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1.  A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization 
This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982). We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity. First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes. The rates of sperm freezing and thawing (δ°C/time) are probably the two most critical variables to control in this procedure. For this reason, do not substitute different tubes for those specified. Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs. Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.
PMCID: PMC2785217  PMID: 19581874
2.  A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization 
This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982). We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity. First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes. The rates of sperm freezing and thawing (δ°C/time) are probably the two most critical variables to control in this procedure. For this reason, do not substitute different tubes for those specified. Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs. Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.
PMCID: PMC2785217  PMID: 19581874
3.  Reverse genetics in zebrafish by TILLING 
TILLING, for Targeting Induced Local Lesions in Genomes, is a reverse genetics strategy that identifies mutations in specific genes of interest in chemically mutagenized populations. First described in 2000 for mutation detection in Arabidopsis, TILLING is now used in a wide range of plants including soybean, rice, barley and maize as well as for animal model systems, including Arabidopsis, Drosophila, Caenorhabditis elegans, rat, medaka and zebrafish and for the discovery of naturally occurring polymorphisms in humans. This review summarizes current TILLING methodologies as they have been applied to the zebrafish, ongoing TILLING projects and resources in the zebrafish community, and the future of zebrafish TILLING.
PMCID: PMC2899843  PMID: 19028802
zebrafish; TILLING; Cel1 mismatch cleavage; resequencing; reverse genetics
4.  Rapid identification and recovery of ENU-induced mutations with next-generation sequencing and Paired-End Low-Error analysis 
BMC Genomics  2015;16(1):83.
Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics approach to directly identify point mutations in specific genes of interest in genomic DNA from a large chemically mutagenized population. Classical TILLING processes, based on enzymatic detection of mutations in heteroduplex PCR amplicons, are slow and labor intensive.
Here we describe a new TILLING strategy in zebrafish using direct next generation sequencing (NGS) of 250bp amplicons followed by Paired-End Low-Error (PELE) sequence analysis. By pooling a genomic DNA library made from over 9,000 N-ethyl-N-nitrosourea (ENU) mutagenized F1 fish into 32 equal pools of 288 fish, each with a unique Illumina barcode, we reduce the complexity of the template to a level at which we can detect mutations that occur in a single heterozygous fish in the entire library. MiSeq sequencing generates 250 base-pair overlapping paired-end reads, and PELE analysis aligns the overlapping sequences to each other and filters out any imperfect matches, thereby eliminating variants introduced during the sequencing process. We find that this filtering step reduces the number of false positive calls 50-fold without loss of true variant calls. After PELE we were able to validate 61.5% of the mutant calls that occurred at a frequency between 1 mutant call:100 wildtype calls and 1 mutant call:1000 wildtype calls in a pool of 288 fish. We then use high-resolution melt analysis to identify the single heterozygous mutation carrier in the 288-fish pool in which the mutation was identified.
Using this NGS-TILLING protocol we validated 28 nonsense or splice site mutations in 20 genes, at a two-fold higher efficiency than using traditional Cel1 screening. We conclude that this approach significantly increases screening efficiency and accuracy at reduced cost and can be applied in a wide range of organisms.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1263-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4457992  PMID: 25886285
TILLING; Zebrafish; Next-generation sequencing; PELE analysis; Rare mutation detection
5.  Zebrafish Neural Tube Morphogenesis Requires Scribble-Dependent Oriented Cell Divisions 
Current biology : CB  2010;21(1):79-86.
How control of subcellular events in single cells determines morphogenesis on the scale of the tissue is largely unresolved. The stereotyped cross-midline mitoses of progenitors in the zebrafish neural keel [1–4] provide a unique experimental paradigm for defining the role and control of single cell orientation for tissue-level morphogenesis in vivo.
We show that the coordinated orientation of individual progenitor cell division in the neural keel is the cellular determinant required for morphogenesis into a neural tube epithelium with a single straight lumen. We find that Scribble is required for oriented cell division and that its function in this process is independent of canonical apico-basal and planar polarity pathways. We identify a role for Scribble in controlling clustering of α-Catenin foci in dividing progenitors. Loss of either Scrib or of N-cadherin results in abnormally oriented mitoses, reduced cross-midline cell divisions and similar neural tube defects. We propose that Scribble-dependent nascent cell-cell adhesion clusters between neuroepithelial progenitors contribute to define orientation of their cell division. Finally, our data demonstrate that while oriented mitoses of individual cells determine neural tube architecture, the tissue can in turn feed back on its constituent cells to define their polarization and cell division orientation to ensure robust tissue morphogenesis.
PMCID: PMC4308621  PMID: 21185191
6.  Role of mef2ca in developmental buffering of the zebrafish larval hyoid dermal skeleton 
Developmental biology  2013;385(2):189-199.
Phenotypic robustness requires a process of developmental buffering that is largely not understood, but which can be disrupted by mutations. Here we show that in mef2cab1086 loss of function mutant embryos and early larvae, development of craniofacial hyoid bones, the opercle (Op) and branchiostegal ray (BR), becomes remarkably unstable; the large magnitude of the instability serves as a positive attribute to learn about features of this developmental buffering. The OpBR mutant phenotype variably includes bone expansion and fusion, Op duplication, and BR homeosis. Formation of a novel bone strut, or a bone bridge connecting the Op and BR together occurs frequently. We find no evidence that the phenotypic stability in the wild type is provided by redundancy between mef2ca and its co-ortholog mef2cb, or that it is related to the selector (homeotic) gene function of mef2ca. Changes in dorsal-ventral patterning of the hyoid arch also might not contribute to phenotypic instability in mutants. However, subsequent development of the bone lineage itself, including osteoblast differentiation and morphogenetic outgrowth, shows marked variation. Hence, steps along the developmental trajectory appear differentially sensitive to the loss of buffering, providing focus for future study.
PMCID: PMC3892954  PMID: 24269905
canalization; developmental instability; osteoblast; skeleton; pharyngeal arch; mef2c
7.  Retinal Regeneration in Adult Zebrafish Requires Regulation of TGFβ Signaling 
Glia  2013;61(10):1687-1697.
Müller glia are the resident radial glia in the vertebrate retina. The response of mammalian Müller glia to retinal damage often results in a glial scar and no functional replacement of lost neurons. Adult zebrafish Müller glia, in contrast, are considered tissue-specific stem cells that can self-renew and generate neurogenic progenitors to regenerate all retinal neurons after damage. Here, we demonstrate that regulation of TGFβ signaling by the corepressors Tgif1 and Six3b is critical for the proliferative response to photoreceptor destruction in the adult zebrafish retina. When function of these corepressors is disrupted, Müller glia and their progeny proliferate less, leading to a significant reduction in photoreceptor regeneration. Tgif1 expression and regulation of TGFβ signaling are implicated in the function of several types of stem cells, but this is the first demonstration that this regulatory network is necessary for regeneration of neurons.
PMCID: PMC4127981  PMID: 23918319
Müller glia; photoreceptor; tgif1; six3b; stem cell
8.  Zebrafish Mef2ca and Mef2cb are essential for both first and second heart field cardiomyocyte differentiation 
Developmental biology  2012;369(2):199-210.
Mef2 transcription factors have been strongly linked with early heart development. D-mef2 is required for heart formation in Drosophila, but whether Mef2 is essential for vertebrate cardiomyocyte (CM) differentiation is unclear. In mice, although Mef2c is expressed in all CMs, targeted deletion of Mef2c causes lethal loss of second heart field (SHF) derivatives and failure of cardiac looping, but first heart field CMs can differentiate. Here we examine Mef2 function in early heart development in zebrafish. Two Mef2c genes exist in zebrafish, mef2ca and mef2cb. Both are expressed similarly in the bilateral heart fields but mef2cb is strongly expressed in the heart poles at the primitive heart tube stage. By using fish mutants for mef2ca and mef2cb and antisense morpholinos to knock down either or both Mef2cs, we show that Mef2ca and Mef2cb have essential but redundant roles in myocardial differentiation. Loss of both Mef2ca and Mef2cb function does not interfere with early cardiogenic markers such as nkx2.5, gata4 and hand2 but results in a dramatic loss of expression of sarcomeric genes and myocardial markers such as bmp4, nppa, smyd1b and late nkx2.5 mRNA. Rare residual CMs observed in mef2ca;mef2cb double mutants are ablated by a morpholino capable of knocking down other Mef2s. Mef2cb over-expression activates bmp4 within the cardiogenic region, but no ectopic CMs are formed. Surprisingly, anterior mesoderm and other tissues become skeletal muscle. Mef2ca single mutants have delayed heart development, but form an apparently normal heart. Mef2cb single mutants have a functional heart and are viable adults. Our results show that the key role of Mef2c in myocardial differentiation is conserved throughout the vertebrate heart.
PMCID: PMC3927553  PMID: 22750409
Second heart field; mef2c; mef2ca; mef2cb; mef2a; Heart; Hand2; Myl7; bulbus arteriosus; outflow tract; cardiomyocyte; differentiation
9.  Hoxb1b controls oriented cell division, cell shape and microtubule dynamics in neural tube morphogenesis 
Development (Cambridge, England)  2014;141(3):639-649.
Hox genes are classically ascribed to function in patterning the anterior-posterior axis of bilaterian animals; however, their role in directing molecular mechanisms underlying morphogenesis at the cellular level remains largely unstudied. We unveil a non-classical role for the zebrafish hoxb1b gene, which shares ancestral functions with mammalian Hoxa1, in controlling progenitor cell shape and oriented cell division during zebrafish anterior hindbrain neural tube morphogenesis. This is likely distinct from its role in cell fate acquisition and segment boundary formation. We show that, without affecting major components of apico-basal or planar cell polarity, Hoxb1b regulates mitotic spindle rotation during the oriented neural keel symmetric mitoses that are required for normal neural tube lumen formation in the zebrafish. This function correlates with a non-cell-autonomous requirement for Hoxb1b in regulating microtubule plus-end dynamics in progenitor cells in interphase. We propose that Hox genes can influence global tissue morphogenesis by control of microtubule dynamics in individual cells in vivo.
PMCID: PMC3899817  PMID: 24449840
Hoxb1b; Hoxa1; Cell polarity; In vivo tissue morphogenesis; Neural tube formation; Oriented cell division; Zebrafish
10.  Wnt-dependent epithelial transitions drive pharyngeal pouch formation 
Developmental cell  2013;24(3):296-309.
The pharyngeal pouches, which form by budding of the foregut endoderm, are essential for segmentation of the vertebrate face. To date, the cellular mechanism and segmental nature of such budding have remained elusive. Here, we find that Wnt11r and Wnt4a from the head mesoderm and ectoderm, respectively, play distinct roles in the segmental formation of pouches in zebrafish. Time-lapse microscopy, combined with mutant and tissue-specific transgenic experiments, reveal requirements of Wnt signaling in two phases of endodermal epithelial transitions. Initially, Wnt11r and Rac1 destabilize the endodermal epithelium to promote the lateral movement of pouch-forming cells. Next, Wnt4a and Cdc42 signaling induce the rearrangement of maturing pouch cells into bilayers through junctional localization of the Alcama immunoglobulin-domain protein, which functions to restabilize adherens junctions. We propose that this dynamic control of epithelial morphology by Wnt signaling may be a common theme for the budding of organ anlagen from the endoderm.
PMCID: PMC3656592  PMID: 23375584
11.  Zebrafish sox9b is crucial for hepatopancreatic duct development and pancreatic endocrine cell regeneration 
Developmental biology  2012;366(2):268-278.
Recent zebrafish studies have shown that the late appearing pancreatic endocrine cells derive from pancreatic ducts but the regulatory factors involved are still largely unknown. Here, we show that the zebrafish sox9b gene is expressed in pancreatic ducts where it labels the pancreatic Notch-responsive cells previously shown to be progenitors. Inactivation of sox9b disturbs duct formation and impairs regeneration of beta cells from these ducts in larvae. sox9b expression in the midtrunk endoderm appears at the junction of the hepatic and ventral pancreatic buds and, by the end of embryogenesis, labels the hepatopancreatic ductal system as well as the intrapancreatic and intrahepatic ducts. Ductal morphogenesis and differentiation are specifically disrupted in sox9b mutants, with the dysmorphic hepatopancreatic ducts containing misdifferentiated hepatocyte-like and pancreatic-like cells. We also show that maintenance of sox9b expression in the extrapancreatic and intrapancreatic ducts requires FGF and Notch activity, respectively, both pathways known to prevent excessive endocrine differentiation in these ducts. Furthermore, beta cell recovery after specific ablation is severely compromised in sox9b mutant larvae. Our data position sox9b as a key player in the generation of secondary endocrine cells deriving from pancreatic ducts in zebrafish.
PMCID: PMC3364407  PMID: 22537488
duct; beta cell regeneration; pancreas; sox9; Notch; FGF
12.  Tardbpl splicing rescues motor neuron and axonal development in a mutant tardbp zebrafish 
Human Molecular Genetics  2013;22(12):2376-2386.
Mutations in the transactive response DNA binding protein-43 (TARDBP/TDP-43) gene, which regulates transcription and splicing, causes a familial form of amyotrophic lateral sclerosis (ALS). Here, we characterize and report the first tardbp mutation in zebrafish, which introduces a premature stop codon (Y220X), eliminating expression of the Tardbp protein. Another TARDBP ortholog, tardbpl, in zebrafish is shown to encode a Tardbp-like protein which is truncated compared with Tardbp itself and lacks part of the C-terminal glycine-rich domain (GRD). Here, we show that tardbp mutation leads to the generation of a novel tardbpl splice form (tardbpl-FL) capable of making a full-length Tardbp protein (Tardbpl-FL), which compensates for the loss of Tardbp. This finding provides a novel in vivo model to study TDP-43-mediated splicing regulation. Additionally, we show that elimination of both zebrafish TARDBP orthologs results in a severe motor phenotype with shortened motor axons, locomotion defects and death at around 10 days post fertilization. The Tardbp/Tardbpl knockout model generated in this study provides an excellent in vivo system to study the role of the functional loss of Tardbp and its involvement in ALS pathogenesis.
PMCID: PMC3658164  PMID: 23427147
13.  Differential regulation of epiboly initiation and progression by zebrafish Eomesodermin A 
Developmental biology  2011;362(1):11-23.
The T-box transcription factor Eomesodermin (Eomes) has been implicated in patterning and morphogenesis in frog, fish and mouse. In zebrafish, one of the two Eomes homologs, Eomesa, has been implicated in dorsal-ventral patterning, epiboly and endoderm specification in experiments employing over-expression, dominant-negative constructs and antisense morpholino oligonucleotides. Here we report for the first time the identification and characterization of an Eomesa mutant generated by TILLING. We find that Eomesa has a strictly maternal role in the initiation of epiboly, which involves doming of the yolk cell up into the overlying blastoderm. By contrast, epiboly progression is normal, demonstrating for the first time that epiboly initiation is genetically separable from progression. The yolk cell microtubules, which are required for epiboly, are defective in maternal-zygotic eomesa mutantembryos. In addition, the deep cells of the blastoderm are more tightly packed and exhibit more bleb-like protrusions than cells in control embryos. We postulate that the doming delay may be the consequence both of overly stabilized yolk cell microtubules and defects in the adhesive properties or motility of deep cells. We also show that Eomesa is required for normal expression of the endoderm markers sox32, bon and og9x; however it is not essential for endoderm formation.
PMCID: PMC3259739  PMID: 22142964
zebrafish; epiboly; eomesodermin; endoderm; T-box; gastrulation
14.  Regulation of intrahepatic biliary duct morphogenesis by Claudin 15-like b 
Developmental biology  2011;361(1):68-78.
The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis.
PMCID: PMC3235368  PMID: 22020048
Claudin; liver development; zebrafish; biliary duct remodeling; biliary cells; biliary duct morphogenesis; tight junctions; cholestasis
15.  The first mecp2-null zebrafish model shows altered motor behaviors 
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder and one of the most common causes of mental retardation in affected girls. Other symptoms include a rapid regression of motor and cognitive skills after an apparently early normal development. Sporadic mutations in the transcription factor MECP2 has been shown to be present in more than 90% of the patients and several models of MeCP2-deficient mice have been created to understand the role of this gene. These models have pointed toward alterations in the maintenance of the central nervous system rather than its development, in line with the late onset of the disease in humans. However, the exact functions of MeCP2 remain difficult to delineate and the animal models have yielded contradictory results. Here, we present the first mecp2-null allele mutation zebrafish model. Surprisingly and in contrast to MeCP2-null mouse models, mecp2-null zebrafish are viable and fertile. They present nonetheless clear behavioral alterations during their early development, including spontaneous and sensory-evoked motor anomalies, as well as defective thigmotaxis.
PMCID: PMC3712905  PMID: 23874272
zebrafish; motor behavior; Rett syndrome; mecp2; early development; thigmotaxis
16.  sox9b Is a Key Regulator of Pancreaticobiliary Ductal System Development 
PLoS Genetics  2012;8(6):e1002754.
The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9bfh313, to dissect its function in the morphogenesis of this structure. Strikingly, sox9bfh313 homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9bfh313 mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9bfh313 mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9bfh313 mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies.
Author Summary
The liver and pancreas function as exocrine glands that secrete bile and pancreatic juice, respectively, to aid the digestion and absorption of nutrients. These fluids reach the intestine via the pancreaticobiliary ductal system, a complex network of ducts. Despite its pivotal role, the development of this ductal system is poorly understood. We have discovered that the zebrafish transcription factor gene sox9b, like its mammalian ortholog, is specifically expressed in the pancreaticobiliary ductal system. The perinatal lethality of Sox9 heterozygous mice makes the analysis of SOX9 function challenging; thus, we turned to the zebrafish to analyze the role of SOX9 in pancreaticobiliary ductal system development. We found that zebrafish sox9b mutants, which survive to adulthood, display defects in the morphogenesis of this ductal network: the intrahepatic and intrapancreatic ducts fail to form a branched network, whereas the ducts connecting the liver and pancreas to the intestine are malformed. These ductal defects affect bile transport and lead to cholestasis in adult mutant fish. At the molecular level, Sox9b interacts with the Notch signaling pathway to regulate the development of the intrahepatic biliary network. Therefore, our work in zebrafish reveals a broad and complex role for SOX9 in pancreaticobiliary ductal system morphogenesis.
PMCID: PMC3375260  PMID: 22719264
17.  Defective cranial skeletal development, larval lethality and haploinsufficiency in Myod mutant zebrafish 
Developmental biology  2011;358(1):102-112.
Myogenic regulatory factors of the myod family (MRFs) are transcription factors essential for mammalian skeletal myogenesis. Here we show that a mutation in the zebrafish myod gene delays and reduces early somitic and pectoral fin myogenesis, reduces miR-206 expression, and leads to a persistent reduction in somite size until at least the independent feeding stage. A mutation in myog, encoding a second MRF, has little obvious phenotype at early stages, but exacerbates the loss of somitic muscle caused by lack of Myod. Mutation of both myod and myf5 ablates all skeletal muscle. Haploinsufficiency of myod leads to reduced embryonic somite muscle bulk. Lack of Myod causes a severe reduction in cranial musculature, ablating most muscles including the protractor pectoralis, a putative cucullaris homologue. This phenotype is accompanied by a severe dysmorphology of the cartilaginous skeleton and failure of maturation of several cranial bones, including the opercle. As myod expression is restricted to myogenic cells, the data show that myogenesis is essential for proper skeletogenesis in the head.
PMCID: PMC3360969  PMID: 21798255
muscle; zebrafish; myosin; slow; fiber; fast; myod; myogenin; myf5; miR-206; skeleton; bone; cartilage; head; fin; haploinsufficiency
18.  Planar polarity pathway and Nance-Horan syndrome-like 1b have essential cell-autonomous functions in neuronal migration 
Development (Cambridge, England)  2011;138(14):3033-3042.
Components of the planar cell polarity (PCP) pathway are required for the caudal tangential migration of facial branchiomotor (FBM) neurons, but how PCP signaling regulates this migration is not understood. In a forward genetic screen, we identified a new gene, nhsl1b, required for FBM neuron migration. nhsl1b encodes a WAVE-homology domain-containing protein related to human Nance-Horan syndrome (NHS) protein and Drosophila GUK-holder (Gukh), which have been shown to interact with components of the WAVE regulatory complex that controls cytoskeletal dynamics and with the polarity protein Scribble, respectively. Nhsl1b localizes to FBM neuron membrane protrusions and interacts physically and genetically with Scrib to control FBM neuron migration. Using chimeric analysis, we show that FBM neurons have two modes of migration: one involving interactions between the neurons and their planar-polarized environment, and an alternative, collective mode involving interactions between the neurons themselves. We demonstrate that the first mode of migration requires the cell-autonomous functions of Nhsl1b and the PCP components Scrib and Vangl2 in addition to the non-autonomous functions of Scrib and Vangl2, which serve to polarize the epithelial cells in the environment of the migrating neurons. These results define a role for Nhsl1b as a neuronal effector of PCP signaling and indicate that proper FBM neuron migration is directly controlled by PCP signaling between the epithelium and the migrating neurons.
PMCID: PMC3119310  PMID: 21693519
Facial branchiomotor neuron; Nance-Horan syndrome-like 1b; Planar cell polarity; Neuron migration; Zebrafish
19.  Asymmetric inhibition of Ulk2 causes left-right differences in habenular neuropil formation 
Studies of the zebrafish epithalamus have provided recent insights into the development of left-right brain asymmetry, which is crucial to normal human brain function. The habenular nuclei (Hb) of zebrafish are robustly asymmetric, with dense elaboration of neuropil only in the left lateral subnucleus. Because this feature is tightly correlated with asymmetric expression of K+ channel tetramerization domain-containing proteins 12.1 and 12.2 (Kctd12.1/12.2), we screened for Kctd12.1-interacting proteins to identify molecular mechanisms leading to neuropil asymmetry, and uncovered a novel interaction between Kctd12.1 and Unc-51-like kinase 2 (Ulk2). We show here that knockdown of Ulk2 or overexpression of Kctd12 proteins reduce asymmetric neuropil elaboration. Conversely, overexpression of Ulk2 or mutation of kctd12 genes cause excess neuropil elaboration. We conclude that Ulk2 activity promotes neuropil elaboration while Kctd12 proteins limit Ulk2 activity asymmetrically. This work describes a regulatory mechanism for neuronal process extension that may be conserved in other developmental contexts in addition to the epithalamus.
PMCID: PMC3142468  PMID: 21734278
Kctd12; zebrafish; epithalamus; habenular nuclei
20.  The ciliopathy gene cc2d2a controls zebrafish photoreceptor outer segment development through a role in Rab8-dependent vesicle trafficking 
Human Molecular Genetics  2011;20(20):4041-4055.
Ciliopathies are a genetically and phenotypically heterogeneous group of human developmental disorders whose root cause is the absence or dysfunction of primary cilia. Joubert syndrome is characterized by a distinctive hindbrain malformation variably associated with retinal dystrophy and cystic kidney disease. Mutations in CC2D2A are found in ∼10% of patients with Joubert syndrome. Here we describe the retinal phenotype of cc2d2a mutant zebrafish consisting of disorganized rod and cone photoreceptor outer segments resulting in abnormal visual function as measured by electroretinogram. Our analysis reveals trafficking defects in mutant photoreceptors affecting transmembrane outer segment proteins (opsins) and striking accumulation of vesicles, suggesting a role for Cc2d2a in vesicle trafficking and fusion. This is further supported by mislocalization of Rab8, a key regulator of opsin carrier vesicle trafficking, in cc2d2a mutant photoreceptors and by enhancement of the cc2d2a retinal and kidney phenotypes with partial knockdown of rab8. We demonstrate that Cc2d2a localizes to the connecting cilium in photoreceptors and to the transition zone in other ciliated cell types and that cilia are present in these cells in cc2d2a mutants, arguing against a primary function for Cc2d2a in ciliogenesis. Our data support a model where Cc2d2a, localized at the photoreceptor connecting cilium/transition zone, facilitates protein transport through a role in Rab8-dependent vesicle trafficking and fusion.
PMCID: PMC3177654  PMID: 21816947
21.  The lta4h Locus Modulates Susceptibility to Mycobacterial Infection in Zebrafish and Humans 
Cell  2010;140(5):717-730.
Exposure to Mycobacterium tuberculosis produces varied early outcomes, ranging from resistance to infection to progressive disease. Here we report results from a forward genetic screen in zebrafish larvae that identify multiple mutant classes with distinct patterns of innate susceptibility to Mycobacterium marinum. A hypersusceptible mutant maps to the lta4h locus encoding leukotriene A4 hydrolase, which catalyzes the final step in the synthesis of leukotriene B4 (LTB4), a potent chemoattractant and proinflammatory eicosanoid. lta4h mutations confer hypersusceptibility independent of LTB4 reduction, by redirecting eicosanoid substrates to anti-inflammatory lipoxins. The resultant anti-inflammatory state permits increased mycobacterial proliferation by limiting production of tumor necrosis factor. In humans, we find that protection from both tuberculosis and multibacillary leprosy is associated with heterozygosity for LTA4H polymorphisms that have previously been correlated with differential LTB4 production. Our results suggest conserved roles for balanced eicosanoid production in vertebrate resistance to mycobacterial infection.
PMCID: PMC2907082  PMID: 20211140
22.  Zebrafish survival motor neuron mutants exhibit presynaptic neuromuscular junction defects 
Human Molecular Genetics  2009;18(19):3615-3625.
Spinal muscular atrophy (SMA), a recessive genetic disease, affects lower motoneurons leading to denervation, atrophy, paralysis and in severe cases death. Reduced levels of survival motor neuron (SMN) protein cause SMA. As a first step towards generating a genetic model of SMA in zebrafish, we identified three smn mutations. Two of these alleles, smnY262stop and smnL265stop, were stop mutations that resulted in exon 7 truncation, whereas the third, smnG264D, was a missense mutation corresponding to an amino acid altered in human SMA patients. Smn protein levels were low/undetectable in homozygous mutants consistent with unstable protein products. Homozygous mutants from all three alleles were smaller and survived on the basis of maternal Smn dying during the second week of larval development. Analysis of the neuromuscular system in these mutants revealed a decrease in the synaptic vesicle protein, SV2. However, two other synaptic vesicle proteins, synaptotagmin and synaptophysin were unaffected. To address whether the SV2 decrease was due specifically to Smn in motoneurons, we tested whether expressing human SMN protein exclusively in motoneurons in smn mutants could rescue the phenotype. For this, we generated a transgenic zebrafish line with human SMN driven by the motoneuron-specific zebrafish hb9 promoter and then generated smn mutant lines carrying this transgene. We found that introducing human SMN specifically into motoneurons rescued the SV2 decrease observed in smn mutants. Our analysis indicates the requirement for Smn in motoneurons to maintain SV2 in presynaptic terminals indicating that Smn, either directly or indirectly, plays a role in presynaptic integrity.
PMCID: PMC2742401  PMID: 19592581
23.  Pbx acts with Hand2 in early myocardial differentiation 
Developmental biology  2009;333(2):409-418.
Transcription factors of the basic helix-loop-helix (bHLH) family are critical regulators of muscle cell differentiation. For example, Myod drives skeletal muscle differentiation, and Hand2 potentiates cardiac muscle differentiation. Understanding how these bHLH factors regulate distinct transcriptional targets in a temporally and spatially controlled manner is critical for understanding their activity in cellular differentiation. We previously showed that Pbx homeodomain proteins modulate the activity of Myod to promote the differentiation of fast-twitch skeletal muscle. Here, we test the hypothesis that Pbx proteins are also necessary for cardiac muscle differentiation through interacting with Hand2. We show that Pbx proteins are required for the activation of cardiac muscle differentiation in zebrafish embryos. Loss of Pbx activity leads to delay of myocardial differentiation and subsequent defective cardiac morphogenesis, similar to reduced Hand2 activity. Genetic interaction experiments support the hypothesis that Pbx proteins modulate the activity of Hand2 in myocardial differentiation. Furthermore, we show that Pbx proteins directly bind the promoter of the myocardial differentiation gene myl7 in vitro, supporting a direct role for Pbx proteins in promoting cardiac muscle differentiation. Our findings demonstrate new roles for Pbx proteins in vertebrate cardiac development and also provide new insight into connections between the transcriptional regulation of skeletal and cardiac muscle differentiation programs.
PMCID: PMC2752274  PMID: 19607825
pbx; hand2; zebrafish; heart development; muscle differentiation
24.  EphA4 and EfnB2a maintain rhombomere coherence by independently regulating intercalation of progenitor cells in the zebrafish neural keel 
Developmental biology  2008;327(2):313-326.
During vertebrate development, the hindbrain is transiently segmented into 7 distinct rhombomeres (r). Hindbrain segmentation takes place within the context of the complex morphogenesis required for neurulation, which in zebrafish involves a characteristic cross-midline division that distributes progenitor cells bilaterally in the forming neural tube. The Eph receptor tyrosine kinase EphA4 and the membrane-bound Ephrin (Efn) ligand EfnB2a, which are expressed in complementary segments in the early hindbrain, are required for rhombomere boundary formation. We showed previously that EphA4 promotes cell-cell affinity within r3 and r5, and proposed that preferential adhesion within rhombomeres contributes to boundary formation. Here we show that EfnB2a is similarly required in r4 for normal cell affinity and that EphA4 and EfnB2a regulate cell affinity independently within their respective rhombomeres. Live imaging of cell sorting in mosaic embryos shows that both proteins function during cross-midline cell divisions in the hindbrain neural keel. Consistent with this, mosaic EfnB2a over-expression causes widespread cell sorting and disrupts hindbrain organization, but only if induced at or before neural keel stage. We propose a model in which Eph and Efn-dependent cell affinity within rhombomeres serve to maintain rhombomere organization during the potentially disruptive process of teleost neurulation.
PMCID: PMC2861865  PMID: 19135438
Eph; Ephrin; Efn; hindbrain; boundary; zebrafish; rhombomere; neuroepithelium; cell affinity; cell sorting
25.  A G Protein-Coupled Receptor is Essential for Schwann Cells to Initiate Myelination 
Science (New York, N.Y.)  2009;325(5946):1402-1405.
The myelin sheath allows axons to rapidly conduct action potentials in the vertebrate nervous system. Incompletely understood axonal signals activate specific transcription factors, including Oct6 and Krox20, that initiate myelination in Schwann cells. Elevation of cAMP can mimic axonal contact in vitro, but the mechanisms that regulate cAMP levels in vivo are unknown. Using mutational analysis in zebrafish, we report that Gpr126 is required autonomously in Schwann cells for myelination. In gpr126 mutants, Schwann cells failed to express oct6 and krox20, and were arrested at the promyelinating stage. Elevation of cAMP in gpr126 mutants, but not krox20 mutants, could restore myelination. We propose that Gpr126 drives the differentiation of promyelinating Schwann cells by elevating cAMP levels, thereby triggering Oct6 expression and myelination.
PMCID: PMC2856697  PMID: 19745155

Results 1-25 (31)