Haigen Huang - Academia.edu (original) (raw)

Papers by Haigen Huang

Proceedings of the National Academy of Sciences, 2006

During embryogenesis, stereotypic vascular patterning requires guidance cues from neighboring tis... more 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 cou...

Cell Reports, 2012

Transgenic zebrafish embryos expressing tissuespecific green fluorescent protein (GFP) can provid... more Transgenic zebrafish embryos expressing tissuespecific green fluorescent protein (GFP) can provide an unlimited supply of primary embryonic cells. Agents that promote the differentiation of these cells may be beneficial for therapeutics. We report a highthroughput approach for screening small molecules that regulate cell differentiation using lineagespecific GFP transgenic zebrafish embryonic cells. After validating several known regulators of the differentiation of endothelial and other cell types, we performed a screen for proangiogenic molecules using undifferentiated primary cells from flk1-GFP transgenic zebrafish embryos. Cells were grown in 384well plates with 12,128 individual small molecules, and GFP expression was analyzed by means of an automated imaging system, which allowed us to screen thousands of compounds weekly. As a result, 23 molecules were confirmed to enhance angiogenesis, and 11 of them were validated to promote the proliferation of mammalian human umbilical vascular endothelial cells and induce Flk1+ cells from murine embryonic stem cells. We demonstrated the general applicability of this strategy by analyzing additional cell lineages using zebrafish expressing GFP in pancreatic, cardiac, and dopaminergic cells.

NPJ Regenerative medicine, 2018

Regenerative medicine holds great promise for both degenerative diseases and traumatic tissue inj... more Regenerative medicine holds great promise for both degenerative diseases and traumatic tissue injury which represent significant challenges to the health care system. Hearing loss, which affects hundreds of millions of people worldwide, is caused primarily by a permanent loss of the mechanosensory receptors of the inner ear known as hair cells. This failure to regenerate hair cells after loss is limited to mammals, while all other non-mammalian vertebrates tested were able to completely regenerate these mechanosensory receptors after injury. To understand the mechanism of hair cell regeneration and its association with regeneration of other tissues, we performed a guided mutagenesis screen using zebrafish lateral line hair cells as a screening platform to identify genes that are essential for hair cell regeneration, and further investigated how genes essential for hair cell regeneration were involved in the regeneration of other tissues. We created genetic mutations either by retrov...

Nature Cell Biology, 2000

Stimulation of erythropoiesis by inhibiting a new hematopoietic death receptor in transgenic zebr... more Stimulation of erythropoiesis by inhibiting a new hematopoietic death receptor in transgenic zebrafish

PLoS genetics, 2017

ATP6V1H is a component of a large protein complex with vacuolar ATPase (V-ATPase) activity. We id... more ATP6V1H is a component of a large protein complex with vacuolar ATPase (V-ATPase) activity. We identified two generations of individuals in which short stature and osteoporosis co-segregated with a mutation in ATP6V1H. Since V-ATPases are highly conserved between human and zebrafish, we generated loss-of-function mutants in atp6v1h in zebrafish through CRISPR/Cas9-mediated gene knockout. Homozygous mutant atp6v1h zebrafish exhibited a severe reduction in the number of mature calcified bone cells and a dramatic increase in the expression of mmp9 and mmp13. Heterozygous adults showed curved vertebra that lack calcified centrum structure and reduced bone mass and density. Treatment of mutant embryos with small molecule inhibitors of MMP9 and MMP13 significantly restored bone mass in the atp6v1h mutants. These studies have uncovered a new, ATP6V1H-mediated pathway that regulates bone formation, and defines a new mechanism of disease that leads to bone loss. We propose that MMP9/MMP13 co...

Blood, Jan 24, 2015

Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood ce... more Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept, a.k.a ACE-011, (and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with β-thalasemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 lik...

The American Journal of Human Genetics, 2015

Ablepharon macrostomia syndrome (AMS) and Barber-Say syndrome (BSS) are rare congenital ectoderma... more Ablepharon macrostomia syndrome (AMS) and Barber-Say syndrome (BSS) are rare congenital ectodermal dysplasias characterized by similar clinical features. To establish the genetic basis of AMS and BSS, we performed extensive clinical phenotyping, whole exome and candidate gene sequencing, and functional validations. We identified a recurrent de novo mutation in TWIST2 in seven independent AMS-affected families, as well as another recurrent de novo mutation affecting the same amino acid in ten independent BSS-affected families. Moreover, a genotype-phenotype correlation was observed, because the two syndromes differed based solely upon the nature of the substituting amino acid: a lysine at TWIST2 residue 75 resulted in AMS, whereas a glutamine or alanine yielded BSS. TWIST2 encodes a basic helix-loop-helix transcription factor that regulates the development of mesenchymal tissues. All identified mutations fell in the basic domain of TWIST2 and altered the DNA-binding pattern of Flag-TWIST2 in HeLa cells. Comparison of wild-type and mutant TWIST2 expressed in zebrafish identified abnormal developmental phenotypes and widespread transcriptome changes. Our results suggest that autosomal-dominant TWIST2 mutations cause AMS or BSS by inducing protean effects on the transcription factor's DNA binding.

BMC developmental biology, Jan 24, 2005

NXT2 is a member of NXT family proteins that are generally involved in exporting nuclear RNA in e... more NXT2 is a member of NXT family proteins that are generally involved in exporting nuclear RNA in eukaryotic cells. It is not known if NXT2 has any function in specific biological processes. A zebrafish mutant exhibiting specific heart defects during embryogenesis was generated by animal cloning-mediated retroviral insertions. Molecular analysis indicated that the mutant phenotype was caused by a disruption of NXT2. Whole-mount RNA in situ hybridization showed that NXT2 transcripts were clearly detectable in embryonic heart as well as other tissues. Further analysis revealed that expression level of one form of alternative splicing NXT2 mRNA transcripts was significantly reduced, resulting in deficient myocardial cell differentiation and the malformation of cardiac valve at the atrioventricular boundary. The defects could be reproduced by morpholino anti-sense oligo knockdown of NXT2. NXT2 has a critical role in maintaining morphogenetic integrity of embryonic heart in vertebrate spec...

Postdoc Journal, 2015

This comparative study used the latest Trends in International Mathematics and Science Study (TIM... more This comparative study used the latest Trends in International Mathematics and Science Study (TIMSS) data sets and examined the relationship between professional development and student achievement. It found that although the national levels of access for students at the fourth and eighth grade levels to teachers who participated in professional learning in the United States were higher than the other countries, one third to one half of the fourth grades were taught by teachers who had no professional learning focusing on math instruction or curriculum. In addition, teachers' participation in professional development was positively associated with higher student math achievement. This cross-national study provided empirical evidence highlighting the importance of investing in teacher learning for improving national educational quality.

PLoS biology, 2013

Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vert... more Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly gen...

Methods in Cell Biology, 2004

Publisher Summary This chapter focuses on cloning zebrafish by nuclear transfer. Cloning of zebra... more Publisher Summary This chapter focuses on cloning zebrafish by nuclear transfer. Cloning of zebrafish by nuclear transfer is a complex procedure involving cell culture, egg selection, and micromanipulation of the eggs and cells, each step affecting the overall efficiency of cloning. The chapter provides a detailed description of the nuclear transfer procedure developed in laboratory. For nuclear transfer, long-term cultured cells are subjected to serum starvation by culturing them in DMEM medium supplemented with 0.5% FBS for 4 days and are then dissociated with trypsin/EDTA (0.25% trypsin/1 mM EDTA in PBS), washed once with PBS, and centrifuged at 1000 rpm for 5 min. Cells are suspended in DMEM containing 0.5% FBS and kept on ice until nuclear transfer. During nuclear transfer, both the recipient eggs and donor cells are placed into an inverted cover of a Falcon Tissue culture dish. The eggs are kept in a large drop of precooled Hank's solution supplemented with 1.5% BSA, and the donor cells are kept nearby in a drop of precooled DMEM medium supplemented with 0.5% FBS (v/v). Cloned zebrafish can be excellent models for studying effects of cloning on animal development. Developmental abnormalities in cloned zebrafish can be easily found because of in vitro and transparent embryogenesis. Short generation time and easy access to a large number of progenies mean that the effects of cloning can be monitored thoroughly in multiple generations in a relatively short time.

Nature Biotechnology, 2002

Molecular Genetics and Metabolism, 2014

Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the b... more Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dosedependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly.

Molecular BioSystems, 2009

Supplemental Data Fig S1. 3F8 does not cause abnormity outside forebrain. 2 dpf control (upper) a... more Supplemental Data Fig S1. 3F8 does not cause abnormity outside forebrain. 2 dpf control (upper) and treated (lower) embryos are shown here. White arrows point to the metencephalic artery and medial cerebral vein. White stars (*) indicate the eye of control embryo or the forebrain residue of treated embryo. Similar observations have been obtained for liver, blood cells and pancreas (data not shown).

Molecular and Cellular Endocrinology, 2001

Using DNA constructs containing regulatory sequences of the zebrafish Pdx-1 and insulin genes, ge... more Using DNA constructs containing regulatory sequences of the zebrafish Pdx-1 and insulin genes, germline transgenic zebrafish expressing the green fluorescent protein (GFP) reporter gene in the pancreas were generated. For both constructs, the GFP expression patterns in transgenic embryos were consistent with the mRNA expression patterns detected by RNA in situ hybridization. A deletion promoter analysis revealed that positive and negative cis-acting elements were involved in regulation of insulin gene expression. Three-dimensional reconstructions imaged from living embryos using two-photon laser-scanning microscopy (TPLSM) demonstrated that the zebrafish pancreas is formed from a single dorsal pancreatic cell mass. This is in contrast to mammals where the pancreas derives from both dorsal and ventral anlage. These transgenic fish should be useful for in vivo studies of factors involved in specifying and regulating pancreatic development and function.

Genome Research, 2013

With the completion of the zebrafish genome sequencing project, it becomes possible to analyze th... more With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1's predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ∼0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and shou...

Cloning and Stem Cells, 2003

Cell Research, 2011

Blood vessels normally maintain stereotyped lumen diameters and their stable structures are cruci... more Blood vessels normally maintain stereotyped lumen diameters and their stable structures are crucial for vascular function. However, very little is known about the molecular mechanisms controlling the maintenance of vessel diameters and the integrity of endothelial cells. We investigated this issue in zebrafish embryos by a chemical genetics approach. Small molecule libraries were screened using live Tg(kdrl:GRCFP) zn1 transgenic embryos in which endothelial cells are specifically labeled with GFP. By analyzing the effects of compounds on the morphology and function of embryonic blood vessels after lumen formation, PP1, a putative Src kinase inhibitor, was identified as capable of specifically reducing vascular lumen size by interrupting endothelial-cell integrity. The inhibitory effect is not due to Src or general VEGF signaling inhibition because another Src inhibitor and Src morpholino as well as several VEG-FR inhibitors failed to produce a similar phenotype. After profiling a panel of 22 representative mammalian kinases and surveying published data, we selected a few possible new candidates. Combinational analysis of these candidate kinase inhibitors established that PP1 induced endothelial collapse by inhibiting both the VEGFR2 and MAP kinase pathways. More importantly, combinatory use of two clinically approved drugs Dasatinib and Sunitinib produced the same phenotype. This is the first study to elucidate the pathways controlling maintenance of endothelial integrity using a chemical genetics approach, indicating that endothelial integrity is controlled by the combined action of the VEGFR2 and MAP kinase pathways. Our results also suggest the possible side effect of the combination of two anticancer drugs on the circulatory system.