatening subarachnoid hemorrhage. Genetic susceptibility has been implicated in IAs, but the causative genes remain elusive. METHODS: We performed next-generation sequencing in a discovery cohort of 20 Chinese IA patients. Bioinformatics filters were exploited to search for candidate deleterious variants with rare and low allele frequency. We further examined the candidate variants in a multiethnic sample collection of 86 whole exome sequenced unsolved familial IA cases from 3 previously published studies. RESULTS: We identified that the low-frequency variant c.4394C>A_p. Ala1465Asp (rs2298808) of ARHGEF17 was significantly associated with IA in our Chinese discovery cohort (P=7.3x10(-4); odds ratio=7.34). It was subsequently replicated in Japanese familial IA patients (P=0.039; odds ratio=4.00; 95% confidence interval=0.832-14.8) and was associated with IA in the large Chinese sample collection comprising 832 sporadic IA-affected and 599 control individuals (P=0.041; odds ratio=1.51; 95% confidence interval=1.02-Inf). When combining the sequencing data of all familial IA patients from 4 different ethnicities (ie, Chinese, Japanese, European American, and French-Canadian), we identified a significantly increased mutation burden for ARHGEF17 (21/106 versus 11/306; P=8.1x10(-7); odds ratio=6.6; 95% confidence interval=2.9-15.8) in cases as compared with controls. In zebrafish, arhgef17 was highly expressed in the brain blood vessel. arhgef17 knockdown caused blood extravasation in the brain region. Endothelial lesions were identified exclusively on cerebral blood vessels in the arhgef17-deficient zebrafish. CONCLUSIONS: Our results provide compelling evidence that ARHGEF17 is a risk gene for IA.

Vascular endothelial growth factor A (Vegfa) signaling regulates vascular development during embryogenesis and organ formation. However, the signaling mechanisms that govern the formation of various arteries/veins in various tissues are incompletely understood. In this study, we utilized transcription activator-like effector nuclease (TALEN) to generate zebrafish vegfaa mutants. vegfaa(-/-) embryos are embryonic lethal, and display a complete loss of the dorsal aorta (DA) and expansion of the cardinal vein. Activation of Vegfa signaling expands the arterial cell population at the expense of venous cells during vasculogenesis of the axial vessels in the trunk. Vegfa signaling regulates endothelial cell (EC) proliferation after arterial-venous specification. Vegfa deficiency and overexpression inhibit the formation of tip cell filopodia and interfere with the pathfinding of intersegmental vessels (ISVs). In the head vasculature, vegfaa(-/-) causes loss of a pair of mesencephalic veins (MsVs) and central arteries (CtAs), both of which usually develop via sprouting angiogenesis. Our results indicate that Vegfa signaling induces the formation of the DA at the expense of the cardinal vein during the trunk vasculogenesis, and that Vegfa is required for the angiogenic formation of MsVs and CtAs in the brain. These findings suggest that Vegfa signaling governs the formation of diverse arteries/veins by distinct cellular mechanisms in vertebrate vasculatures. Copyright (C) 2017, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.

Prostaglandins regulate a wide variety of physiological and pathological processes, including inflammation, reproduction, cardiovascular homeostasis, and cancer progression. Cyclooxygenase (COX) catalyzes the rate-limiting step in the production of prostaglandins from arachidonic acid (Fig. 1). In 2 reaction steps, arachidonic acid is firstly converted to PGH2, subsequently metabolized to form structurally related prostanoids in various tissues, including PGE2, PGD2, PGF2α, PGI2 and Thromboxane A2 (TxA2).1 Until now, most of the studies evaluating prostaglandin signaling have come from mammalian models. We propose that zebrafish provides a useful model system to elucidate the roles of prostaglandin signaling in embryogenesis and organ development. Figure 1. Prostaglandin signaling regulates ciliogenesis by modulating intraflagellar transport. PGE2 is synthesized by COX1 and COX2 and then exported by LKT/ABCC4 transporter. Released PGE2 binds to EP4 receptor on the cilium, which activates adenylate cyclase ... Through analyzing a zebrafish mutant leakytail (lkt), we demonstrate that cilia formation and elongation require prostaglandin signaling during development.2 Our findings indicate that Lkt/ABCC4-mediated PGE2 signaling affects cAMP level and promotes ciliogenesis. A model is proposed where PGE2 is exported from cells via Lkt/ABCC4 transporter on the plasma cell membrane and signals through the G-protein coupled receptor EP4 on the cilium or at the base of the cilium, thereby activating adenylate cyclase (AC) and cAMP signaling to promote the anterograde intraflagellar transport (IFT)2 (Fig. 1). In support of this model, we found that PGE2 treatment causes an increase of intracellular cAMP but not Ca2+ in inner medullary collecting duct 3 (IMCD3) cells during ciliogenesis.2 PGE2 treatment increases anterograde velocities of IFT particles but has no obvious effects on retrograde velocities2 (Fig. 1). We further observed that deficiency in Lkt/Abcc4 or Ep4 activities failed to alter expression of the ciliary transcriptional factor Foxj1,2 suggesting that PGE2 signaling regulates ciliogenesis through facilitating the axonemal assembly rather than cilia biosynthesis. These findings linking PGE2 signaling to the second messenger represent a key regulatory step in the control of the anterograde IFT during ciliogenesis. Cilia formation and elongation require coordinate regulation of bidirectional traffic of IFT particles, including frequency and speed. One plausible mechanism in control of IFT speed during ciliogenesis can be inferred from studies of the nematode cilium, where 2 motors, kinesin-II and OSM3, act in a concerted fashion and produce an intermediate speed in lengthening cilia3 (Fig. 1). Deletion of the fast motor OSM3 causes IFT particles to move at the slow velocity and ablation of the slow motor Kinesin-II results in IFT particles to move at the fast velocity.3 A mechanism to enzymatically modulates motor or IFT proteins could be used to regulate particle velocity. It is unknown whether the motors can be served as substrates of PKA in response to PGE2 (Fig. 1). During the anterograde axonal transport of vesicle in the leg giant axon, several axoplasmic proteins including kinesin can be phosphorylated by cAMP/PKA activation.4 Alternatively, kinesin or cytoplasmic dynein proteins form complexes with PKA subunits in response to PGE2 during IFT. In melanophores, cAMP/PKA regulates intracellular organelle transport during aggregation and dispersion of pigment granules. Motor proteins dynein and kinesin II form 2 separate complexes with PKA regulatory subunits. Removal of PKA from granules causes disruption of dynein-dependent pigment aggregation, leading to kinesin II-dependent pigment dispension.5 This direct contact model appears to be the efficient way to switch between various kinds of intracellular transport. The roles of PGE2 signaling in ciliary development were not apparent from previous murine genetic studies. This is likely due to the maternal contribution of prostaglandins in the placenta allowing PGE2 deficient mouse embryos to develop normally. We circumvented the maternal interference by using externally developing zebrafish embryos to reveal the roles of Lkt/ABCC4-mediated PGE2 signaling in regulating ciliogenensis and organ laterality. In agreement with animal model studies, cultured mammalian cells deficient in PGE2 signaling display defective ciliogenesis.2 Zebrafish are an attractive model system to reveal the roles of prostaglandins in embryogenesis, stem cell formation and organ development.1 PGE2 signaling has been identified to regulate morphogenetic movements of convergence and extension and is essential for gastrulation movements in zebrafish embryos.1 Importantly, activation of PGE2 signaling expands haematopoietic stem cells in zebrafish and mouse, thereby providing potential therapy for human patients with depleted haematopoietic stem cells. Recent studies indicate that PGE2 activity has a critical role in the specification and outgrowth of liver and pancreas, and can determine the fate of liver versus pancreas progenitor cells.6 These findings place prostaglandin signaling among the canonical protein signaling pathways in regulating embryo development and organ formation, in addition to its long-standing roles in physiology and pathology. From an evolution perspective, prostaglandins have been found in all major phyla, and can regulate development of metazoans similar to protein signaling pathways.7 Prostaglandins offer unique ancestral advantages compared to protein signaling pathways. Prostaglandins are synthesized de novo from membrane-released arachidonic acid when cells are activated, and they do not need the energy and storage requirements of proteins. The important properties of prostaglandins are their stereochemical precision in recognition, their potency in the nanomolar range and their evanescent half-life. This enables them to have more quick and accurate response in spatial and temporal manners in the setting of development and physiology. Prostaglandin molecules are truly a conundrum. New insight into their roles in development and disease, and the potential therapeutics based on the novel findings will undoubtedly arise in the near future.

Cilia are microtubule-based organelles that mediate signal transduction in a variety of tissues. Despite their importance, the signalling cascades that regulate cilium formation remain incompletely understood. Here we report that prostaglandin signalling affects ciliogenesis by regulating anterograde intraflagellar transport (IFT). Zebrafish leakytail (lkt) mutants show ciliogenesis defects, and the lkt locus encodes an ATP-binding cassette transporter (ABCC4). We show that Lkt/ABCC4 localizes to the cell membrane and exports prostaglandin E2 (PGE2), a function that is abrogated by the Lkt/ABCC4(T804M) mutant. PGE2 synthesis enzyme cyclooxygenase-1 and its receptor, EP4, which localizes to the cilium and activates the cyclic-AMP-mediated signalling cascade, are required for cilium formation and elongation. Importantly, PGE2 signalling increases anterograde but not retrograde velocity of IFT and promotes ciliogenesis in mammalian cells. These findings lead us to propose that Lkt/ABCC4-mediated PGE2 signalling acts through a ciliary G-protein-coupled receptor, EP4, to upregulate cAMP synthesis and increase anterograde IFT, thereby promoting ciliogenesis.

The Hippo tumor suppressor pathway plays important roles in organ size control through Lats1/2 mediated phosphorylation of the YAP/TAZ transcription co-activators. However, YAP/TAZ independent functions of the Hippo pathway are largely unknown. Here we report a novel role of the Hippo pathway in angiogenesis. Angiomotin p130 isoform (AMOTp130) is phosphorylated on a conserved HXRXXS motif by Lats1/2 downstream of GPCR signaling. Phosphorylation disrupts AMOT interaction with F-actin and correlates with reduced F-actin stress fibers and focal adhesions. Furthermore, phosphorylation of AMOT by Lats1/2 inhibits endothelial cell migration in vitro and angiogenesis in zebrafish embryos in vivo. Thus AMOT is a direct substrate of Lats1/2 mediating functions of the Hippo pathway in endothelial cell migration and angiogenesis.

The effect of BHC80 (a component of BRAF-HDAC complex) on development was not well studied, because BHC80 gene knock-out mice died in one day after birth. Interestingly, zebrafish embryos can live, even if their important organs like cardiac system has severe dysfunction, as 25%-40% O2 are supplied through their skin. Therefore, a model of BHC80 gene knock-down zebrafish embryos was established to explore the effect of BHC80 on the early embryonic development. BHC80-morpholino antisense oligonucleotides 2 (BHC80-MO2) was designed and injected into zebrafish embryos to interrupt the correct translation of BHC80 mRNA at one or two cells stage, which was proved by RT-PCR analysis. Two control groups, including non-injection group and control-MO (con-MO) injection group, and four different doses of BHC80-MO2 injection groups, including 4 ng, 6 ng, 8 ng and 10 ng per embryo were set up. The embryonic heart phenotype and cardiac function were monitored, analyzed and compared between con-MO and BHC80-MO2 groups by fluorescence microscope in vmhc:gfp transgenic zebrafish which express green fluorescent protein in ventricle. The results showed that BHC80-MO2 microinjection effectively knocked down the BHC80 gene expression, because the BHC80-MO2 group emerged a new 249 bp band which reduced 51 bp compared to 300 bp band of con-MO group in RT-PCR analysis, and the 51 bp was the extron 10. The abnormal embryo rate rose with the increase of BHC80-MO2 dosage. The proper BHC80-MO2 injection dosage was 8 ng per embryo, as minor embryos had abnormal phenotype in 4 ng and 6 ng per embryo groups and most embryos died in 10 ng per embryo group. BHC80-MO2 embryos exhibited abnormal cardiac phenotype, including imbalance of the proportion of heart ventricle to atrium, incomplete D-loop, even tubular heart, slow heart rates and cardiac dysfunction. The results from a model of BHC80 gene knock-down zebrafish embryos show that the abnormal cardiac phenotype and cardiac dysfunction of BHC80-MO2 embryos may be one of the probable reasons for the BHC80 gene knock-out mice death, which would provide a good research model to clarify the mechanism of cardiac development.