Converse conformational control of smoothened activity by structurally related small molecules.

The seven-pass transmembrane protein Smoothened (Smo) is an essential component of the Hedgehog (Hh) signaling pathway that is critically involved in normal animal development as well as pathological malignancies. In studying Hh-related biological processes, it would be highly desirable if Smo activity could be instantly switched between activation and inhibition. Using Gli1-dependent GFP transgenic zebrafish and in vitro biochemical assays, we identified and characterized two potent Smo inhibitors, SANT74 and 75 (Smoothened antagonist 74 and 75), by screening a small molecule library designed based on the scaffold of Smo agonist SAG.

Discovery and characterization of novel vascular and hematopoietic genes downstream of etsrp in zebrafish.

The transcription factor Etsrp is required for vasculogenesis and primitive myelopoiesis in zebrafish. When ectopically expressed, etsrp is sufficient to induce the expression of many vascular and myeloid genes in zebrafish. The mammalian homolog of etsrp, ER71/Etv2, is also essential for vascular and hematopoietic development.

The mob as tumor suppressor (mats1) gene is required for growth control in developing zebrafish embryos.

The mob as tumor suppressor (mats) family genes are highly conserved in evolution. The Drosophila mats gene functions in the Hippo signaling pathway to control tissue growth by regulating cell proliferation and apoptosis. However, nothing is known about whether mats family genes are required for the normal development of vertebrates.

Identifying tumor cell growth inhibitors by combinatorial chemistry and zebrafish assays.

Cyclin-dependent kinases (CDKs) play important roles in regulating cell cycle progression, and altered cell cycles resulting from over-expression or abnormal activation of CDKs observed in many human cancers. As a result, CDKs have become extensive studied targets for developing chemical inhibitors for cancer therapies; however, protein kinases share a highly conserved ATP binding pocket at which most chemical inhibitors bind, therefore, a major challenge in developing kinase inhibitors is achieving target selectivity. To identify cell growth inhibitors with potential applications in cancer therapy, we used an integrated approach that combines one-pot chemical synthesis in a combinatorial manner to generate diversified small molecules with new chemical scaffolds coupled with growth inhibition assay using developing zebrafish embryos.

Essential role of spi-1-like (spi-1l) in zebrafish myeloid cell differentiation.

The ETS protein Spi-1/Pu.1 plays a pivotal and widespread role throughout hematopoiesis in many species. This study describes the identification, characterization, and functional analysis of a new zebrafish spi transcription factor spi-1-like (spi-1l) that is expressed in primitive myeloid cells, erythro-myelo progenitor cells, and in the adult kidney.

Structural Basis for the Immunogenic Properties of the Meningococcal Vaccine Candidate LP2086.

LP2086 is a family of outer membrane lipoproteins from Neisseria meningitidis, which elicits bactericidal antibodies and are currently undergoing human clinical trials in a bivalent formulation where each antigen represents one of the two known LP2086 subfamilies. Here we report the NMR structure of the recombinant LP2086 variant B01, a representative of the LP2086 subfamily B. The structure reveals a novel fold composed of two domains: a "taco-shaped" N-terminal beta-sheet and a C-terminal beta-barrel connected by a linker.

Exdpf is a key regulator of exocrine pancreas development controlled by retinoic acid and ptf1a in zebrafish.

Both endocrine and exocrine pancreatic cells arise from pancreatic-duodenal homeobox 1 (pdx1)-positive progenitors. The molecular mechanisms controlling cell fate determination and subsequent proliferation, however, are poorly understood. Unlike endocrine cells, less is known about exocrine cell specification.

Skeletal muscle-specific ablation of raptor, but not of rictor, causes metabolic changes and results in muscle dystrophy.

Mammalian target of rapamycin (mTOR) is a central controller of cell growth. mTOR assembles into two distinct multiprotein complexes called mTOR complex 1 (mTORC1) and mTORC2. Here we show that the mTORC1 component raptor is critical for muscle function and prolonged survival.

p53 family in development.

The p53 family network is a unique cellular processor that integrates information from various pathways and determines cellular choices between proliferation, replication arrest/repair, differentiation, senescence, or apoptosis. The most studied role of the p53 family is the regulation of stress response and tumor suppression. By removing damaged cells from the proliferating pool, p53 family members preserve the integrity of the genome.

Role of p53 family in birth defects: lessons from zebrafish.

p53 Protein family is an important teratologic suppressor, but in certain conditions it can cause congenital abnormalities. p53 Family performs this dual role in development by integrating information from cell's interior with that from the environment to determine the choice between life and death. Understanding of p53 family developmental functions may lead to new therapeutic approaches for treatment and prevention of birth defects.

Muscle-wide secretion of a miniaturized form of neural agrin rescues focal neuromuscular innervation in agrin mutant mice.

Agrin and its receptor MuSK are required for the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). In the current model the local deposition of agrin by the nerve and the resulting local activation of MuSK are responsible for creating and maintaining the postsynaptic apparatus including clusters of acetylcholine receptors (AChRs). Concomitantly, the release of acetylcholine (ACh) and the resulting depolarization disperses those postsynaptic structures that are not apposed by the nerve and thus not stabilized by agrin-MuSK signaling.

Zebrafish as a developmental model organism for pediatric research.

Zebrafish has many advantages as a model of human pediatric research. Given the physical and ethical problems with performing experiments on human patients, biomedical research has focused on using model organisms to study biologic processes conserved between humans and lower vertebrates. The most common model organisms are small mammals, usually rats and mice.

Ribosomal protein S19 deficiency in zebrafish leads to developmental abnormalities and defective erythropoiesis through activation of p53 protein family.

Mutations in several ribosomal proteins (RPs) lead to Diamond-Blackfan anemia (DBA), a syndrome characterized by defective erythropoiesis, congenital anomalies, and increased frequency of cancer. RPS19 is the most frequently mutated RP in DBA. RPS19 deficiency impairs ribosomal biogenesis, but how this leads to DBA or cancer remains unknown.

In vivo time-lapse imaging delineates the zebrafish pituitary proopiomelanocortin lineage boundary regulated by FGF3 signal.

The anterior pituitary gland (adenohypophysis) comprises anterior and intermediate lobes (the pars distalis and pars intermedia) arising from placodal ectoderm at the anterior neural ridge. Signaling molecules including SHH, FGF, WNT, BMP and Notch are involved in regulating primordial pituitary proliferation and lineage determination. However, morphogenic events and molecular mechanisms governing anterior and intermediate lobe specification are not clear.

C1q-like inhibits p53-mediated apoptosis and controls normal hematopoiesis during zebrafish embryogenesis.

Except for the complement C1q, the immunological functions of other C1q family members have remained unclear. Here we describe zebrafish C1q-like, whose transcription and translation display a uniform distribution in early embryos, and are restricted to mid-hind brain and eye in later embryos. In vitro studies showed that C1q-like could inhibit the apoptosis induced by ActD and CHX in EPC cells, through repressing caspase 3/9 activities.

Gfi1.1 regulates hematopoietic lineage differentiation during zebrafish embryogenesis.

Growth factor independence 1 (GFI1) is important for maturation of mammalian lymphocytes and neutrophils and maintenance of adult hematopoietic stem cells (HSCs). The role of GFI1 in embryonic hematopoiesis is less well characterized. Through an enhancer trap screen and bioinformatics analysis, we identified a zebrafish homolog of Gfi1 (named gfi1.

ER71 acts downstream of BMP, Notch, and Wnt signaling in blood and vessel progenitor specification.

FLK1-expressing (FLK1(+)) mesoderm generates blood and vessels. Here, we show that combined BMP, Notch, and Wnt signaling is necessary for efficient FLK1(+) mesoderm formation from embryonic stem cells (ESCs). Inhibition of BMP, Notch, and Wnt signaling pathways greatly decreased the generation of FLK1(+) mesoderm and expression of the Ets transcription factor Er71.

The role of nerve- versus muscle-derived factors in mammalian neuromuscular junction formation.

Neuromuscular junctions (NMJs) normally form in the central region of developing muscle. In this process, agrin released from motor neurons has been considered to initiate the formation of synaptic acetylcholine receptor (AChR) clusters (neurocentric model). However, in muscle developing in the absence of nerves and thus of agrin, AChR clusters still form in the muscle center.

Interplay among Etsrp/ER71, Scl, and Alk8 signaling controls endothelial and myeloid cell formation.

Vascular endothelial and myeloid cells have been proposed to originate from a common precursor cell, the hemangioblast. The mechanism of endothelial and myeloid cell specification and differentiation is poorly understood. We have previously described the endothelial-specific zebrafish Ets1-related protein (Etsrp), which was both necessary and sufficient to initiate vasculogenesis in the zebrafish embryos.

Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish.

We describe an enhancer trap transgenic zebrafish line, ETvmat2:GFP, in which most monoaminergic neurons are labeled by green fluorescent protein (GFP) during embryonic development. The reporter gene of ETvmat2:GFP was inserted into the second intron of vesicular monoamine transporter 2 (vmat2) gene, and the GFP expression pattern recapitulates that of the vmat2 gene. The GFP positive neurons include the large and pear-shaped tyrosine hydroxylase positive neurons (TH populations 2 and 4) in the posterior tuberculum of ventral diencephalon (PT neurons), which are thought to be equivalent to the midbrain dopamine neurons in mammals.

A highly conserved regulatory element controls hematopoietic expression of GATA-2 in zebrafish.

Background: GATA-2 is a transcription factor required for hematopoietic stem cell survival as well as for neuronal development in vertebrates. It has been shown that specific expression of GATA-2 in blood progenitor cells requires distal cis-acting regulatory elements. Identification and characterization of these elements should help elucidating transcription regulatory mechanisms of GATA-2 expression in hematopoietic lineage.

Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions.

Using a combination of techniques we developed, we infected zebrafish embryos using pseudotyped retroviruses and mapped the genomic locations of the proviral integrations in the F(1) offspring of the infected fish. From F(1) fish, we obtained 2,045 sequences representing 933 unique retroviral integrations. A total of 599 were mappable to the current genomic assembly (Zv6), and 233 of the integrations landed within genes.

Synapse loss in cortex of agrin-deficient mice after genetic rescue of perinatal death.

Agrin-deficient mice die at birth because of aberrant development of the neuromuscular junctions. Here, we examined the role of agrin at brain synapses. We show that agrin is associated with excitatory but not inhibitory synapses in the cerebral cortex.

Tyrosine phosphatases such as SHP-2 act in a balance with Src-family kinases in stabilization of postsynaptic clusters of acetylcholine receptors.

Background: Development of neural networks requires that synapses are formed, eliminated and stabilized. At the neuromuscular junction (NMJ), agrin/MuSK signaling, by triggering downstream pathways, causes clustering and phosphorylation of postsynaptic acetylcholine receptors (AChRs). Postnatally, AChR aggregates are stabilized by molecular pathways that are poorly characterized.

Identification of a lectin causing the degeneration of neuronal processes using engineered embryonic stem cells.

Unlike the mechanisms involved in the death of neuronal cell bodies, those causing the elimination of processes are not well understood owing to the lack of suitable experimental systems. As the neurotrophin receptor p75(NTR) is known to restrict the growth of neuronal processes, we engineered mouse embryonic stem (ES) cells to express an Ngfr (p75(NTR)) cDNA under the control of the Mapt locus (the gene encoding tau), which begins to be active when ES cell-derived progenitors start elongating processes. This caused a progressive, synchronous degeneration of all processes, and a prospective proteomic analysis showed increased levels of the sugar-binding protein galectin-1 in the p75(NTR)-engineered cells.