Macrophages enhance regeneration of lateral line neuromast derived from interneuromast cells through TGF-ß in zebrafish.

Macrophages play a pivotal role in the response to injury, contributing significantly to the repair and regrowth of damaged tissues. The external lateral line system in aquatic organisms offers a practical model for studying regeneration, featuring interneuromast cells connecting sensory neuromasts. Under normal conditions, these cells remain dormant, but their transformation into neuromasts occurs when overcoming inhibitory signals from Schwann cells and posterior lateral line nerves. The mechanism enabling interneuromast cells to evade inhibition by Schwann cells remains unclear. Previous observations suggest that macrophages physically interact with neuromasts, nerves, and Schwann cells during regeneration. This interaction leads to the regeneration of neuromasts in a subset of zebrafish with ablated neuromasts. To explore whether macrophages achieve this effect through secreted cytokines, we conducted experiments involving tail amputation in zebrafish larvae and tested the impact of cytokine inhibitors on neuromast regeneration. Most injured larvae remarkably regenerated a neuromast within 4 days post-amputation. Intriguingly, removal of macrophages and inhibition of the anti-inflammatory cytokine transforming growth factor-beta (TGF-ß) significantly delayed neuromast regeneration. Conversely, inhibition of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) had minor effects on the regeneration process. This study provides insights into how macrophages activate interneuromast cells, elucidating the pathways underlying neuromast regeneration.

Macrophages Break Interneuromast Cell Quiescence by Intervening in the Inhibition of Schwann Cells in the Zebrafish Lateral Line.

In the zebrafish lateral line system, interneuromast cells (INCs) between neuromasts are kept quiescent by underlying Schwann cells (SWCs). Upon severe injuries that cause the complete loss of an entire neuromast, INCs can occasionally differentiate into neuromasts but how they escape from the inhibition by SWCs is still unclear. Using a genetic/chemical method to ablate a neuromast precisely, we found that a small portion of larvae can regenerate a new neuromast. However, the residual regeneration capacity was hindered by inhibiting macrophages. Using in toto imaging, we further discovered heterogeneities in macrophage behavior and distribution along the lateral line. We witnessed the crawling of macrophages between the injured lateral line and SWCs during regeneration and between the second primordium and the first mature lateral line during development. It implies that macrophages may physically alleviate the nerve inhibition to break the dormancy of INCs during regeneration and development in the zebrafish lateral line.

GSTM3 variant is a novel genetic modifier in Brugada syndrome, a disease with risk of sudden cardiac death.

BACKGROUND: Brugada syndrome (BrS) is a rare inherited disease causing sudden cardiac death (SCD). Copy number variants (CNVs) can contribute to disease susceptibility, but their role in Brugada syndrome (BrS) is unknown. We aimed to identify a CNV associated with BrS and elucidated its clinical implications. METHODS: We enrolled 335 unrelated BrS patients from 2000 to 2018 in the Taiwanese population. Microarray and exome sequencing were used for discovery phase whereas Sanger sequencing was used for the validation phase. HEK cells and zebrafish were used to characterize the function of the CNV variant. FINDINGS: A copy number deletion of GSTM3 (chr1:109737011-109737301, hg38) containing the eighth exon and the transcription stop codon was observed in 23.9% of BrS patients versus 0.8% of 15,829 controls in Taiwan Biobank (P < 0.001), and 0% in gnomAD. Co-segregation analysis showed that the co-segregation rate was 20%. Patch clamp experiments showed that in an oxidative stress environment, GSTM3 down-regulation leads to a significant decrease of cardiac sodium channel current amplitude. Ventricular arrhythmia incidence was significantly greater in gstm3 knockout zebrafish at baseline and after flecainide, but was reduced after quinidine, consistent with clinical observations. BrS patients carrying the GSTM3 deletion had higher rates of sudden cardiac arrest and syncope compared to those without (OR: 3.18 (1.77-5.74), P<0.001; OR: 1.76 (1.02-3.05), P = 0.04, respectively). INTERPRETATION: This GSTM3 deletion is frequently observed in BrS patients and is associated with reduced INa, pointing to this as a novel potential genetic modifier/risk predictor for the development of the electrocardiographic and arrhythmic manifestations of BrS. FUNDING: This work was supported by the Ministry of Science and Technology (107-2314-B-002-261-MY3 to J.M.J. Juang), and by grants HL47678, HL138103 and HL152201 from the National Institutes of Health to CA.

Wnt5b integrates Fak1a to mediate gastrulation cell movements via Rac1 and Cdc42.

Focal adhesion kinase (FAK) mediates vital cellular pathways during development. Despite its necessity, how FAK regulates and integrates with other signals during early embryogenesis remains poorly understood. We found that the loss of Fak1a impaired epiboly, convergent extension and hypoblast cell migration in zebrafish embryos. We also observed a clear disturbance in cortical actin at the blastoderm margin and distribution of yolk syncytial nuclei. In addition, we investigated a possible link between Fak1a and a well-known gastrulation regulator, Wnt5b, and revealed that the overexpression of fak1a or wnt5b could cross-rescue convergence defects induced by a wnt5b or fak1a antisense morpholino (MO), respectively. Wnt5b and Fak1a were shown to converge in regulating Rac1 and Cdc42, which could synergistically rescue wnt5b and fak1a morphant phenotypes. Furthermore, we generated several alleles of fak1a mutants using CRISPR/Cas9, but those mutants only revealed mild gastrulation defects. However, injection of a subthreshold level of the wnt5b MO induced severe gastrulation defects in fak1a mutants, which suggested that the upregulated expression of wnt5b might complement the loss of Fak1a. Collectively, we demonstrated that a functional interaction between Wnt and FAK signalling mediates gastrulation cell movements via the possible regulation of Rac1 and Cdc42 and subsequent actin dynamics.

The first complete mitochondrial genome of the sand dollar Sinaechinocyamus mai (Echinoidea: Clypeasteroida).

Morphologic and molecular data often lead to different hypotheses of phylogenetic relationships. Such incongruence has been found in the echinoderm class Echinoidea. In particular, the phylogenetic status of the order Clypeasteroida is not well resolved. Complete mitochondrial genomes are currently available for 29 echinoid species, but no clypeasteroid had been sequenced to date. DNA extracted from a single live individual of Sinaechinocyamus mai was sequenced with 10× Genomics technology. This first complete mitochondrial genome (mitogenome) for the order Clypeasteroida is 15,756 base pairs in length. Phylogenomic analysis based on 34 ingroup taxa belonging to nine orders of the class Echinoidea show congruence between our new genetic inference and published trees based on morphologic characters, but also includes some intriguing differences that imply the need for additional investigation.

MicroRNAs regulate gene plasticity during cold shock in zebrafish larvae.

BACKGROUND: MicroRNAs (miRNAs) are critical regulators responding to acute environmental stresses in both plants and animals. By modulating gene expression, miRNAs either restore or reconstitute a new expression program to enhance cell tolerance to stresses. Cold shock is one of the stresses that can induce acute physiological responses and transcriptional changes in aquatic creatures. Previous genomic studies have revealed many cold-affected genes in fish larvae and adults, however, the role of miRNAs in acute cold response is still ambiguous. To elucidate the regulatory roles of miRNAs in the cold-inducible responses, we performed small RNA-seq and RNA-seq analyses and found potential cold regulatory miRNAs and genes. We further investigated their interactions and involvements in cold tolerance. RESULTS: Small RNA-seq and RNA-seq identified 29 up-/26 down-regulated miRNAs and 908 up-/468 down-regulated genes, respectively, in responding to cold shock for 4 h at 18 °C. miRNA and transcriptomic analyses showed these miRNAs and mRNAs are involved in similar biological processes and pathways. Gene ontology enrichment analyses revealed the cold-induced genes were enriched in pathways, including melanogenesis, GnRH pathway, circadian rhythm, etc. We were particularly interested in the changes in circadian clock genes that affect daily metabolism. The enrichment of circadian clock genes was also observed in previous fish cold acclimation studies, but have not been characterized. To characterize the functional roles of circadian clock genes in cold tolerance, we individually overexpressed selected clock genes in zebrafish larvae and found one of the core clock genes per2 resulted in better recovery from cold shock. In addition, we validated the interaction of per2 with its associate miRNA, dre-mir-29b, which is also cold-inducible. It suggests the transcription of per2 can be modulated by miRNA upon cold shock. CONCLUSIONS: Collectively, our observations suggest that miRNAs are fine turners for regulating genomic plasticity against cold shock. We further showed that the fine tuning of core clock gene per2 via its associated miRNA, dre-mir-29b, can enhance the cold tolerance of zebrafish larvae.

Stathmin-like 4 is critical for the maintenance of neural progenitor cells in dorsal midbrain of zebrafish larvae.

A delicate balance between proliferating and differentiating signals is necessary to ensure proper growth and neuronal specification. By studying the developing zebrafish brain, we observed a specific and dynamic expression of a microtubule destabilizer gene, stathmin-like 4 (stmn4), in the dorsal midbrain region. The expression of stmn4 was mutually exclusive to a pan-neuronal marker, elavl3 that indicates its role in regulating neurogenesis. We showed the knockdown or overexpression of stmn4 resulted in premature neuronal differentiation in dorsal midbrain. We also generated stmn4 maternal-zygotic knockout zebrafish by the CRISPR/Cas9 system. Unexpectedly, only less than 10% of stmn4 mutants showed similar phenotypes observed in that of stmn4 morphants. It might be due to the complementation of the increased stmn1b expression observed in stmn4 mutants. In addition, time-lapse recordings revealed the changes in cellular proliferation and differentiation in stmn4 morphants. Stmn4 morphants displayed a longer G2 phase that could be rescued by Cdc25a. Furthermore, the inhibition of Wnt could reduce stmn4 transcripts. These results suggest that the Wnt-mediated Stmn4 homeostasis is crucial for preventing dorsal midbrain from premature differentiation via the G2 phase control during the neural keel stage.

Dynamic regulation of the microtubule and actin cytoskeleton in zebrafish epiboly.

Gastrulation is a key developmental stage with striking changes in morphology. Coordinated cell movements occur to bring cells to their correct positions in a timely manner. Cell movements and morphological changes are accomplished by precisely controlling dynamic changes in cytoskeletal proteins, microtubules, and actin filaments. Among those cellular movements, epiboly produces the first distinct morphological changes in teleosts. In this review, I describe epiboly and its mechanics, and the dynamic changes in microtubule networks and actin structures, mainly in zebrafish embryos. The factors regulating those cytoskeletal changes will also be discussed.

SCUBE3 (signal peptide-CUB-EGF domain-containing protein 3) modulates fibroblast growth factor signaling during fast muscle development.

SCUBE3 (signal peptide CUB-EGF-like domain-containing protein 3) belongs to a newly identified secreted and cell membrane-associated SCUBE family, which is evolutionarily conserved in vertebrates. Scube3 is predominantly expressed in a variety of developing tissues in mice such as somites, neural tubes, and limb buds. However, its function during development remains unclear. In this study, we first showed that knockdown of SCUBE3 in C2C12 myoblasts inhibited FGF receptor 4 expression and FGF signaling, thus resulting in reduced myogenic differentiation. Furthermore, knockdown of zebrafish scube3 by antisense morpholino oligonucleotides specifically suppressed the expression of the myogenic marker myod1 within the lateral fast muscle precursors, whereas its expression in the adaxial slow muscle precursors was largely unaffected. Consistent with these findings, immunofluorescent staining of fast but not slow muscle myosin was markedly decreased in scube3 morphants. Further genetic studies identified fgf8 as a key regulator in scube3-mediated fast muscle differentiation in zebrafish. Biochemical and molecular analysis showed that SCUBE3 acts as a FGF co-receptor to augment FGF8 signaling. Scube3 may be a critical upstream regulator of fast fiber myogenesis by modulating fgf8 signaling during zebrafish embryogenesis.

The Nogo-C2/Nogo receptor complex regulates the morphogenesis of zebrafish lateral line primordium through modulating the expression of dkk1b, a Wnt signal inhibitor.

The fish lateral line (LL) is a mechanosensory system closely related to the hearing system of higher vertebrates, and it is composed of several neuromasts located on the surface of the fish. These neuromasts can detect changes in external water flow, to assist fish in maintaining a stationary position in a stream. In the present study, we identified a novel function of Nogo/Nogo receptor signaling in the formation of zebrafish neuromasts. Nogo signaling in zebrafish, like that in mammals, involves three ligands and four receptors, as well as three co-receptors (TROY, p75, and LINGO-1). We first demonstrated that Nogo-C2, NgRH1a, p75, and TROY are able to form a Nogo-C2 complex, and that disintegration of this complex causes defective neuromast formation in zebrafish. Time-lapse recording of the CldnB::lynEGFP transgenic line revealed that functional obstruction of the Nogo-C2 complex causes disordered morphogenesis, and reduces rosette formation in the posterior LL (PLL) primordium during migration. Consistent with these findings, hair-cell progenitors were lost from the PLL primordium in p75, TROY, and Nogo-C2/NgRH1a morphants. Notably, the expression levels of pea3, a downstream marker of Fgf signaling, and dkk1b, a Wnt signaling inhibitor, were both decreased in p75, TROY, and Nogo-C2/NgRH1a morphants; moreover, dkk1b mRNA injection could rescue the defects in neuromast formation resulting from knockdown of p75 or TROY. We thus suggest that a novel Nogo-C2 complex, consisting of Nogo-C2, NgRH1a, p75, and TROY, regulates Fgf signaling and dkk1b expression, thereby ensuring stable organization of the PLL primordium.

Aromatic L-amino acid decarboxylase (AADC) is crucial for brain development and motor functions.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare pediatric neuro-metabolic disease in children. Due to the lack of an animal model, its pathogenetic mechanism is poorly understood. To study the role of AADC in brain development, a zebrafish model of AADC deficiency was generated. We identified an aadc gene homolog, dopa decarboxylase (ddc), in the zebrafish genome. Whole-mount in situ hybridization analysis showed that the ddc gene is expressed in the epiphysis, locus caeruleus, diencephalic catecholaminergic clusters, and raphe nuclei of 36-h post-fertilization (hpf) zebrafish embryos. Inhibition of Ddc by AADC inhibitor NSD-1015 or anti-sense morpholino oligonucleotides (MO) reduced brain volume and body length. We observed increased brain cell apoptosis and loss of dipencephalic catecholaminergic cluster neurons in ddc morphants (ddc MO-injected embryos). Seizure-like activity was also detected in ddc morphants in a dose-dependent manner. ddc morphants had less sensitive touch response and impaired swimming activity that could be rescued by injection of ddc plasmids. In addition, eye movement was also significantly impaired in ddc morphants. Collectively, loss of Ddc appears to result in similar phenotypes as that of ADCC deficiency, thus zebrafish could be a good model for investigating pathogenetic mechanisms of AADC deficiency in children.

Quantitative proteomics reveals diverse roles of miR-148a from gastric cancer progression to neurological development.

MicroRNAs (miRNAs) are noncoding RNAs that control gene expression either by degradation of mRNAs or inhibition of protein translation. miR-148a has been reported to have the impacts on tumor progression. Here, a quantitative proteomics combined with stable isotope labeling was applied to identify the global profile of miR-148a-regulated downstream proteins. The data have been deposited to the ProteomeXchange with identifier PXD000190. A total of 2938 proteins were quantified, and 55 proteins were considered to be regulated by miR-148a. We found that not only proteins associated with cancer progression but also molecules involved in neural development were regulated by miR-148a. This study is the first to identify the function of miR-148a in neural development by using a proteomic approach. Analysis of a public clinical database also showed that the patients with neural diseases could display abnormal expression of miR-148a. Moreover, silencing of miR-148a led to the abnormal morphology and decreased expression of neuron-related markers in the developing brain of zebrafish. These results provided important insight into the regulation of neurological development elicited by miR-148a.

Calnexin is required for zebrafish posterior lateral line development.

The lateral line is a mechanosensory system in fish and amphibians to detect local water flow and pressure. Development of the posterior lateral line (PLL) originates from the migrating PLL primordium (PLLP). The PLLP deposits neuromasts on the trunk during migration to the tail. Molecular dissection revealed that PLL development is associated with genes mediating cell adhesion, morphogenesis, neurogenesis and development, but the regulatory signaling network is far from completion. To further investigate candidate regulatory genes for lateral line development, we found using whole-mount in situ hybridization that calnexin, an endoplasmic reticular (ER) calcium-binding protein gene, is expressed in PLL neuromasts. Knockdown of calnexin using antisense morpholino oligonucleotides resulted in a dose-dependent reduction in neuromasts and hair cells of the PLL. Using a transgenic claudin b:gfp line, we observed a notably reduced PLLP size, but no significant migration defect in calnexin morphants. Finally, we discovered that the reduced PLLP is associated with a reduction in cell proliferation and an increase in ER stress-dependent apoptosis. These results suggest that calnexin is essential for neuromast formation during lateral line development in the zebrafish.

Zebrafish scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF (epidermal growth factor) domain-containing protein 1) is involved in primitive hematopoiesis.

scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF domain-containing protein 1), the founding member of a novel secreted and cell surface SCUBE protein family, is expressed predominantly in various developing tissues in mice. However, its function in primitive hematopoiesis remains unknown. In this study, we identified and characterized zebrafish scube1 and analyzed its function by injecting antisense morpholino-oligonucleotide into embryos. Whole-mount in situ hybridization revealed that zebrafish scube1 mRNA is maternally expressed and widely distributed during early embryonic development. Knockdown of scube1 by morpholino-oligonucleotide down-regulated the expression of marker genes associated with early primitive hematopoietic precursors (scl) and erythroid (gata1 and hbbe1), as well as early (pu.1) and late (mpo and l-plastin) myelomonocytic lineages. However, the expression of an early endothelial marker fli1a and vascular morphogenesis appeared normal in scube1 morphants. Overexpression of bone morphogenetic protein (bmp) rescued the expression of scl in the posterior lateral mesoderm during early primitive hematopoiesis in scube1 morphants. Biochemical and molecular analysis revealed that Scube1 could be a BMP co-receptor to augment BMP signaling. Our results suggest that scube1 is critical for and functions at the top of the regulatory hierarchy of primitive hematopoiesis by modulating BMP activity during zebrafish embryogenesis.

Autotaxin in embryonic development.

Autotaxin (ATX) is a secreted lysophospholipase D that generates the multifunctional lipid mediator lysophosphatidic acid (LPA). LPA signals through six distinct G protein-coupled receptors, acting alone or in concert to activate multiple effector pathways. The ATX-LPA signaling axis is implicated in a remarkably wide variety of physiological and pathological processes and plays a vital role in embryonic development. Disruption of the ATX-encoding gene (Enpp2) in mice results in intrauterine death due to vascular defects in the extra-embryonic yolk sac and embryo proper. In addition, Enpp2 (-/-) embryos show impaired neural development. The observed angiogenic defects are attributable, at least in part, to loss of LPA signaling through the Gα(12/13)-linked RhoA-ROCK-actin remodeling pathway. Studies in zebrafish also have uncovered a dual role for ATX in both vascular and neural development; furthermore, they point to a key role for ATX-LPA signaling in the regulation of left-right asymmetry. Here we discuss our present understanding of the role of ATX-LPA signaling in vertebrate development. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Autotaxin/Lpar3 signaling regulates Kupffer's vesicle formation and left-right asymmetry in zebrafish.

Left-right (L-R) patterning is essential for proper organ morphogenesis and function. Calcium fluxes in dorsal forerunner cells (DFCs) are known to regulate the formation of Kupffer's vesicle (KV), a central organ for establishing L-R asymmetry in zebrafish. Here, we identify the lipid mediator lysophosphatidic acid (LPA) as a regulator of L-R asymmetry in zebrafish embryos. LPA is produced by Autotaxin (Atx), a secreted lysophospholipase D, and triggers various cellular responses through activation of specific G protein-coupled receptors (Lpar1-6). Knockdown of Atx or LPA receptor 3 (Lpar3) by morpholino oligonucleotides perturbed asymmetric gene expression in lateral plate mesoderm and disrupted organ L-R asymmetries, whereas overexpression of lpar3 partially rescued those defects in both atx and lpar3 morphants. Similar defects were observed in embryos treated with the Atx inhibitor HA130 and the Lpar1-3 inhibitor Ki16425. Knockdown of either Atx or Lpar3 impaired calcium fluxes in DFCs during mid-epiboly stage and compromised DFC cohesive migration, KV formation and ciliogenesis. Application of LPA to DFCs rescued the calcium signal and laterality defects in atx morphants. This LPA-dependent L-R asymmetry is mediated via Wnt signaling, as shown by the accumulation of ß-catenin in nuclei at the dorsal side of both atx and lpar3 morphants. Our results suggest a major role for the Atx/Lpar3 signaling axis in regulating KV formation, ciliogenesis and L-R asymmetry via a Wnt-dependent pathway.

Lysophosphatidic acid induces erythropoiesis through activating lysophosphatidic acid receptor 3.

Lysophosphatidic acid (LPA), an extracellular lipid mediator, exerts multiple bioactivities through activating G protein-coupled receptors. LPA receptor 3 (LPA(3)) is a member of the endothelial differentiation gene family, which regulates differentiation and development of the circulation system. However, the relationship among the LPA receptors (LPARs) and erythropoiesis is still not clear. In this study, we found that erythroblasts expressed both LPA(1) and LPA(3), and erythropoietic defects were observed in zLPA(3) antisense morpholino oligonucleotide-injected zebrafish embryos. In human model, our results showed that LPA enhanced the erythropoiesis in the cord blood-derived human hematopoietic stem cells (hHSCs) with erythropoietin (EPO) addition in the plasma-free culture. When hHSCs were treated with Ki16425, an antagonist of LPA(1) and LPA(3), erythropoietic process of hHSCs was also blocked, as detected by mRNA and protein expressions of CD71 and GlyA. In the knockdown study, we further demonstrated that specific knockdown of LPA(3), not LPA(1), blocked the erythropoiesis. The translocation of ß-catenin into the nucleus, a downstream response of LPAR activation, was blocked by Ki16425 treatment. In addition, upregulation of erythropoiesis by LPA was also blocked by quercetin, an inhibitor of the ß-catenin/T-cell factor pathway. Furthermore, the enhancement of LPA on erythropoiesis was diminished by blocking c-Jun-activated kinase/signal transducer and activator of transcription and phosphatidylinositol 3-kinase/AKT activation, the downstream signaling pathways of EPO receptor, suggested that LPA might play a synergistic role with EPO to regulate erythropoietic process. In conclusion, we first reported that LPA participates in EPO-dependent erythropoiesis through activating LPA(3).

Establishment of a transgenic zebrafish line for superficial skin ablation and functional validation of apoptosis modulators in vivo.

BACKGROUND: Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)(cy17) (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR(+) fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR(+) signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR(+) fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR(+) fluorescent signaling. CONCLUSION/SIGNIFICANCE: The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.

Ptenb mediates gastrulation cell movements via Cdc42/AKT1 in zebrafish.

Phosphatidylinositol 3-kinase (PI3 kinase) mediates gastrulation cell migration in zebrafish via its regulation of PIP(2)/PIP(3) balance. Although PI3 kinase counter enzyme PTEN has also been reported to be essential for gastrulation, its role in zebrafish gastrulation has been controversial due to the lack of gastrulation defects in pten-null mutants. To clarify this issue, we knocked down a pten isoform, ptenb by using anti-sense morpholino oligos (MOs) in zebrafish embryos and found that ptenb MOs inhibit convergent extension by affecting cell motility and protrusion during gastrulation. The ptenb MO-induced convergence defect could be rescued by a PI3-kinase inhibitor, LY294002 and by overexpressing dominant negative Cdc42. Overexpression of human constitutively active akt1 showed similar convergent extension defects in zebrafish embryos. We also observed a clear enhancement of actin polymerization in ptenb morphants under cofocal microscopy and in actin polymerization assay. These results suggest that Ptenb by antagonizing PI3 kinase and its downstream Akt1 and Cdc42 to regulate actin polymerization that is critical for proper cell motility and migration control during gastrulation in zebrafish.