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In vivo cell biology in zebrafish – providing insights into vertebratedevelopment and disease

Ana M. Vacaru1,2, Gokhan Unlu3, Marie Spitzner4, Marina Mione4, Ela W. Knapik3 and Kirsten C. Sadler1,2,5,*

ABSTRACT

Over the past decades, studies using zebrafish have significantly

advanced our understanding of the cellular basis for development

and human diseases. Zebrafish have rapidly developing transparent

embryos that allow comprehensive imaging of embryogenesis

combined with powerful genetic approaches. However, forward

genetic screens in zebrafish have generated unanticipated findings

that are mirrored by human genetic studies: disruption of genes

implicated in basic cellular processes, such as protein secretion or

cytoskeletal dynamics, causes discrete developmental or disease

phenotypes. This is surprising because many processes that were

assumed to be fundamental to the function and survival of all cell

types appear instead to be regulated by cell-specific mechanisms.

Such discoveries are facilitated by experiments in whole animals,

where zebrafish provides an ideal model for visualization and

manipulation of organelles and cellular processes in a live

vertebrate. Here, we review well-characterized mutants and newly

developed tools that underscore this notion. We focus on the

secretory pathway and microtubule-based trafficking as illustrative

examples of how studying cell biology in vivo using zebrafish has

broadened our understanding of the role fundamental cellular

processes play in embryogenesis and disease.

KEY WORDS: Zebrafish, Protein secretion, Vesicular transport,

Microtubule transport

IntroductionClassical cell biologists studied cells in organisms abundantly

available in their environments, such as algae, plants and marineanimals. As the field evolved, cultured cells became de rigueur,and thus in vitro studies have generated much of the modern cell

biology lexicon. The genetic advantages and expanding tools forstudying subcellular structures in the small transparent zebrafishembryo offer the opportunity for cell biologists to return to our

roots and to address fundamental cell biological questions in thecontext of a whole organism.

Forward genetic screens to identify genes underlyingdevelopmental events are a mainstay of zebrafish research.These screens have generated an extensive repertoire of mutants

where a gene implicated in a basic cell biological process has been

disrupted. Surprisingly, many of these mutants have specificphenotypes that involve only a few tissues or cell types.This is reminiscent of human genetic disorders such asmitochondriopathies (Schapira, 2006), ciliopathies (Hildebrandt

et al., 2011) and diseases of protein trafficking (De Matteis andLuini, 2011) where a defect in a protein involved in a fundamentalorganelle function results in a discrete clinical syndrome.

Similarly, researchers were surprised by the finding that manygenes thought to be essential for the function or survival of cells inculture, and that were assumed to be ubiquitously expressed, were

instead revealed to have spatio-temporally restricted expressionpatterns during zebrafish development. These findings led to thehypothesis that fundamental cellular processes are regulated by

cell-type-specific mechanisms.The use of zebrafish and other animals – both invertebrate and

vertebrate – (see Box 1) provides valuable insights into how basiccellular processes are regulated during development and howdisrupting these processes can impact on embryogenesis. This is

elegantly illustrated by a recent investigation into the formationof the notochord, which forms the embryonic axial skeleton (Elliset al., 2013). Notochord cells appear hollow owing to a large

cytoplasmic vacuole found to be a lysosomal-derived organellegenerated by the endosomal trafficking machinery. Rab32a is aGTPase which, in cultured cells, has been found to be involved inmitochondrial dynamics (Alto et al., 2002; Bui et al., 2010),

trafficking to autophagosomes (Hirota and Tanaka, 2009) andother lysosome-related functions (Bultema et al., 2012; Wasmeieret al., 2006), and, in zebrafish, Rab32a has been found to be

essential for vacuole formation and hence for notochorddevelopment. This exemplifies how pairing traditional cellbiological approaches with advances in microscopy, genetics

and pharmacology in zebrafish leads to unprecedentedunderstanding of how basic cellular processes drive specificdevelopmental events.

Here, we review the advantages of using zebrafish to study theintersection between cell biology, vertebrate development and

pathology, and highlight some well-established imaging andgenetic tools. We focus on the protein secretory pathway andmicrotubule-directed vesicular traffic to exemplify how merging

cell biology, genetics and embryology has provided uniqueinsight into how cells work in the context of tissues and organs ina living organism.

The zebrafish toolbox – microscopes, genetics and drugsZebrafish overviewZebrafish produce large numbers of externally fertilized eggs,

which rapidly develop into transparent embryos and progress tofree-swimming feeding larvae within 5 days post fertilization(dpf). Gastrulation is complete within hours of fertilization and by

1 dpf, embryonic axes are established and neural development is

1Department of Developmental and Regenerative Biology, Icahn School ofMedicine at Mount Sinai, 1 Gustave L. Levy Place, Box 1020, New York, NY10029, USA. 2Department of Medicine/Division of Liver Diseases, Icahn School ofMedicine at Mount Sinai, 1 Gustave L. Levy Place, Box 1020, New York, NY10029, USA. 3Division of Genetic Medicine, Department of Medicine, andDepartment of Cell and Developmental Biology, Vanderbilt University MedicalCenter, Nashville, TN 37232, USA. 4Institute of Toxicology and Genetics,Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany. 5Graduate Schoolof Biomedical Sciences, Icahn School of Medicine at Mount Sinai, 1 Gustave L.Levy Place, Box 1020, New York, NY 10029, USA.

*Author for correspondence (Kirsten.edepli@mssm.edu)

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 485–495 doi:10.1242/jcs.140194

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underway. An exquisite map of cell division and movementduring these early developmental events has been achieved bytracking individually labeled nuclei using advanced microscopy

(Keller et al., 2008; Schmid et al., 2013). By the end of 2 dpf,organogenesis is underway throughout the embryo and, by 5 dpf,most organs carry out specialized functions. Although zebrafish

have traditionally been used to study embryogenesis, areas suchas behavior, pathology, infectious diseases and drug screening areactively investigated. Here, we focus on zebrafish tools usedto study cell biology in vivo, with the aim to understand

development and disease.

Labeling subcellular structures in zebrafishHigh-resolution studies of embryonic development and diseasemodels require analysis of the behaviors of individual cells,subcellular structures and proteins in the context of an intact

tissue or organism. Fluorescent proteins have revolutionized cellbiology. However, the relative small size and dynamic nature ofsubcellular components present challenges for imaging. This isexacerbated by the cellular complexity of whole animals. Thus, in

contrast to the elegant studies describing organelle dynamics incultured cells or yeast, relatively little is known about these

processes in vertebrates.Developing a zebrafish toolkit that labels, tracks and measures

intracellular structures comparable to that available in mammalsis a work in progress. A recent and concerted effort by the

zebrafish community and several companies has improved thelibrary of antibodies recognizing zebrafish proteins, which arecataloged in the zebrafish model organism database (http://zfin.

org). However, specific antibodies for many structures remainunavailable, and antibody staining does not allow live imaging, astrength of the zebrafish system. Developing transgenics

(Kawakami, 2005; Kwan et al., 2007) and vital dyes provides agood alternative. Well-characterized fluorescent markers relevantto topics discussed here are listed in Table 1 and illustrated in

Fig. 1F. Fig. 1 shows two examples of such transgenics:Tg(bact:GalT-GFP) fish express a portion of Galactose-1-phosphate uridyl transferase (GALT) fused to GFP under thesemi-ubiquitous b-actin promoter (Gerhart et al., 2012) to enable

imaging of the trans-Golgi in the developing embryo using both alow-resolution fluorescence stereomicroscope (Fig. 1A–C) andconfocal imaging (Fig. 1D). The second, Tg(ef1a:dclk-GFP) line

(Tran et al., 2012), labels microtubules with GFP and, when usedin combination with the vesicle marker clathrin fused to dsRed(M.S. and M.M., unpublished data; Fig. 1E), it provides a real-

time view of microtubule-based vesicular transport.Fluorescent vital dyes also allow for imaging subcellular

structures in vivo. BODIPY, Alizarin Red, quantum dots and

dyes, such as MitoTracker or LysoTracker, are versatilecounterstains to visualize dynamic cellular processes in live fish(Cooper et al., 2005; He et al., 2009; Koster and Fraser, 2006;Rieger et al., 2005; Zhang et al., 2008). In combination, transgenics

and dyes provide a microscopic view of intracellular processes anda macroscopic view of how disrupting basic cellular processesinterrupt development or cause disease.

ImagingMany zebrafish imaging studies are carried out using microscopes

that are available to most researchers, whereas an in-depthanalysis of dynamic movements or morphometric analyses ofsubcellular structures requires the advanced microscopytechniques limited to researchers who have specialized training

and access to facilities with these capabilities.Low-resolution epifluorescent and stereomicroscopes can be

used to detect gross changes in localization or intensity of many

markers, and standard confocal microscopy can image cells that areclose to the surface of live embryos but imaging deeper cellsrequires histological sections. Unprecedented views of how cells

move individually and in concert in a developing embryo havebeen provided using light-sheet microscopy to track individualnuclei labeled with fluorescently-tagged histones (Huisken and

Stainier, 2009; Keller et al., 2008). Selective plane illuminationmicroscopy has provided a three-dimensional view of endodermalcell movement in older live embryos (Schmid et al., 2013).Multiphoton imaging has been used to investigate how neuronal

circuits assemble during retinal development by tracking dynamicchanges in cell structure and connectivity (Williams et al., 2013),and super-resolution microscopy has been used to image a single

receptor molecule uncovering the role for receptor clustering in theantiviral immune response (Gabor et al., 2013). Many morespectacular zebrafish images are being generated as imaging

technologies continue to improve.

Box 1. Comparative analysis of common model organisms

Zebrafish are the most widely used non-mammalian vertebrateorganism, with thousands of laboratories devoted to zebrafishresearch worldwide. What distinguishes zebrafish from the fly(Drosophila melanogaster) and worm (Caenorhabditis elegans) –commonly used invertebrate organisms – is that, as a vertebrate, itshares properties with most organs found in mammals. Althoughzebrafish development is slower than that of invertebrates, it isconsiderably faster (5 days) than mice (19 days). As zebrafishdevelopment is external, like invertebrates, live embryos can beobserved and manipulated; however, these same features makethis system less than ideal for studies focused on in utero

development or the transition from pre- to post-nataldevelopment. Finally, the high fecundity provides sufficientsample sizes to enable large-scale screening, and drug screensare facilitated by the ability to add compounds directly to theirculture water.The zebrafish genome is diploid and has been fully sequenced,

with annotation being underway on Ensembl, and the SangerCenter and other genome browsers, providing advantages over thetetraploid Xenopus genome. Genome duplication occurred duringteleost development so that ,30% of zebrafish genes areduplicated (Howe et al., 2013; Woods et al., 2000; Woods et al.,2005). The advantage of this is that orthologs might have divergedin function and allow for emergence of specific phenotypes whenone is targeted; however, there is the potential for duplicated genesthat act redundantly, and hence phenotypes might not emergeunless both genes are targeted (Roest Crollius and Weissenbach,2005).The transparency and accessibility of zebrafish embryos is a

significant advantage for cell biology, allowing visualization offluorescent proteins in vivo, as in worms. A low-power microscopeis sufficient for observing organotypic defects or living embryos at asingle-cell resolution. Thus, zebrafish provide an excellent systemfor live imaging in a whole vertebrate and are comparable toXenopus in the ease of morpholino knockdown. However, therapidity of the system and the repertoire of tools for manipulatinggene function and expression have not yet achieved the same levelthat is available for flies and worms. Thus, there is value in eachanimal model and the strengths of each can be leveraged incomparative approaches to fully understand gene function.

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GeneticsThe zebrafish genome has been sequenced and annotated, andmost zebrafish genes are highly conserved in mammals, with azebrafish ortholog identified for ,70% of human genes (Howe

et al., 2013). Transgenesis and forward genetic screens areimportant widely used methods in zebrafish, and recent advancesin reverse genetic approaches are having a major impact on thefield. Box 2 details methods of generating transgenics and

targeted mutagenesis using TALENs and the Crispr/Cas systems.Forward genetic screens allow the unbiased discovery of genes

that contribute to a specific phenotype and have generated thousands

of mutants in the zebrafish genome. One drawback, however, is thatgenerating and maintaining stable lines can be laborious and anotheris that the genome duplication that occurred during teleost evolution

(Postlethwait et al., 1998) can generate genes that act redundantly,

necessitating that both are targeted before revealing a phenotype. Inaddition, maternal stores of mRNA and proteins can delay theemergence of mutant phenotypes until after these are exhausted, and

thus mutant phenotypes often reflect the impact of gene loss on laterdevelopmental stages.

Morpholinos provide a complementary approach, wherebyoligonucleotides that are injected into the fertilized egg block

target mRNA translation or splicing. Although the use ofmorpholinos is plagued by fears of off-target effects andtransient effects, translation-blocking morpholinos deplete

proteins derived from both maternal and zygotic mRNA, so thatthe effects of target gene loss on early embryonic events can bestudied. Additionally, by titrating the amount of morpholino

Table 1. List of markers and tools of intracellular and extracellular structures used in vivo in zebrafish

Cellular structure or process Tool type Tool name Application References

Nucleus Vital dye Acridine orange Visualize the nuclei of dying cells Paquet et al., 2009; Peri andNusslein-Volhard, 2008

Transgenic H2B–mRFP Visualize nuclei in vivo Distel et al., 2011Transgenic H2B–EGFP Visualize nuclei in vivo Keller et al., 2008Transgenic NLS–EGFP Visualize the nuclei in vivo Kim et al., 2011Transgenic NLS–mCherry Visualize the nuclei in vivo Lam et al., 2010

Chromosomes Transgenic H2afz–GFP Visualize chromosomes during mitosis andtrack single nuclei during early development

Laguerre et al., 2009

Cytoplasm Vital dye BODIPY TR methylester dye

Cooper et al., 2005

Secretion Transgenic Dbp–EGFP Monitoring glycoprotein secretion fromhepatocytes

Xie et al., 2010

Endoplasmic reticulum stress Transgenic huORFZ huORFchop–GFP [expresses GFP fused tothe upstream open reading frame (uORF)from the 5’UTR of the human CHOP ]*

Lee et al., 2011

Golgi apparatus Transgenic GalT–GFP Trans-Golgi marker Gerhart et al., 2012Endosomes Vital dye F4-64 Endosomal marker Fischer-Parton et al., 2000

Transgenic Rab3–YFP Secretory endosomal marker Campbell et al., 2007Transgenic Rab5c–GFP Early endosomal marker Clark et al., 2011Transgenic Rab5c-Q81L–mCherry Constitutively active form of Rab5 Clark et al., 2011Transgenic Rab5c-S36N–mCherry Dominant-negative form of Rab5 Clark et al., 2011Transgenic Rab7–GFP Late endosomal marker Clark et al., 2011Transgenic Rab7-Q67L–mCherry Constitutively active form of Rab7 Clark et al., 2011Transgenic Rab7-T22N–mCherry Dominant-negative form of Rab7 Clark et al., 2011Transgenic Rab11a–GFP Recycling endosomal marker Clark et al., 2011Transgenic Rab11a-Q70L–mCherry Constitutively active form of Rab11 Clark et al., 2011Transgenic Rab11a-S25N-mCherry Dominant-negative form of Rab11 Clark et al., 2011Transgenic Rab32a–GFP Endosomal marker Ellis et al., 2013

Rab32a-T27N–GFP Dominant-negative form of Rab32 Ellis et al., 2013Transgenic Rab38b-T23N-GFP Dominant-negative form of Rab38 Ellis et al., 2013

Plasma membrane Vital dye Annexin–Cy5 Visualize early membrane of apoptotic cells Peri and Nusslein-Volhard, 2008Transgenic GFP–CaaX

(mem–GFP)Plasma membrane marker Ellis et al., 2013

Transgenic Mem–CFP Plasma membrane marker Distel et al., 2011Microtubules and motor

proteinsTransgenic Dclk–GFP Microtubule marker Tran et al., 2012

Transgenic Tau–GFP Marker for plus-end of microtubules in axons Yanicostas et al., 2009; Yoshidaet al., 2002

Transgenic a-tubulin–GFP Microtubule marker Asakawa and Kawakami, 2010Transgenic Kif17–GFP Marker for plus-end directed vesicle trafficking Bader et al., 2012; Insinna

et al., 2009Transgenic EB1–GFP Labels microtubules plus-end to visualize

growth directionTran et al., 2012

Transgenic EB3–GFP Labels microtubules plus-end to visualizegrowth direction

Tran et al., 2012

Lysosomes Transgenic Lamp2–GFP Lysosomal marker Ellis et al., 2013

*This regulates GFP translation to reflect endogenous Chop levels and monitor the ER stress response. GFP is found in neural tissues during ER and thermalstress.

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injected, the degree of knockdown can be finely tuned. This hasproved particularly useful for our work to model one type of

congenital disorder of glycosylation (CDG), which, in humans iscaused by a hypomorphic mutation in one of the genes requiredfor N-linked protein glycosylation (Freeze, 2007). Homozygous

null mutations of these genes are lethal in mammals (Thiel andKorner, 2011) and injecting high morpholino concentrations causessevere embryonic phenotypes and high mortality in zebrafish (Chuet al., 2013; Cline et al., 2012). However, fine-tuning of the

knockdown was facilitated by injecting lower morpholinoconcentrations so that residual enzyme activity in the zebrafishmorphants matched that measured in samples from patients,

improving survival and revealing novel phenotypes (Chu et al.,2013; Cline et al., 2012). Although the breadth and speed ofzebrafish genetic approaches do not match those available in

invertebrates, they are accomplished at a fraction of the costs of, andwith sample sizes exceeding, typical rodent experiments (see Box 1).

DrugsZebrafish have a rich history in toxicology research, as

compounds can be simply added to their water. For example,zebrafish are proving useful for alcohol research (Jang et al.,2012; Monk et al., 2013; North et al., 2010; Passeri et al., 2009;

Tsedensodnom et al., 2013; Yin et al., 2012). By using transgeniczebrafish expressing a GFP-tagged secreted glycoprotein inhepatocytes (Howarth et al., 2013; Tsedensodnom et al., 2013;Xie et al., 2010), and zebrafish that express fluorescent protein

markers of the hepatocyte secretory organelles and of other cellsin the liver (Yin et al., 2012), the mechanisms by which alcoholand other drugs cause organ-specific and organelle-mediated

toxicity can be uniquely addressed. Moreover, large-scale drugscreens exploit the ease of treating zebrafish with drugs (Petersonand Macrae, 2012) and have identified compounds modulating

processes ranging from metabolism (Nath et al., 2013) to sleep(Rihel and Schier, 2012).

200 µm

200 µm

2 dpfTg(bact:GalT-GFP)

A

B

C

Muscle cells

DAPITg(bact:GalT-GFP)

10 µm

10 µm

4 dpfHepatocytes

clathrin-DS-RedTg(ef1a:dclk-GFP)

1 dpf

D

E

COPII

ER

ERGIC

Golgi

COPI

LE

EE

PM

Lysosome

NLS–cherry h2afz–mRFP

EEEEEEEEERab5–GFP

Microtubules

GFP–α–tubulin dclk–GFP Tau–GFP

Kif17–GFP EB1–GFP EB2–GFP

Rab7–GFP

Rab11–GFP

GFP–CaaX mem–CFP

Lamp2–GFP

NucleusRE

GalT–EGFP

NLS–EGFP

F

Fig. 1. Fluorescent protein markers of subcellularstructures in zebrafish. (A–C) Live 2 dpf Tg(bact:GalT-

GFP) embryo highlighting the trans-Golgi complex. Theboxed region in B is magnified in the C. (D) Confocalprojection through a cryosection of the liver from 5 dpfTg(bact:GalT-GFP) larva. Nuclei are stained with DAPI(gray). (E) Muscle in a live 1 dpf Tg(ef1a:dclk-GFP)

embryo with GFP-tagged microtubule-associated protein(DCK) and vesicles marked with clathrin-DS-Red(magenta). (F) Zebrafish fluorescent-protein-taggedorganelle markers that can be expressed withtransgenics. PM, plasma membrane; LE, late endosomes;EE, early endosomes; RE, recycling endosomes.

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The protein secretory pathwayThe secretory pathway generates, trafficks and processes proteinsdestined for the extracellular space or the plasma membrane. Itcomprises the endoplasmic reticulum (ER), the ER-Golgiintermediate compartment (ERGIC), the Golgi complex and the

vesicles that carry cargo between them (Fig. 2). Protein synthesisand glycosylation occur in the ER and Golgi, respectively. Thecoat protein II (COPII) complex facilitates cargo selection,

vesicle formation and anterograde trafficking from the ER to theGolgi, whereas retrograde transport occurs in COPI vesicles(Fig. 1F; Fig. 2). Diverse protein complexes function at each step

of this pathway to recruit Rab GTPases and SNARE proteins,which direct and tether vesicles to target organelles and facilitatemembrane fusion.

Work in unicellular organisms and cultured cells created anassumption that protein secretion is uniformly regulated across celltypes. Studies in human, zebrafish and other vertebrates, however,revealed that this pathway is regulated in a spatio-temporal and

paralog-specific manner (Melville and Knapik, 2011; Unlu et al.,2013). Although all cells secrete proteins, some – including B-

cells, chondrocytes, hepatocytes and the endocrine and exocrinepancreatic cells – are considered ‘professional’ secretory cellsand these cells were assumed to be particularly sensitive tosecretory pathway disruption. Zebrafish mutants in secretory

pathway genes both supported and refuted this hypothesis: somemutants show phenotypes in most highly secretory cells,whereas others have phenotypes that are restricted to only a

subset of cells.The emerging view is that the secretory machinery is integral to

morphogenesis and organ function in a cell-specific fashion. The

availability of zebrafish genetic mutants and fluorescent in vivo

reporters provides novel insight into organismal functions of thesecretory pathway. The effects of secretory pathway disruption

relevant to development and disease are discussed below.

Developmental consequences of secretory pathway disruptionThe COPII complex comprises the Sar1 GTPase, Sec23–Sec24

dimers of the inner coat, and Sec13–Sec31 heterotetramers of theouter coat (Fig. 2; Kaiser and Schekman, 1990; Novick et al.,1980). Zebrafish mutants of sec23a (crusher) and sec24d

(bulldog) develop craniofacial dysmorphology, kinked pectoralfins and short body length (Lang et al., 2006; Sarmah et al.,2010). These are attributed to a failure of extracellular matrix

(ECM) secretion during chondrocyte differentiation. Sec23A- andSec24D-deficient animals fail to export collagen and other N-glycosylated proteins from the chondrocyte ER, arresting

differentiation and ultimately causing cell death (Lang et al.,2006; Sarmah et al., 2010; Unlu et al., 2013), whereas collagensecretion and skeletal development are intact upon depletion ofthe close paralog Sec24C (Sarmah et al., 2010). Sec23B

mutations in humans and zebrafish disrupt erythropoesis(Bianchi et al., 2009; Schwarz et al., 2009), a differentphenotype from the chondrocyte defects observed in crusher

and bulldog mutants. These COPII phenotypes are distinct fromthe dwarf mutants sneezy, happy and dopey, which disrupt genesthat encode the a, b and b9 subunits of the COPI complex,

respectively (Fig. 2), which are characterized by defects innotochord and melanosome formation (Coutinho et al., 2004).These data suggest that although COPI and COPII are requiredfor efficient secretion and membrane recycling in all cells, loss of

specific members of each complex have profound and disparateeffects on a subset of cells. Mutations in individual COPIIcomponents cause an array of phenotypes in highly secretory cell

types in organs such as cartilage, notochord, eye and gut (Niuet al., 2012; Schmidt et al., 2013; Townley et al., 2008; Townleyet al., 2012), and in erythrocytes (Bianchi et al., 2009; Schwarz

et al., 2009; Unlu et al., 2013), whereas cells that depend onvacuole formation are most sensitive to COPI depletion.

So how do transcriptional regulatory mechanisms direct the

secretory pathway to assure a timely availability of cargo-specificcoats? A large-scale screen in zebrafish identified the feelgood

mutant, which carries a missense variant in the creb3L2 gene(Driever et al., 1996; Knapik, 2000; Neuhauss et al., 1996) – the

first known transcription factor that regulates availability of theCOPII components sec24d and sec23a, but not sec24c (Melvilleet al., 2011). Similarities between feelgood, crusher and bulldog

mutant phenotypes suggest that a ‘secretory module’ consisting ofCreb3L2–Sec23A–Sec24D specializes in procollagen secretion.Given that zebrafish depleted of Sec24C do not manifest skeletal

dysmorphology and the gene is not a target of Creb3L2, it is

Box 2. Genetic approaches – a summary

Transgenics are a common means to overexpress genes inzebrafish and, now, both transient knockdown by morpholinosand gene targeting are routine methods to induce gene loss.Random targeting of a transgene and promoter in the genome isfacilitated using the tol2 transposon (Suster et al., 2009). Cell-specific promoters are available for many cell types to directtransgene expression and bacterial artificial chromosomerecombineering can recapitulate endogenous gene expressionpatterns and levels; the heat shock promoter (hsp70) is also awidely used inducible system (Halloran et al., 2000). Multiple linesof fish expressing the same transgene in different tissues can bequickly generated by crossing fish in which the transgene is underthe UAS promoter, to lines that express the Gal4 transcriptionalactivator under a tissue-specific promoter (Distel et al., 2009;Halpern et al., 2008).Chemical and gene-breaking transposons (http://zfishbook.org/)

have generated a large library of non-targeted mutants.Homologous recombination to generate targeted gene mutationshas eluded the zebrafish community for years. However, use ofTALENs (transcription activator-like effector nucleases) (Bedell etal., 2012; Huang et al., 2011; Sander et al., 2011) and the type IIprokaryotic CRISPR/Cas (clustered regularly interspaced shortpalindromic repeats/CRISPR-associated) system (Hwang et al.,2013; Jao et al., 2013; Xiao et al., 2013) has recently beensuccessful in specific gene targeting to generate germ-linemutants. These systems use chimeric nucleases composed ofprogrammable sequence-specific DNA-binding modules linked tononspecific DNA nucleases to induce double-stranded DNA breaksthat are inefficiently repaired by error-prone non-homologous end-joining or homology-directed repair (Gaj et al., 2013). TALEN pairsthat have been engineered by a zebrafish consortium arecommercially available from http://www.addgene.com for manygenes. Resources for TALEN design include https://groups.google.com/group/talengineering, http://www.addgene.org/talengineering/TALENzebrafish/ and http://www.TALengineering.org TheCRISPR/Cas system introduces single nucleotide substitutions,insertions and deletions ranging from 1 to 20 bp. The relative easeof synthesis makes the CRISPR/Cas systems an attractiveapproach for genome editing (see Hwang et al., 2013; http://www.addgene.org; http://www.crispr-cas.org). It is anticipated thatwithin the near future, nearly all protein-coding genes in zebrafishwill have been disrupted by either forward genetic screens or oneof these reverse genetic approaches.

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likely that other cargo-specific secretory modules regulate sec24c

and other genes in this pathway. Future studies in zebrafish andother animal models will be needed to crack the code ofphysiologically relevant, cargo-specific secretory networks.

Diseases caused by secretory pathway disruption in zebrafishand humansSeveral human syndromes are associated with secretory pathway

defects (De Matteis and Luini, 2011), some of which arerecapitulated in mutations in orthologous zebrafish genes (Fig. 2).The concurrent identification of crusher/sec23a mutants in

zebrafish and patients with SEC23A/cranio-lenticulo-suturaldysplasia (CLSD) variants (Boyadjiev et al., 2006; Lang et al.,2006) provides an excellent example of convergence between

human genetics and zebrafish developmental biology to uncoverphysiological ramifications caused by disrupting basic cellbiological processes. Both crusher mutants and CLSD patientspresent with craniofacial dysmorphology and axial skeleton

defects attributed to backlog of ECM proteins in the ER(Boyadjiev et al., 2006; Lang et al., 2006). The closely relatedSEC23B gene is mutated in congenital dyserythropoietic anemia

type II patients who have multinucleated erythroblasts in bonemarrow, a phenotype recapitulated in zebrafish sec23b morphants(Bianchi et al., 2009; Schwarz et al., 2009).

It is unclear why mutations in SEC23A and SEC23B paralogs,which differ only by an 18 amino acid stretch, cause such differentphenotypes. One possibility is that spatio-temporal differences in

expression confer cell-specific functions of some COPII complexgenes. However, as sar1a and sar1b are ubiquitously expressedearly in development and become enriched in distinct tissues lateron (E.W.K., unpublished observations), it is unlikely that their

gene expression pattern is solely responsible for the divergentphenotypes observed in these mutants.

The craniofacial phenotypes of COPII mutants suggest that

chondrocytes are highly sensitive to ECM secretory defects. Thispredicts that other manipulations that block the secretory pathway

would also cause craniofacial dysmorphology. This, however, is

not supported by data from zebrafish or humans when factorsfunctioning at other steps in the secretory pathway are depleted.The transport protein particle (TRAPP) complex tethers ER-derived vesicles to the cis-Golgi membrane (Fig. 2 and Sacher

et al., 2008). Fibroblasts from patients with TRAPPC11 orTRAPPC2 mutations (Bogershausen et al., 2013; Scrivens et al.,2009), and cultured cells depleted of TRAPPC11 (Scrivens et al.,

2011; Wendler et al., 2010) display Golgi fragmentationand secretory protein retention. However, when these proteinsare depleted in whole organisms their cell-specific roles

are uncovered: patients with TRAPPC2 mutation developspondyloepiphyseal dysplasia tarda (SEDT) distinguished byskeletal defects, short stature and microcephaly (Gedeon et al.,

1999; Huson et al., 1993). This is recapitulated in trappc2

zebrafish morphants, which have a short trunk and microcephaly(A. M. Vacaru and K. C. Sadler, unpublished). The short statureand/or trunk phenotype might reflect a defect in chondrocyte

formation or in ECM deposition, as in COPII mutants, butpatients and zebrafish with TRAPPC11 mutation presentvery differently. TRAPPC11 mutation in humans causes

myopathy, intellectual impairment and hyperkinetic movements(Bogershausen et al., 2013). Interstingly, zebrafish foie gras

(foigr) mutants carrying a mutagenic viral insertion in the

trappc11 gene display a different phenotype from other modelsof TRAPP complex disruption: they develop fatty liver,hepatomegaly, smaller gut and jaw, and fin defects (Cinaroglu

et al., 2011; Sadler et al., 2005). The foigr/trappc11 fatty liverphenotype is partially attributed to activation of the unfoldedprotein response (Cinaroglu et al., 2011); however, neithermammalian cultured cells that are depleted of TRAPPC11 nor

trappc2 morphants induce this response (A.M.V. and K.C.S.,unpublished observations), highlighting the utility of in vivo cellbiology studies in vertebrates.

The unique phenotypes that differentiate TRAPP complexdisruption from COPII or COPI mutations indicate that a global

COPI

SEC24D (?)Sec24D (bulldog)

SEC13-SEC31 (?)Sec13 (sec13sq198), sec31a (mo)

SAR1B (CMRD)Sar1B (mo)

Exocyticvesicle

SEC23B (CDAII)Sec23B (mo)

NucleussER

COPIIvesicle

αCOP (?)COP (sneezy)

β�COP (?)βCOP (?)βCOP (happy)

TRAPPC11 (LGMD)Trappc11 (foie gras)

Golgi

TRAPPC2 (SEDT)Trappc2 (?)

TRAPPC9 (ID)Trappc9 (?)

SEC24B (NTD)Sec24B (?)

SEC23A (CLSD)Sec23A (crusher)

SE9C24C (?)Sec24C (mo)

TRAPP complex

COPI complex

Plasmamembrane

β�COP (dopey)

Fig. 2. Secretory pathwaycomponents studied in zebrafishand implicated in human diseases.Secretory pathway proteinsimplicated in human disease areshown in black with correspondingzebrafish tools to study these in red.NTD, neural tube defects; CLSD,cranio-lenticular-sutural dysplasia;CDAII: congenital dyserythropoieticanemia II; CMRD, chylomicronretention disease; LGMD, limb girdlemuscular dystrophy; SEDL, X-linkedspondyloepiphyseal dysplasia tarda;ID, intellectual disability.

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block in protein secretion is not the only mechanism thatunderlies their associated phenotypes. Moreover, although

depleting individual TRAPP or COP complex factors hassimilar effects in isolated cells, the physiological consequencescould not be predicted without use of whole animals. Thesefindings point to cell- and developmental-specific roles for each

gene involved in protein secretion and underscore the need forcomparative whole animal models to decipher the cellular andphysiological functions of this pathway.

Microtubule highways in vesicular transportMicrotubules serve as tracks for organelles and vesicle movement

(Figs 1, 3), an especially crucial task for the long axons of nerve cells.Microtubules are formed by a-tubulin–b-tubulin heterodimers thatgenerate a polarized structure – the primary determinant of motor

protein directional movement. The fast growing plus-end (b-tubulincapped) and a degrading minus-end (a-tubulin capped) havecentrosomes that serve as microtubule-organizing centers fromwhich microtubules undergo assembly and disassembly (Wade, 2007).

Zebrafish tools to study microtubulesSeveral transgenic zebrafish lines have been developed to image

microtubules in vivo (Table 1; Figs 1, 3). A GAL4–UAS system hasbeen used to spatially and temporally express GFP-tagged a-tubulin(Asakawa and Kawakami, 2010). GFP-tagged doublecortin-like

kinase (York et al., 2012), a ubiquitously expressed microtubule-associated protein, uncovered the dynamics and polarity ofmicrotubules in early-stage embryos (Tran et al., 2012). Markers

of the growing plus-end of microtubules, including GFP-tagged Tau(Yanicostas et al., 2009; Yoshida et al., 2002) and the plus-endmicrotubule components Eb1 and Eb3 (Tran et al., 2012), have beencombined with other lines that express markers of specific vesicle

populations (Fig. 1C) for live imaging of vesicular transport alongmicrotubules in whole animals.

Cargos are transported along microtubules by members of twomotor protein superfamilies, the kinesins (Kifs) and the dyneins(Fig. 3). Although Kifs are cargo-specific, only cytoplasmicdynein1 (Dync1) is involved in directed microtubule transport of

cargos (Roberts et al., 2013). Dync1 is a multi-protein complexthat achieves specificity and versatility through distinct subunitsand interaction with regulatory proteins, including dynactin

(Karki and Holzbaur, 1999; King and Schroer, 2000; King, 2000).Like vesicle trafficking in the secretory pathway, cargo–motor

interactions are regulated by Rab GTPases which serve to

regulate, connect and attach cargo to microtubule motors. Rab3Aconnects vesicles to Kif1Bb and Kif1A in neuronal cells (Niwaet al., 2008), whereas Rab11A regulates endosomal trafficking

events through interaction with Kif3B (Schonteich et al., 2008)and Rab7 controls late endosomal transport (Jordens et al., 2001).The available markers for Rabs and their dominant-negativeversions in zebrafish are included in Table 1. GFP-tagged Rabs in

zebrafish colocalize to vesicles with lipophilic dye FM4-64(Fischer-Parton et al., 2000), and experiments using this systemhave shown that different Rabs associate with different types of

vesicles (Clark et al., 2011). This analysis was aided by anautomated tracking program (https://pantherfile.uwm.edu/cohena/www/rabtools.html), which enabled the in-depth analysis of

recycling endosomes and their intracellular dynamics, anunprecedented feat in whole organisms.

Collectively, work on microtubule-based transport in zebrafish

has led to several important conclusions: (1) microtubule-basedcargo transport shapes embryonic development from fertilizationonwards; (2) different motor proteins control transport in differentcell compartments – for instance, Kif17 in the photoreceptor outer

Minus-end

Plus-end

RAB7 (CMT2B)Rab7 (mCherry–Rab7DN,

mCherry–Rab7CA)

KIF18 (CMT2A)Kif1b (kif1bst43)

Dynein (NDD) Dync1h1 (cnb, mikreoko, ale oko)

Cargo

KIF5 (HSP)Kif5 (?)

Cargo

CargoRAB11Rab11 (mCherry–Rab11DN,

mCherry–Rab11CA)

Cargo

Cargo

Microtubule Dynein Rab3 Kif1B Dynactin Rab7 Kif3 DENN/MAD Rab11A Kif5 RIF11 Rab27

Key

Fig. 3. Microtubules, motor proteins and motor- and microtubule-binding proteins studied in zebrafish and involved in human diseases. Microtubuleassociated proteins implicated in human disease are shown in black with corresponding zebrafish tools to study these are in red. HSP, hereditary spasticparaplegia; NDD, neurodegenerative disease.

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segment and Kif3 in the cytoplasm; and (3) zebrafish Rabhomologs recapitulate the behavior of their counterparts in

mammalian cells. Thus, the expanding set of tools in zebrafish isenabling organism-wide functional analysis of proteins involvedin directed vesicular transport.

Off-track – microtubule-based diseases in humans and zebrafishAlthough most cells rely on microtubules for transporting theircargo, disruption of this process profoundly affects the central

and peripheral nervous system due to the long-distance thatcargos travel from cell bodies to axonal tips. Alzheimer,Huntington disease (HD) and Charcot–Marie–Tooth disease

(CMT) (De Vos et al., 2008) are some neurodegenerativediseases (NDDs) caused by defects in vesicular transport. Fig. 3illustrates the orthologous genes underlying NDDs in humans and

mutant phenotypes in zebrafish.CMT type 2A is an axonal sensorimotor neuropathy

characterized by severe peripheral muscle weakness andatrophy induced by loss of function mutations in the kinesin-3

family member, Kif1B (Zhao et al., 2001). In zebrafish, the pointmutation kif1bst43 affects the microtubule interaction site and hasbeen used to study kif1b functions in the developing central

nervous system. The use of chimeric embryos, generated bytransplanting kif1b-deficient neuronal cells into wild-type hosts(and vice versa), has revealed that there is a reduced growth

potential in mutant neuronal axons (Lyons et al., 2009). Thus,kif1b is required in a cell autonomous manner for axonaldevelopment in the central and peripheral nervous system.

Shprintzen–Goldberg syndrome is characterized by central andenteric nervous system defects and caused by homozygousmutations of the Kif1-binding protein Kbp (Brooks et al., 2005).The role of kbp in axonal outgrowth and maintenance, microtubule

organization and localization of axonal mitochondria and vesicleswas elucidated using kbpst23 zebrafish line that expresses a mutatedversion of kbp (Lyons et al., 2008). These studies uncovered a

reduced number of axons in the enteric nervous system of kbpst23

mutants, providing a new animal model for this syndrome.Several mutations in cytoplasmic dynein 1 heavy chain 1

(DYNC1H1) are linked to hereditary motor neuropathiesincluding CMT and spinal muscular atrophy with lowerextremity predominance (Harms et al., 2012; Weedon et al.,2011). Mutations in axonemal dynein or cytoplasmic dynein 2

have been linked to a large number of ciliopathies (Leigh et al.,2009; Huber and Cormier-Daire, 2012). Zebrafish morphants foraxonemal dyneins are used to study left–right asymmetry

(Kawakami et al., 2005); (Essner et al., 2005), and morphantsof cytoplasmic dynein 2 subunits revealed ciliary abnormalities inzebrafish kidney, eye and nose (Krock et al., 2009).

Although the mechanism of pathogenesis caused by dync1h1

mutations remains unclear, experiments in mice have suggestedthat there are defects in axonal transport of mitochondria

(Eschbach et al., 2013). This is also reflected in the zebrafishdync1h1 (cannonball) mutant, which displays abnormal organellepositioning and accumulation of Golgi-associated vesicles in theinner segment of the retina (Insinna et al., 2010) and Schwann

cell deficient myelination (Langworthy and Appel, 2012), asymptom common to NDDs. Additionally, dynactin mutationshave been found in patients with amyotrophic lateral sclerosis and

Perry syndrome (Stockmann et al., 2013). Death of sensoryneurons and axonal degeneration is also reported in zebrafishwith morpholino knockdown of dctn1a, dctn1b and dctn2a

(Insinna et al., 2010) and the mutants dctn1a (mikre oko)

(Tsujikawa et al., 2007) and dctn2 (ale oko) (Jing and Malicki,2009). This suggests that depletion of Dync1 or Dync2 has a

dose-dependent effect on photoreceptor cell organization and onvesicle transport.

Mutations in Rab GTPases can also lead to NDDs. It has beenshown that Rab11-dependent vesicle formation and transport is

influenced by the huntingtin (Htt) protein, which causes HD (Liet al., 2009a; Li et al., 2009b). Zebrafish expressing a GFP-taggedHtt mutant protein (Williams et al., 2008) are a valuable tool to

identify the Rabs and other factors that are required for Htttrafficking. The transgenics described above with dominant-negative and constitutively active versions of fluorescently

tagged Rab5, Rab7 and Rab11 proteins (Clark et al., 2011; Elliset al., 2013) will be valuable systems for dissecting specificcontributions of Dync1, dynactin and Rab GTPases in NDDs.

All cells are not created equal – conclusions and futuredirectionsIdentifying novel genes that regulate embryogenesis is a common aim

of zebrafish forward genetic screens, which continue to supplylibraries of mutants with disease-related phenotypes. Historically,researchers have focused on those mutants caused by disrupting genes

encoding transcription factors or signaling molecules that are thoughtto serve key roles in directing specific developmental processes.However, similar phenotypes result from mutations in genes that serve

basic cellular functions. These have been largely overlooked. Thevacuolar sorting protein 18 (vps18a) mutant serves to illustrate thispoint: VPS18 is required for endosomal and lysosomal trafficking in

yeast (Poupon et al., 2003) but in zebrafish, vps18a mutation causesspecific defects in the hepatobiliary system, notochord andmelanocytes (Ellis et al., 2013; Maldonado et al., 2006; Sadleret al., 2005). This recurring theme – whereby a gene is assumed to

serve a universal and essential cellular function based on studies inyeast or isolated cells but instead is revealed to play cell-specific rolesduring development or in a pathology – is an important and largely

underexplored topic in cell biology, and can be addressed by usingzebrafish.

Other lines of evidence that challenge the widely held assumption

that genes that carry out basic cellular functions must beubiquitously expressed and function equivalently in all cellsinclude an in situ screen to document the expression patterns ofthe zebrafish transcriptome during development, with many such

genes found to be expressed in a spatio-temporally specific fashion.For instance, the zebrafish orthologs of members of theheterotrimeric Sec61 translocon complex – sec61al1, sec61al2,

sec61b and sec61 – which threads newly synthesized proteins fromthe ribosome to ER lumen, all have distinct in situ expressionpatterns [Thisse, B. and Thisse, C. (2004) Fast Release Clones:

A High Throughput Expression Analysis. ZFIN Direct DataSubmission (http://zfin.org/)]. Interestingly, mutation in sec61al1

causes defects in the jaw (Nissen et al., 2006), liver and gut (K.C.S.,

unpublished observations) and brain ventricle laterality (Doll et al.,2011), corresponding to the organs where sec61al1 is highly expressed,indicating tissue-specific roles for this translocon component.

Screens typically yield more mutants than can be examined in

detail. Although much has been learned from mutants that haveobvious ties to well-studied transcriptional or signaling networks, wepropose that focusing instead on those mutants resulting from defects

in genes that have been previously classified as fundamental tocellular homeostasis are equally valuable because such mutants canclarify how cells are integrated into the function and formation of the

organism.

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In summary, in vivo cell biology in zebrafish is uncoveringtissue- and organ-specific functions for genes that previously

have been assumed to serve the same function across cell types.The multicolor imaging tools and multiple genetic approachesallow zebrafish researchers to address both developmental anddisease-related questions pertinent to cell biology. Because of

these advances, we are coming full circle back to the topics thatwere of interest to the cell biology founders who pioneeredstudies of cells in situ.

AcknowledgementsWe are grateful to Jaime Chu and Michel Bagnat for critical reading of themanuscript.

Competing interestsThe authors declare no competing interests.

FundingThe work of our laboratories is supported by the Association for InternationalCancer Research (to M.M.); the National Institutes of Health (to K.C.S. andE.W.K.); the Zebrafish Initiative of the Vanderbilt University Academic VentureCapital Fund (to E.W.K.); and the Vanderbilt International Scholar Program (toG.U.). Deposited in PMC for release after 12 months.

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