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Shuo Lin

Publications and source records attributed to Shuo Lin.

At least 19 recordsLinked to original sources
PubMed2006

Vhnf1 acts downstream of Bmp, Fgf, and RA signals to regulate endocrine beta cell development in zebrafish.

Bmp, Fgf, and retinoic acid (RA) signals have been implicated as regulators of pancreas development. However, the integration of these signaling pathways in vivo is not fully understood. Variant hnf1 (Vhnf1) is a transcription factor involved in pancreas, liver, and kidney development and its mutation in zebrafish causes underdeveloped pancreas and liver. We investigated the signaling pathways that regulate vhnf1 expression during pancreas development. First, we showed that Bmp activity is required for vhnf1 expression in the endoderm. In chordin (a Bmp antagonist) morpholino (MO)-injected embryos, vhnf1 expression in endoderm and in endocrine beta cells is expanded. On the other hand, in alk8 (a type I TGFbeta receptor) MO-injected embryos, vhnf1 expression in the endoderm is significantly reduced. Second, we showed that Fgf signaling participates in regulation of pancreas development through the vhnf1 pathway. Third, we demonstrated that RA fails to rescue reduction of insulin expression in vhnf1 mutants, whereas overexpression of vhnf1 restores insulin expression that is repressed by treatment with a RA receptor inhibitor. And finally, we revealed that both Bmp and Fgf signals act genetically upstream of RA in directing pancreas development. Taken together, our data establish that vhnf1 acts downstream of the signaling pathways of RA, Bmp, and Fgf to regulate pancreas development in zebrafish.

Animals↗
PubMed2006

Vertebrate MAX-1 is required for vascular patterning in zebrafish.

During embryogenesis, stereotypic vascular patterning requires guidance cues from neighboring tissues. However, key molecules involved in this process still remain largely elusive. Here, we report molecular cloning, expression, and functional studies of zebrafish max-1, a homolog of Caenorhabditis elegans max-1 that has been implicated in motor neuron axon guidance. During early embryonic development, zebrafish max-1 is specifically expressed in subsets of neuronal tissues, epithelial cells, and developing somites through which vascular endothelial cells migrate from large ventral axial vessels to form stereotypic intersegmental blood vessels (ISV). Blocking zebrafish max-1 mRNA splicing by morpholino injection led to aberrant ISV patterning, which could be rescued by injection of either C. elegans or zebrafish max-1 mRNA. Analysis of motor neurons in the same region showed normal neuronal axon pathfinding. Further studies suggested that the ISV defect caused by max-1 knockdown could be partially rescued by overexpression of ephrinb3 and that max-1 was involved in mediating membrane localization of ephrin proteins, which have been shown to provide guidance cues for endothelial cell migration. Our findings therefore suggest that max-1, acting upstream of the ephrin pathway, is critically required in vascular patterning in vertebrate species.

Animals↗
PubMed2006

The neurite outgrowth inhibitor Nogo-A promotes denervation in an amyotrophic lateral sclerosis model.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss and muscle wasting. In muscles of ALS patients, Nogo-A-a protein known to inhibit axon regeneration-is ectopically expressed at levels that correlate with the severity of the clinical symptoms. We now show that the genetic ablation of Nogo-A extends survival and reduces muscle denervation in a mouse model of ALS. In turn, overexpression of Nogo-A in wild-type muscle fibres leads to shrinkage of the postsynapse and retraction of the presynaptic motor ending. This suggests that the expression of Nogo-A occurring early in ALS skeletal muscle could cause repulsion and destabilization of the motor nerve terminals, and subsequent dying back of the axons and motor neurons.

Amyotrophic Lateral Sclerosis↗
PubMed2006

Initiation of V(D)J recombination in zebrafish (Danio rerio) ovaries.

The assembly of mammalian antigen receptor genes is a lymphoid-specific process. However, rearranged immunoglobulin genes can also be recovered from non-lymphoid tissues of cartilaginous fish. This event, known as germline rearrangement, has been speculated to arise from recombination-activating gene (RAG)-mediated recombination in germ cells. In this report, we demonstrate that zebrafish (Danio rerio) oocytes expressing high levels of RAG-RNA can readily initiate recombination cleavage at immunoglobulin gene loci, providing direct evidence for an ongoing process of attempted germline rearrangement in zebrafish ovaries. This attempted rearrangement is largely unproductive, yielding no accumulation of germline-joined immunoglobulin genes in zebrafish, which is consistent with their general absence in this species. Our data, therefore, substantiate the speculation that RAG might have been derived from a transposase, invading germ cells of ancient species, and later become a dedicated recombinase only expressed in developing lymphocytes.

Animals↗
PubMed2006

Isolation and expression of zebrafish zinc-finger transcription factor gene tsh1.

We report the expression patterns of tsh1, a zebrafish homologue of the Drosophila homeotic gene teashirt. Expression of tsh1 is first detected at the 2-somite stage (10h post-fertilization, hpf) at the anterior end of the spinal cord. Expression expands toward the posterior spinal cord, and by the prim-5 stage (24 hpf) tsh1 transcripts are detected throughout spinal cord. Between the 14- and 25-somite stage (16-24 hpf), spinal cord expression shows a clear anterior boundary at the rostral margin of rhombomere 7. Around the prim-25 stage (36 hpf), while the spinal expression of tsh1 decreases, new expression is detected in the pectoral fin buds and dorsal forebrain. By the long-pec stage (48 hpf), spinal cord expression is undetectable, but strong expression is observed in the rhombencephalon, telencephalon, tectum opticum, midbrain-hindbrain boundary, in the first pharyngeal arch and in the eyes. This expression persists at least until the larval stages. Retinoic acid signaling influences tsh1 expression. Zebrafish tsh1 expression was induced in the anterior neural tube in embryos treated briefly with exogenous retinoic acid. Furthermore, tsh1 expression was down-regulated in the spinal cord in the zebrafish neckless mutant in which RA signaling is disrupted due to a missense mutation in the gene encoding retinaldehyde dehydrogenase type 2.

Amino Acid Sequence↗
PubMed2006

Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter.

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development.

Amino Acid Sequence↗
PubMed2006

Fluorescent tagged analysis of neural gene function using mosaics in zebrafish and Xenopus laevis.

An important question in the neurosciences is the role of specific gene expression in the control of neural morphology and connectivity. To address this question, methods are needed for expression of exogenous genes in a subset of neurons. This limited and mosaic expression allows the assessment of gene expression in a cell autonomous fashion without environmental contributions from neighboring expressing cells. These methods must also label neurons so that detailed morphology and neural connections can be evaluated. The labeling method should label only a subset of neurons so that neuronal morphology can be viewed upon a non-stained background, in a Golgi staining fashion. Here, we report methods using plasmids called pTAGUM (tagged analysis of genes using mosaics) that accomplish these goals. These methods should prove useful for the analysis of neural gene function in two important model organisms, the zebrafish and Xenopus laevis.

Animals↗
PubMed2006

Regulation of primitive hematopoiesis in zebrafish embryos by the death receptor gene.

OBJECTIVE: We investigated the regulatory mechanism of primitive hematopoiesis in zebrafish (Danio rerio) embryos with particular reference to the role of a death receptor (zDR) gene, based on a morpholino (MO) knockdown approach. METHODS: MOs targeting the zDR and chordin (Chd) were injected into naturally spawned embryos at one- to four-cell stage. A random sequence (RS) MO was used as a control. Effects on hemoglobin formation (Hb), apoptosis, and lineage-specific gene expression were examined. Embryos injected with zDR, Chd, and RS-MOs were denoted zDR(mo), zChd(mo), and zRS(mo), respectively. Those co-injected with Chd+zDR-MOs and Chd+RS-MOs were abbreviated zChd+DR(mo) and zChd+RS(mo). RESULTS: zDR mRNA expression was restricted to the intermediate cell mass of wild-type (WT) and zChd(mo) embryos. At 48 hours postfertilization, zDR(mo) embryos showed increased Hb compared with WT or zRS(mo) embryos (2.36 x 10(-2) +/- 1.13 x 10(-3) vs 1.85 x 10(-2) +/- 5.60 x 10(-4) vs 1.79 x 10(-2) +/- 1.31 x 10(-3) U, p < 0.05). zChd+DR(mo) embryos also showed increased Hb compared with zChd(mo) or zChd+RS(mo) embryos (4.60 x 10(-2) +/- 2.79 x 10(-3) vs 3.17 x 10(-2) +/- 1.07 x 10(-3) vs 3.05 x 10(-2) +/- 1.25 x 10(-3) U, p < 0.05). zDR-MO reduced apoptosis, as shown by reduced terminal transferase-mediated dUTP nick end-labeling staining in zChd+DR(mo) compared with zChd+RS(mo) embryos and caspase-3 activity in zDR(mo) vs zRS(mo) (0.525 +/- 0.094 vs 0.953 +/- 0.113 U, p < 0.05), and zChd+DR(mo) vs zChd+RS(mo) embryos (0.247 +/- 0.121 vs 1.180 +/- 0.082, p < 0.05). zChd+DR(mo) embryos showed upregulation of erythroid-specific embryonic hemoglobin gene expression but not that of a myeloid-specific myeloperoxidase gene. CONCLUSION: Knockdown of zDR in zebrafish embryos decreased apoptosis and increased Hb, suggesting that zDR may regulate primitive hematopoiesis during development.

Animals↗
PubMed2006

Modified bacterial artificial chromosomes for zebrafish transgenesis.

Transgenesis using bacterial artificial chromosomes (BAC) offers greater fidelity in directing desirable expression of foreign genes. Application of this technology in the optically transparent zebrafish with fluorescent protein reporters enables unparalleled visual analysis of regulation of gene expression in a living organism. Here we describe a streamlined procedure of direct selecting multiple BAC clones based on public sequence databases followed by rapid modification with GFP or RFP for transgenic analysis in zebrafish. Experimental procedures for BAC DNA preparation, microinjection of zebrafish embryos and screening of transgenic zebrafish carrying GFP/RFP modified BAC clones are detailed.

Animals↗
PubMed2006

Ets1-related protein is a key regulator of vasculogenesis in zebrafish.

During embryonic development, multiple signaling pathways control specification, migration, and differentiation of the vascular endothelial cell precursors, angioblasts. No single gene responsible for the commitment of mesenchymal cells to the angioblast cell fate has been identified as yet. Here we report characterization and functional studies of Etsrp, a novel zebrafish ETS domain protein. etsrp embryonic expression is only restricted to vascular endothelial cells and their earliest precursors. Morpholino knockdown of Etsrp protein function resulted in the complete absence of circulation in zebrafish embryos. Angioblasts in etsrp-morpholino-injected embryos (morphants) failed to undergo migration and differentiation and did not coalesce into functional blood vessels. Expression of all vascular endothelial molecular markers tested was severely reduced in etsrp morphants, whereas hematopoietic markers were not affected. Overexpression of etsrp RNA caused multiple cell types to express vascular endothelial markers. etsrp RNA restored expression of vascular markers in cloche mutants, defective in hematopoietic and endothelial cell formation, arguing that etsrp functions downstream of cloche in angioblast formation. etsrp gene function was also required for endothelial marker induction by the vascular endothelial growth factor (vegf) and stem cell leukemia (scl/tal1). These results demonstrate that Etsrp is necessary and sufficient for the initiation of vasculogenesis.

Animals↗
PubMed2005

Prolactin receptor signaling mediates the osmotic response of embryonic zebrafish lactotrophs.

The pituitary hormone prolactin (PRL) regulates salt and water homeostasis by altering ion retention and water uptake through peripheral osmoregulatory organs. To understand the role of osmotic homeostasis in the development of PRL-secreting lactotrophs, we generated germline transgenic zebrafish coexpressing red fluorescent protein directed by Prolactin regulatory elements (PRL-RFP) and green fluorescent protein by the Pro-opiomelanocortin promoter (POMC-GFP). Transparent embryos expressing fluorescent markers specifically targeted to lactotrophs and corticotrophs, the two pituitary lineages involved in teleost osmotic adaptation, allowed in vivo dynamic tracing of pituitary ontogeny during altered environmental salinity. Physiological osmotic changes selectively regulate lactotroph but not corticotroph proliferation during early ontogeny. These changes are not suppressed by pharmacological dopamine receptor blockade but are completely abrogated by morpholino knockdown of the PRL receptor. PRL receptor signaling exerts robust effects on lactotroph development and plays a permissive role in lactotroph osmo-responsiveness, reflecting the dual peripheral and central interactions required for early pituitary development and embryonic homeostasis.

Amino Acid Sequence↗
PubMed2005

Src-family kinases stabilize the neuromuscular synapse in vivo via protein interactions, phosphorylation, and cytoskeletal linkage of acetylcholine receptors.

Postnatal stabilization and maturation of the postsynaptic membrane are important for development and function of the neuromuscular junction (NMJ), but the underlying mechanisms remain poorly characterized. We examined the role of Src-family kinases (SFKs) in vivo. Electroporation of kinase-inactive Src constructs into soleus muscles of adult mice caused NMJ disassembly: acetylcholine receptor (AChR)-rich areas became fragmented; the topology of nerve terminal, AChRs, and synaptic nuclei was disturbed; and occasionally nerves started to sprout. Electroporation of kinase-overactive Src produced similar but milder effects. We studied the mechanism of SFK action using cultured src(-/-);fyn(-/-) myotubes, focusing on clustering of postsynaptic proteins, their interaction with AChRs, and AChR phosphorylation. Rapsyn and the utrophin-glycoprotein complex were recruited normally into AChR-containing clusters by agrin in src(-/-);fyn(-/-) myotubes. But after agrin withdrawal, clusters of these proteins disappeared rapidly in parallel with AChRs, revealing that SFKs are of general importance in postsynaptic stability. At the same time, AChR interaction with rapsyn and dystrobrevin and AChR phosphorylation decreased after agrin withdrawal from mutant myotubes. Unexpectedly, levels of rapsyn protein were increased in src(-/-);fyn(-/-) myotubes, whereas rapsyn-cytoskeleton interactions were unaffected. The overall cytoskeletal link of AChRs was weak but still strengthened by agrin in mutant cells, consistent with the normal formation but decreased stability of AChR clusters. These data show that correctly balanced activity of SFKs is critical in maintaining adult NMJs in vivo. SFKs hold the postsynaptic apparatus together through stabilization of AChR-rapsyn interaction and AChR phosphorylation. In addition, SFKs control rapsyn levels and AChR-cytoskeletal linkage.

Agrin↗
PubMed2005

Wnt5 signaling in vertebrate pancreas development.

BACKGROUND: Signaling by the Wnt family of secreted glycoproteins through their receptors, the frizzled (Fz) family of seven-pass transmembrane proteins, is critical for numerous cell fate and tissue polarity decisions during development. RESULTS: We report a novel role of Wnt signaling in organogenesis using the formation of the islet during pancreatic development as a model tissue. We used the advantages of the zebrafish to visualize and document this process in living embryos and demonstrated that insulin-positive cells actively migrate to form an islet. We used morpholinos (MOs), sequence-specific translational inhibitors, and time-lapse imaging analysis to show that the Wnt-5 ligand and the Fz-2 receptor are required for proper insulin-cell migration in zebrafish. Histological analyses of islets in Wnt5a(-/-) mouse embryos showed that Wnt5a signaling is also critical for murine pancreatic insulin-cell migration. CONCLUSION: Our results implicate a conserved role of a Wnt5/Fz2 signaling pathway in islet formation during pancreatic development. This study opens the door for further investigation into a role of Wnt signaling in vertebrate organ development and disease.

Animals↗
PubMed2005

Genetic analysis of early endocrine pancreas formation in zebrafish.

Endocrine pancreas of zebrafish consist of at least four different cell types that function similarly to mammalian pancreatic islet. No mutants specifically affecting formation of the endocrine pancreas have been identified during the previous large-scale mutagenesis screens in zebrafish due to invisibility of a pancreatic islet. We combined in situ hybridization method to visualize pancreatic islet with an ethyl-nitroso-urea mutagenesis screen to identify novel genes involved in pancreatic islet formation in zebrafish. We screened 900 genomes and identified 11 mutations belonging to nine different complementation groups. These mutants fall into three major phenotypic classes displaying severely reduced insulin expression, reduced insulin expression with abnormal islet morphology, or abnormal islet morphology with relatively normal number of insulin expressing cells. Seven of these mutants do not have any other visible phenotypes associated. These mutations affect different processes in pancreatic islet development. Additional analysis on glucagon and somatostatin cell specification revealed that somatostatin cells are specified at a separate domain from insulin cells whereas glucagon cells are specified adjacent to insulin cells. Furthermore, glucagon cells and somatostatin cells are always associated with insulin cells in mutants that have scattered insulin expression. These data indicate that there are separate mechanisms regulating endocrine cell migration, proliferation, and differentiation. Further study on these mutants will reveal important information on novel genes involved in pancreatic islet cell specification and morphogenesis.

Animals↗
PubMed2005

15-zinc finger protein Bloody Fingers is required for zebrafish morphogenetic movements during neurulation.

A novel zebrafish gene bloody fingers (blf) encoding a 478 amino acid protein containing fifteen C(2)H(2) type zinc fingers was identified by expression screening. As determined by in situ hybridization, blf RNA displays strong ubiquitous early zygotic expression, while during late gastrulation and early somitogenesis, blf expression becomes transiently restricted to the posterior dorsal and lateral mesoderm. During later somitogenesis, blf expression appears only in hematopoietic cells. It is completely eliminated in cloche, moonshine but not in vlad tepes (gata1) mutant embryos. Morpholino (MO) knockdown of the Blf protein results in the defects of morphogenetic movements. Blf-MO-injected embryos (morphants) display shortened and widened axial tissues due to defective convergent extension. Unlike other convergent extension mutants, blf morphants display a split neural tube, resulting in a phenotype similar to the human open neural tube defect spina bifida. In addition, dorsal ectodermal cells delaminate in blf morphants during late somitogenesis. We propose a model explaining the role of blf in convergent extension and neurulation. We conclude that blf plays an important role in regulating morphogenetic movements during gastrulation and neurulation while its role in hematopoiesis may be redundant.

Amino Acid Sequence↗
PubMed2005

Identification of novel vascular endothelial-specific genes by the microarray analysis of the zebrafish cloche mutants.

The zebrafish cloche (clo) mutation affects the earliest known step in differentiation of blood and endothelial cells in vertebrates. We established clo/gata1-GFP transgenic line with erythroid-specific green fluorescent protein (GFP) expression, which allowed differentiation of clo and wild-type siblings at the midsomitogenesis stages before morphologically visible phenotypes appeared. To discover novel genes potentially involved in hematopoietic and vascular development, we performed microarray analysis of more than 15,000 zebrafish genes or expressed sequence tags (ESTs) in clo mutant embryos. We isolated the full-length sequences and determined the expression patterns for 8 novel cDNAs that were significantly down-regulated in clo-/- embryos. Dual specificity phosphatase 5 (dusp5), cadherin 5 (cdh5; VE-cadherin), aquaporin 8 (aqp8), adrenomedullin receptor (admr), complement receptor C1qR-like (crl), scavenger receptor class F, member 1 (scarf1), and ETS1-like protein (etsrp) were specifically expressed in the vascular endothelial cells, while retinol binding protein 4 (rbp4) was expressed in the yolk syncytial layer and the hypochord. Further functional studies of these novel genes should help to elucidate critical early steps leading to the formation of vertebrate blood vessels.

Amino Acid Sequence↗