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RESEARCH ARTICLE Characterization of harpy/Rca1/emi1 Mutants: Patterning in the Absence of Cell Division Bruce B. Riley, 1 Elly M. Sweet, 1 Rebecca Heck, 1 Adrienne Evans, 1 Karen N. McFarland, 2 Rachel M. Warga, 2 and Donald A. Kane 2 * We have characterized mutations in the early arrest gene, harpy (hrp), and show that they introduce pre- mature stops in the coding region of early mitotic inhibitor1 (Rca1/emi1). In harpy mutants, cells stop dividing during early gastrulation. Lineage analysis confirms that there is little change in cell number af- ter approximately cycle-14. Gross patterning occurs relatively normally, and many organ primordia are produced on time but with smaller numbers of cells. Despite the lack of cell division, some organ systems continue to increase in cell number, suggesting recruitment from surrounding areas. Analysis of bromo- deoxyuridine incorporation shows that endoreduplication continues in many cells well past the first day of development, but cells cease endoreduplication once they begin to differentiate and express cell-type markers. Despite relatively normal gross patterning, harpy mutants show several defects in morphogene- sis, cell migration and differentiation resulting directly or indirectly from the arrest of cell division. Developmental Dynamics 239:828–843, 2010. V C 2010 Wiley-Liss, Inc. Key words: zebrafish; endoreduplication; primary neurons; muscle pioneers; neural crest; otic placode; pronephros; DNA replication Accepted 3 December 2009 INTRODUCTION Development often progresses th- rough an early phase of proliferation of multipotent progenitors followed by specification and differentiation of specific cell types. During this time, the rhythm of the cell cycle undergoes a dramatic slowing, shifting from rapid cleavage divisions to the long regulated cell cycle typical of differen- tiated tissues. In most cases, the shift from proliferation to differentiation is gradual and involves prolongation of one or both ‘‘gap’’ phases as cells react to various environmental signals (Kane et al., 1992; Kane and Kimmel, 1993; Zamir et al., 1997; Reviewed in Baker, 2007; Nogare et al., 2007; Ulloa and Briscoe, 2007; Orford and Scad- den, 2008). Proliferation is necessary to generate a sufficient number of cells to support larvae and then adult func- tions, but it also plays several direct and indirect roles in tissue patterning. For example, proliferation can pro- mote ‘‘community effects’’ whereby the size of a population modifies responses of individual cells to inductive cues (Gurdon et al., 1993; Schilling et al., 2001). In addition, many organ sys- tems develop from morphogenetic fields, groups of cells coordinated by diffusion gradients of growth factors Developmental Dynamics ABBREVIATIONS bmn branchio-motor nuclei dc diencephalon ep epiphysis ed endoderm edc endothelial cell fp floor plate hb hindbrain hbn hindbrain neurons hc hypochord hgl hatching gland ls lens mhb midbrain hindbrain border mp muscle pioneers Mth Mauthner neurons mus muscle nc notochord np nose placode oc otocyst op otic placode os optic stalk ov otic vesicle pcr pancreas pdm periderm pmn primary motor neurons pp pharyngeal pouch prd pronephric duct prn pronephric neck prt pronephric tubule prt pronephros px pharynx r4 fourth rhombomere rb Rohon-Beard sensory neuron rbc red blood cell scn spinal cord neurons sin spinal interneurons tc telencephalon tg trigeminal ganglia vb ventral brain 1 Department of Biology, Texas A&M University, College Station, Texas 2 Department of Biological Sciences, Western Michigan University, Kalamazoo, Michigan Grant sponsor: National Institutes of Health; Grant number: NIDCD-DC003806. *Correspondence to: Donald A. Kane, Department of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008. E-mail: [email protected] DOI 10.1002/dvdy.22227 Published online 3 February 2010 in Wiley InterScience (www.interscience.wiley.com). DEVELOPMENTAL DYNAMICS 239:828–843, 2010 V C 2010 Wiley-Liss, Inc.

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Page 1: Characterization of harpy/Rca1/emi1 Mutants: Patterning in

a RESEARCH ARTICLE

Characterization of harpy/Rca1/emi1 Mutants:Patterning in the Absence of Cell DivisionBruce B. Riley,1 Elly M. Sweet,1 Rebecca Heck,1 Adrienne Evans,1 Karen N. McFarland,2

Rachel M. Warga,2 and Donald A. Kane2*

We have characterized mutations in the early arrest gene, harpy (hrp), and show that they introduce pre-mature stops in the coding region of early mitotic inhibitor1 (Rca1/emi1). In harpy mutants, cells stopdividing during early gastrulation. Lineage analysis confirms that there is little change in cell number af-ter approximately cycle-14. Gross patterning occurs relatively normally, and many organ primordia areproduced on time but with smaller numbers of cells. Despite the lack of cell division, some organ systemscontinue to increase in cell number, suggesting recruitment from surrounding areas. Analysis of bromo-deoxyuridine incorporation shows that endoreduplication continues in many cells well past the first dayof development, but cells cease endoreduplication once they begin to differentiate and express cell-typemarkers. Despite relatively normal gross patterning, harpy mutants show several defects in morphogene-sis, cell migration and differentiation resulting directly or indirectly from the arrest of cell division.Developmental Dynamics 239:828–843, 2010. VC 2010 Wiley-Liss, Inc.

Key words: zebrafish; endoreduplication; primary neurons; muscle pioneers; neural crest; otic placode; pronephros;DNA replication

Accepted 3 December 2009

INTRODUCTION

Development often progresses th-rough an early phase of proliferationof multipotent progenitors followed byspecification and differentiation ofspecific cell types. During this time,the rhythm of the cell cycle undergoesa dramatic slowing, shifting fromrapid cleavage divisions to the longregulated cell cycle typical of differen-tiated tissues. In most cases, the shift

from proliferation to differentiation isgradual and involves prolongation ofone or both ‘‘gap’’ phases as cells reactto various environmental signals(Kane et al., 1992; Kane and Kimmel,1993; Zamir et al., 1997; Reviewed inBaker, 2007; Nogare et al., 2007; Ulloaand Briscoe, 2007; Orford and Scad-den, 2008). Proliferation is necessaryto generate a sufficient number of cellsto support larvae and then adult func-

tions, but it also plays several directand indirect roles in tissue patterning.For example, proliferation can pro-mote ‘‘community effects’’ whereby thesize of a population modifies responsesof individual cells to inductive cues(Gurdon et al., 1993; Schilling et al.,2001). In addition, many organ sys-tems develop from morphogeneticfields, groups of cells coordinated bydiffusion gradients of growth factors

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ABBREVIATIONS bmn branchio-motor nuclei dc diencephalon ep epiphysis ed endoderm edc endothelial cell fp floor platehb hindbrain hbn hindbrain neurons hc hypochord hgl hatching gland ls lens mhb midbrain hindbrain border mp muscle pioneersMth Mauthner neurons mus muscle nc notochord np nose placode oc otocyst op otic placode os optic stalk ov otic vesicle pcrpancreas pdm periderm pmn primary motor neurons pp pharyngeal pouch prd pronephric duct prn pronephric neck prt pronephrictubule prt pronephros px pharynx r4 fourth rhombomere rb Rohon-Beard sensory neuron rbc red blood cell scn spinal cord neuronssin spinal interneurons tc telencephalon tg trigeminal ganglia vb ventral brain

1Department of Biology, Texas A&M University, College Station, Texas2Department of Biological Sciences, Western Michigan University, Kalamazoo, MichiganGrant sponsor: National Institutes of Health; Grant number: NIDCD-DC003806.*Correspondence to: Donald A. Kane, Department of Biological Sciences, Western Michigan University, Kalamazoo, MI49008. E-mail: [email protected]

DOI 10.1002/dvdy.22227Published online 3 February 2010 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 239:828–843, 2010

VC 2010 Wiley-Liss, Inc.

Page 2: Characterization of harpy/Rca1/emi1 Mutants: Patterning in

that, depending on concentration,specify diverse cell fates while spa-tially regulating the pattern ofgrowth (Mariani et al., 2008). On amore local scale, asymmetric celldivision is a common mechanismfor generating cell type diversitywhereby daughter cells adopt differ-ent fates due to partitioning of cyto-plasmic determinants during mitosis(Kuang et al., 2007; Knoblich, 2008;Orford and Scadden, 2008). Thus,proliferation and patterning arefinely balanced and coordinated dur-ing much of early development.

The requirement for early prolifera-tion has been previously studied inXenopus embryos by using hydrox-yurea and aphidicolin to block entryinto S-phase at different stages of de-velopment (Harris and Hartenstein,1991; Rollins and Andrews, 1991).Treatment during blastula stageseverely perturbs embryonic pattern-ing because there are too few cells toexecute normal morphogenetic move-ments of gastrulation. In contrast,treatment during gastrulation resultsin development of a relatively normalbody plan, with each region compris-ing fewer cells. Within the neuraltube, an intricate array of primaryneural cell types still forms, thoughlater aspects of neural developmentare necessarily compromised withoutfurther cell division. Such findingsunderscore the surprising degreeof precision afforded by cell–cellinteractions.

Recently, Zhang et al. (2008)described amutation in zebrafish earlymitotic inhibitor 1 (emi1) that blocksmitosis just after the onset of gastrula-tion. The homologous gene inDrosoph-ila has been identified by the mutationRegulator of Cyclin A (Rca1; Donget al., 1997). Rca1/emi1is an inhibitorof Anaphase Promoting Complex/Cyclosome (APC/C), an E3 ubiquitinligase that targets mitotic regulatorsfor destruction (Reimann et al.,2001a,b; Miller et al., 2006; Di Fioreand Pines, 2007). In the absence ofRca1/emi1, APC/C remains constitu-tively active and blocks entry into mi-tosis. In addition, Rca1/emi1 mutantscontinue to undergo endoreduplica-tion, repeated cycles of DNA synthesiswithout mitosis, through at least earlysegmentation. This likely reflectsAPC/C-mediated destruction of Gemi-

nin, an inhibitor that normally blocksre-replication (Di Fiore and Pines,2007; Machida and Dutta, 2007). Con-sistent with data from mitoticallyblocked Xenopus embryos, gross pat-terning is relatively normal in Rca1/emi1 mutants. Somites initially formnormally and, indeed, the somitogenicclock operates with greater than nor-mal precision, confirming the hypothe-sis that mitosis can interfere with theability of individual cells to synchron-ize with their local cohort (Horikawaet al., 2006). However, patterningwithin somites is perturbed, as cellsthat should form the central mesen-chymal domain instead join the outerepithelial layer (Zhang et al., 2008).Likely there are many other pattern-ing defects arising directly or indi-rectly from the absence of cell division,as might be expected from the knowneffects of growth factors that mediatethe salient cell–cell interactions.

Here, we describe two new muta-tions in Rca1/emi1 and show thatthey are allelic to the ‘‘early arrest’’mutant harpy (hrp; Kane et al., 1996).Using cell counting and lineage analy-sis, we show that the cell cycle arrestoccurs at approximately cycle 14.Using in situ hybridization to analyzemarkers of specification and differen-tiation, we show that although globalpattern formation is normal, thereare many diminutive development-al defects arising from cessation ofcell division. Using transplantationbetween mutant and wild-type em-bryos, we find that the cycling defectsare autonomous although some of theobserved phenotypic perturbationsare not. And from an analysis of bro-modeoxyuridine (BrdU) uptake in theDNA of the mutant embryo, we findthat endoreduplication continues wellpast 24 hr in the cycle arrested cells,with a major exception being cells thatnormally stop cycling early, e.g., cellsthat differentiate as specific neural ormesodermal cell types.

RESULTS

Identification of New

Alleles of harpy

The mutant harpy was initially iden-tified as an early arrest phenotype inthe Tubingen zebrafish screen for

early morphogenetic mutants, repre-sented by a single recessive alleleti245 (Kane et al., 1996). The earliestvisible phenotype seen using a dis-secting scope was an abnormallybumpy head (Fig. 1A,B) accompaniedby a shortened body axis, easily seenby comparing the distance betweenthe nose and tail bud around the ven-tral aspect of the embryo. As develop-ment proceeds, mutant embryos wereslightly smaller than normal at 24 hrand show little or no growth there-after (Fig. 1C). In contrast to theother early arrest mutants, cell deathand necrosis was not apparent before24 hr (Kane et al., 1996), althoughafterward low numbers of cellsappeared to be lysing throughout theembryo, especially in the centralnervous system (CNS). (Evidence oflow level cellular lysis based onpyknotic nuclei can be seen in Fig. 2Hand throughout Fig. 7.)In a screen for neural patterning

mutants, one of us (B.B.R.) recovereda recessive lethal mutation, termedx1, which causes a phenotype inwhich neurons were reduced in num-ber but increased in size (Fig. 1D,E).Further investigation of 40,6-diami-dine-2-phenylidole-dihydrochloride(DAPI) -stained embryos showed sim-ilar changes in cell number and sizethroughout the embryo (Fig. 1F,G),and DAPI staining of dissociated cellsrevealed that cell nuclei were greatlyenlarged in cells from mutantembryos (Fig. 1F0,G0). The early mor-phological defects of x1 mutantsresembled those of hrpti245 and com-plementation testing confirmed themutations were allelic. The new alleleis henceforth designated as hrpx1.

harpy Is a Mutation in

Rca1/emi1

The altered number and size of cells inharpy mutants suggested a defect inthe cell division cycle. The nucleishown in Figure 1G0 are more thantwice the diameter of wild-type cells,suggesting that they might be at 8N orgreater ploidy. In support, staininghrpx1 mutants for phospho-histone H3revealed a gradual cessation of mitosisjust after the onset of gastrulation.Few if any mitotic cells were detectedat any stage after 8 hr (Fig. 2A–F).

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Identical results were seen for thehrpti245 allele (not shown).The mutation tiy121 of Zhang et al.

(2008) that disrupts the mitotic regu-lator Rca1/emi1 produces a pheno-type that closely resembles that ofhrp. We confirmed that harpy mapswithin 1 cM of the map position ofRca1/emi1 (data not shown). Onsequencing the alleles (Fig. 1H), bothcontained nonsense mutations thattruncate the Rca1/emi1 protein mid-way through the transcript: hrpti245

has a stop codon within a putative nu-clear localization signal and hrpx1 hasa stop codon within the F-box domain,as well as two other changes in thecoding region that introduce aminoacid substitutions in the N-terminalhalf of the protein. (The tiy121 allelecontains a premature stop codon thattruncates the protein before the F-box

Fig. 1.

Fig. 2.

Fig. 1. General phenotype of harpy mutants.A,B: Live wild-type (WT) and mutant embryosat the 10 somite stage. Double sided arrowsindicate the distinctive nose–tail bud separa-tion in the mutants. C: Side views of live wild-type and mutant embryos at 24 hr. D,E: Anti-HuC (HU) stained neurons at 16 hr in wild-typeand mutant embryos. The trigeminal ganglia(tg) are indicated, and double sided arrowsindicate the width of the neural keel. Smallarrows indicate single cells; note the largersize of the mutant cells. F,G: The 40,6-dia-midine-2-phenylidole-dihydrochloride (DAPI)staining at 20 hr in wild-type and mutantembryos. F0 and G0 are whole embryo dissoci-ations. Note the cell in mitosis in the WT field;no cells are found in mitosis in the mutants.H: Rca1/emi1 coding region showing individualprotein domains and the locations of pointmutations (asterisks) found in hrpti245 and hrpx1

mutants. Scale bar ¼ 50 mm in D,E, 5 mm inF0,G0.

Fig. 2. Cycle characterization of cells inharpy mutants. A–F: Anti-phospho-histone H3(pH3) staining in wild-type and harpy mutantembryos from 6 through 24 hr showed thattypically by 8 hr no cells are in mitosis. Onlyoccasional cells were pH3 positive at 24 hr(F). G: Mean number of cells per embryo,based on cell counts. Cells were counted afterdissociating individual embryos with collage-nase. Each time point shows the meanand standard deviation from 10 embryos.H–N: Bromodeoxyuridine (BrdU) incorporationin wild-type and harpy mutant embryos. Hshows an harpy mutant injected at 14 hr andfixed 10 hr later. I–N shows embryos injectedand fixed 45 min later. Note that at 30 and 42hr many cells were positive in mutants; how-ever, at 48 hr, mutants were mostly negativeexcept for a small focus of BrdU incorporationin the tail. Scale bar ¼ 100 mm in A–F, 85 mmin H,J,K,M,N, 200 mm in I,L.

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domain. Hereafter, we refer to thetiy121 allele as hrptiy121). Truncationof the C-terminus, which includes theZn-binding region and other domainsthat are indispensable for Rca1/emi1activity (Reimann et al., 2001a; Milleret al., 2006), argues that all threeharpy alleles are likely to be amorphicfor Rca1/emi1 function. There is afourth allele of hrphi2648 that has beenidentified in the insertional screen inNancy Hopkin’s Laboratory at MIT(Amsterdam et al., 2004; Wielletteet al., 2004); this allele is caused byan insertion in the first intron andbehaves as a hypomorph (Zhanget al., 2008; Rhodes et al., 2009).

The Cell Cycle in harpy

Mutants Is Blocked

During Gastrulation at

Approximately Division 14

We performed several experiments tobetter characterize the timing and di-vision characteristics of the cell cycledefect in harpy mutants. To confirmwhether cell division is blocked,embryos were dissociated with collage-nase at various stages of developmentand the number of cells was counteddirectly using a hemocytometer (Fig.2G). Wild-type embryos showed asteady increase from 4,600 6 1,100cells at 6 hr to 82,6006 11,800 cells at24 hr. In contrast, hrpx1 mutantsshowed little change in cell numberduring this period, still comprisingonly 6,4006 2,000 cells at 24 hr. Thesedata are consistent with the phospho-histone H3 staining and confirm thatcell division ceases soon after 6 hr inharpymutants.

It was previously reported thathrptiy121 mutants continue to synthe-size DNA in endoreduplication cyclesthrough at least 14 hr (Zhang et al.,2008). We examined BrdU incorpora-tion in hrpx1 mutants at later stages todetermine the persistence of thesecycles. Despite the lack of cell division,many cells continued to synthesizeDNA throughout the segmentation pe-riod, shown by BrdU incorporationover a 10-hr period (Fig. 2H). Toexplore the duration of endoreduplica-tion, we used shorter pulses (45 min)at times up until 48 hr. We found thatthe intensity of label in mutants wasslightly lower than wild-type embryos

at 30 hr (Fig. 2I,L). Afterward, at 42hr, the apparent number of cells la-beled decreased (Fig. 2J,M), and at 48hr, the only region of substantial label-ing was in the tail (Fig. 2K,N). Thus,endoreduplication continued well intoday 2 of development, but eventuallytended to stop in all but a few cells ofthe embryo. It is interesting to notethat for all embryos, both wild-typeand mutant, individual cells are eitherlabeled intensely or not at all, suggest-ing that the cell cycle dynamics in themutant might be similar to that inwild-type embryos, i.e., cells in theharpy mutants may contain a distinc-tive S-phase. These results are consist-ent with the results by Rhodes et al.(2009) using flow cytometry to docu-ment the appearance of 4 N andgreater nuclei during the segmenta-tion stages.

To precisely assess the cycle that isblocked in harpy mutants, we labeledindividual cells in mutant and wild-type embryos in divisions immediatelyafter the Midblastula Transition, not-ing the cell cycle when the cell waslabeled. We later counted the numberof labeled cells at the beginning of gas-trulation (6 hr) and the next day (atapproximately 30 hr). In these experi-ments, we labeled a single exteriorblastomere with lineage tracer andfollowed the progeny over the nextday of development. Cells at the sur-face of the blastula typically alternatebetween anticlinal and periclinal divi-sions; hence, after two divisions thereare typically two enveloping layer cellsand two deep cells. Because of the fatedifferences in enveloping layer cellsand deep cells, we counted each celltype separately, and from thosecounts, using the starting clone cellcycle and the log to the base 2, wederived the average cycle number thatwas attained by each enveloping layeror deep cell clone. These experimentsare summarized in Figure 3 and Ta-ble 1. In normal embryos, deep cellsadvance to cycle 14 by the beginning ofgastrulation and division 14 (into cycle15) tends to occur during early gastru-lation (Kane et al., 1992; Kane andKimmel, 1993; Kimmel et al., 1994).On the other hand, enveloping layercells typically arrest or slow their divi-sions at the beginning of epiboly: thistemporary cycle arrest occurs in cycle13 or, somewhat less frequently, in

cycle 14. Within experimental error,these results were recapitulated in ourstudies. In both mutant and wild-typeembryos, deep cells reached mid cycle14 and enveloping layer cells only justreached cycle 14 at the beginning ofgastrulation (Table 1; 6-hr time point).Cell counts the next day revealed

nearly a �10 difference between thenumbers of cells in clones in mutantand those in wild-type embryos.Clones in wild-type embryos under-went 2 to 3 rounds of division betweenthe beginning of gastrulation and 30hr, reaching mid cycle 16 for EVLclones and mid cycle 17 for deepclones. Our results at 30 hr are con-sistent with a normal value of 50 to100 thousand cells in the 1-dayembryo. In the same time period, EVLclones in mutant embryos did notdivide at all and deep cell clones ei-ther did not divide or underwent 1additional round of division. Theseresults predict that there are approxi-mately 10 thousand cells in the mu-tant embryo at the time of arrest.For many of the above experiments,

we also time-lapse recorded severalembryos (N ¼ 12) from the blastulastage until after the end of gastrula-tion tail bud stage and reconstructedpartial lineages (Fig. 3). Two pairs ofwild-type and mutant lineages areshown, matched as ventral and dorsalexamples. The wild-type exampleswere typical of this type of analysis:in the deep lineages there are manyearly divisions with major tissuerestrictions occurring in the gastrulastage; in the enveloping layer line-ages, the division rates tended to slowor arrest during epiboly. After gastru-lation—and after recording—both lin-eages went through several divisions;we show the 24–30 hr fates and theirnumbers present on the next day. Inthe mutants, cell divisions occurrednormally before gastrulation andthen arrested just after the onset ofgastrulation; when we checked theembryos the next day, no more divi-sions were apparent.

Global Patterning Was

Normal in harpy Mutants but

Local Patterning Was Not

In terms of the cell types producedby the above lineages, the lineage

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TABLE 1. Average Number of Cycles Completed in EVL and Deep Clonesa

6 Hours 30 Hours

EVL Deep EVL Deep

Cycle inj. n no. of cells Calc. cycles No. of cells Calc. cycles No. of cells Calc. cycles No. of cells Calc. Cycles

harpy 10 4 6.3 13.6 9.3 14.0 6.3 13.6 9.3 14.011 4 4.0 13.9 7.5 14.9 4.0 13.9 11.0 15.312 5 2.2 14.1 4.0 15.0 2.2 14.1 9.8 16.2All 13.9 14.6 13.9 15.1

WT 10 4 8.5 13.9 8.8 14.0 55.5 16.8 62.0 16.911 7 4.0 14.2 8.9 15.0 16.4 16.1 71.3 17.412 7 2.7 14.1 2.1 14.5 16.4 16.7 16.3 17.6All 14.1 14.6 16.4 17.3

aEVL, enveloping layer; Deep, deep cell clone; WT, wild-type.

Fig. 3. harpy mutant cells stop dividing at cell cycle 14. A–D: Dorsally derived fates (A,B), and ventrally derived fates (C,D). In each embryo, anindividual blastomere was labeled at cycle 11 or 12 and followed by time-lapse video microscopy through early segmentation. Each is restrictedto a deep cell lineage (d) or an EVL (e) lineage at the cycle after labeling. The reconstructed cell lineage is displayed by cell cycle. Twenty-fourhour histotypes and cell counts for the clones are shown at the bottom of the lineage tree, and left side views of the 24 hr embryos are shownbelow. E–I: Comparison of different cell types at 24 hr. Whereas notochord cells (E) were normally sized in the mutants, neurons (F) and musclecells (G) were larger, and blood cells (H) and periderm cells (I) were supersized. In our lineage studies we never saw axons sprouted from pre-sumptive neurons in mutants. Wild-type cells, green; harpy mutant cells, red. edc, endothelial cell; hgl, hatching gland; mus, muscle; nc, noto-chord; pdm, periderm; rbc, red blood cell. Scale bar ¼ 100 mm in E–I.

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relationships in harpy mutant em-bryos are simple versions of develop-ment in wild-type embryos. Notochordlineages are normally related to mus-cle lineages and blood lineages are nor-mally related to endothelial lineages,and, respectively, such examples are

shown in Figure 3A,B and Figure3C,D.

At 30 hr, the labeled cells in theclones gave an agnostic glimpse intothe morphology of individual cells inthe mutant embryos. Cells that nor-mally have their terminal mitosis in

the gastrula tended to appear nor-mally sized in the mutants. For exam-ple, notochord cells (Fig. 3E) seemedto be completely normal (although thesheath cells surrounding them werelarger than normal, data not shown).Neurons (Fig. 3F) and muscles (Fig.3G) were slightly larger than normal;however, neurons tended not tosprout axons, and muscle cells wereoften dumpy and did not alwaysspan an entire somite. On the otherhand, cells that normally divide manytimes in the embryo, such as blood(Fig. 3H) and periderm (Fig. 3I),became supersized versions of theirwild-type counterparts. In the case ofthe blood cells, these cells wereactually bigger than the blastomeresthat produced them, suggesting thatsome process—we suggest endoredu-plication—might be occurring inthese particular cells that is causingabnormal cell sizing.Many aspects of early development

are relatively normal in harpymutants: defects do not appear duringcleavage, early epiboly, nor the begin-ning of gastrulation. harpy mutantscan first be identified morphologically

Fig. 4.

Fig. 4. Neural patterning defects in harpymutants. Panels show wild-type referenceembryos and harpy mutants. A–D: Expressionof pax2a at 24 hr. Note that the diminution ofexpression in each domain is the result notof lowered expression in individual cells butof lower total numbers of positive cells.A,C: Whole-mount side views; insets showdorsal views of head-trunk region. B,D: Highresolution dorsal views of hindbrain. E–K:Expression of islet1 at 24 hr. E,H: Whole-mount side views; insets show dorsal views ofhead region. The midline of the central nerv-ous system (CNS; black line) is indicated nearthe epiphysis. F,G,I–K: Transverse sections atlevels indicated in whole-mounts. Note lackingprimary motor neurons, normally a bilaterallypaired structure. L–O: Expression of foxa2(axial) at 24 hr. L,N: Show a whole-mount sideview; insets show dorsal views of head region.M,O: Show high resolution side views oftrunk. Note the gaps between the large cellsof the mutant floor plate and hypochord. bmn,branchio-motor nuclei; dc, diencephalon; ep,epiphysis; ed, endoderm; fp, floor plate; hbn,hindbrain neurons; hc, hypochord; mhb, mid-brain–hindbrain border; nc, notochord; os,optic stalk; ov, otic vesicle; pcr, pancreas; pp,pharyngeal pouch; pmn, primary motor neu-rons; prn pronephric neck; prt pronephrictubule; prd, pronephric duct; px, pharynx; rb,Rohon-Beard sensory neuron; scn, spinalcord neurons; tc, telencephalon; vb, ventralbrain. Scale bar ¼ 100 mm in A,C,E,H,L,N, 40mm in B,D,F,G,I,J,K,M,O.

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at 14 hr when they exhibit shortenedAP axes (Fig. 1A,B). This appears toreflect slowing of convergent extension(Fig. 1D,E) as cells begin to increase insize. Neurogenin staining revealedthat many neural progenitors are al-ready enlarged at 10 hr and the neuralplate is roughly 30% wider than nor-mal (data not shown); anti-HuC stain-ing showed that it still appeared widerthan normal at 16 hr (Fig. 1D,E). Theneural tube approaches normal widthby 24 hr, but in the head region theneural tube becomes thinner than thewild-type especially in the anteriorbrain regions.

To provide a global view of specifica-tion in the mutant, we examined themRNA expression patterns of pax2a,islet1, and foxa2, genes which areexpressed in many different tissues ofthe embryo (Fig. 4).

pax2a mRNA is expressed in cellsin the midbrain–hindbrain border,reticulospinal and spinal interneur-ons, otic placode, and pronephros(Krauss et al., 1991a). In general, all ofthese cell types were produced at thecorrect times and locations in harpymutants (Fig. 4A–D). As expected,based on the division arrest pheno-type, there were fewer cells presentand they were larger than normal;however, there was a distinct impres-sion that there was less tissue express-ing pax2a. This was best seen in theinterneurons of the spinal cord, wheremany regions in the mutant weredevoid of pax2a-expressing cells, andalso in the hindbrain, which normallyhas almost a continuous layer ofexpression in the reticulospinal layers(Fig. 4B,D). There were numerousother defects, notably, the otic vesiclewas smaller (discussed below) andexpression in the proximal portion ofthe pronephric tubule was absent.

islet1 is expressed in cells of theneurectoderm, in the epiphysis, innuclei of the telencephalon and thediencephalon, and in branchiomotorneurons, primary motor neurons andRohan-Beard sensory cells (Korzhet al., 1993; Inoue et al., 1994). Inharpy mutants (Fig. 4E–K) the gen-eral pattern of expression was nor-mal, and, with the caveat mentionedabove due to the lack of cell division,tissue expression was not as dimin-ished as in the case of pax2a expres-sion. A nice example of normal

expression was that of the branchio-motor nuclei overlying the pharynx,which appeared almost normal in themutant (Fig. 4H inset). The majorexception to this general observationwas the complete absence of islet1positive nuclei in the telencephalonand diencephalon of the forebrain,and the almost complete loss of theepiphysis (Fig. 4E,H). Patterning atthe cellular level was also abnormalin many regions. Typically, Rohon-Beard cells and primary motor neu-rons are reiterated paired structuresof the spinal cord (Fig. 4F,G); in themutant there were many missingcells (Fig. 4I–K) often represented byone very large cell on only one side ofthe spinal cord in tissue sections.

islet1 is also expressed in the endo-dermally derived portions of the phar-ynx and the pancreas. These tissuesare dramatically reduced. The endo-dermal portion of the pharynx is thelight expression underneath and cau-dalward of the branchio-motor nuclei,seen in the inset in Figure 4E; thiswas reduced in the mutant. islet1 isexpressed in the endocrine portion ofthe pancreas; this expression wasalso reduced in the mutant (Fig.4F,I). Similar to expression in thepancreas, both of these tissues areamong the earliest tissues to prolifer-ate in the developing embryo; in theabsence of growth, only founder cellswould be present and one wouldexpect the region of expression toappear smaller but not necessarilyweaker, and that is what weobserved.

foxa2 (also called axial) is expressedin the ventral CNS of the forebrain,the floor plate neurons of the spinalcord, in the mesodermally derivedhypochord, and in the endoderm(Strahle et al., 1993; Odenthal andNusslein-Volhard, 1998). Normallythe floor plate and hypochord arearranged as rows of single cells, asseen in Figure 4M. In the mutant, thetwo tissues were patterned normallybut cellular organization was con-strained by the reduction in cell num-ber: both tissues formed a single row ofoversized cells which exhibited largegaps (Fig. 4O). On the other hand, theventral forebrain (Fig. 4L,N) and themutant pharynx (Fig. 4N inset)showed defects in line with the lack ofgrowth in both of these tissues.

Neural Specification

We focused on perturbations in thenervous system using the mRNA ex-pression patterns of deltaA, notch1B,and fgf8, and protein expression pat-terns of Pax7, 3A10, Grasp, and ace-tylated tubulin (Fig. 5).

Notch and Delta control neuronal

cell fate and proliferation in the verte-

brate nervous system much like in

Drosophila. Notch expression is gen-

erally associated with proliferative

zones of undifferentiated cells; these

cells are thought to consist primarily

of neural stem cells (Bierkamp and

Campos-Ortega, 1993). Delta expres-

sion is found in scattered cells within

proliferative zones; these cells have

become post mitotic and are differen-

tiating into neurons (Appel and Eisen,

1998; Haddon et al., 1998).

We found that the expression pat-tern of deltaA was mostly normal inthe CNS of harpy mutants (Fig.5A,B), although the number of posi-tive cells was reduced. The differencesthat were observed seemed caused bythe large cell size and the reducednumber of cells in the mutant CNS.However, notch1b expression wasseverely reduced throughout the CNSof the mutants (Fig. 5C,D), suggestingthat neural stem cell populationsdiminished in the mutant.fgf8 is expressed normally in the

neural plate during gastrulation (notshown) and continues to be expressedin the midbrain–hindbrain borderthrough 24 hr (Reifers et al., 1998), asseen in Figure 5E. However, harpymutants also showed an ectopic stripeof fgf8 expression in rhombomere 4 ofthe hindbrain (Fig. 5F). This repre-sents an expression domain that isnormally lost in wild-type embryos by14 hr (Kwak et al., 2002), but whichwas aberrantly retained in harpymutants.anti-Pax7 marks a population of

neural crest cells that migrate exten-sively and are often seen migratingover the surface of the eye and intolateral regions where cranial placodesand pharyngeal arches form. Thesecells were produced in harpy mutants,as shown by the presence of migratoryPax7-expressing cells in the head andanterior trunk (Fig. 5G,H); however,they were never observed beyond the

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lateral edges of the brain, suggestingthat migration is impaired.

Pathfinding

Axon pathfinding relies on stereotypedmolecular cues provided by other cells.We asked if axonal pathfinding wasperturbed in harpy mutants, becausecues are likely to be missing due toearly cessation of cell division.

3A10 antibody marks Mauthnercells (Metcalfe et al., 1990); these largeneurons normally extend axons acrossthe midline to join the contralateralmedial longitudinal fascicle. Mauth-ner cells always formed in harpymutants. However, in approximately athird of the mutants, Mauthner cellsfailed to extend axons or showedhighly aberrant axon trajectories (Fig.

5I,J), in line with the almost completelack of axons in lesser neurons.

Grasp-positive commissural neu-rons in the hindbrain normally decus-sate near rhombomere boundaries toform a regular array of commissures(Trevarrow et al., 1990), but in harpymutants commissural axons formedhighly chaotic patterns (Fig. 5K,L).

Acetylated tubulin antibody la-bels mature axons, and is stronglyexpressed in axons of the 24 hr embryo(Fig. 5M). In harpy mutants, althoughneurons with axons are present, theyonly expressed acetylated tubulin inthe tail (Fig 5N) or not at all.

Placodal Specification

In Figure 6, we focused on placodalstructures and other structures that

interact with the epidermis, using themRNA expression patterns of dlx3b,pax2a, fgf8, and ncad. Most of thesestructures are induced and their mor-phogenesis requires many cellularinteractions, which could be com-prised by the abnormally large size ofthe mutant cells.dlx3b mRNA is normally expressed

in the nasal, lens, and otic placodes(Akimenko et al., 1994), shown in Fig-ure 6A. In the harpy mutant, dlx3bwas expressed in far fewer cells (Fig.6B), indicating that at 24 hr some pla-codal structures do not contain the cor-rect cell number or that some cells arenot yet completely specified. The lensforms in harpy mutants and gives riseto a small but relatively normal lens,but failure of the eye-cup to expandproperly leads to extrusion of the lensby 24 hr (Fig. 6C,D). The otic vesicleremains small and usually producesonly one or two hair cells with a singleotolith (Fig. 6E,F). Reliable supportcell markers are not available inzebrafish, but the macula remainsessentially a single layer rather thanstratifying into apical hair cell- andbasal support cell-layers. Additionally,the macula shows little or no expres-sion of fgf8 (Fig. 6G,H), which nor-mally marks both hair cells and sup-port cells of the developing maculae(Leger and Brand, 2002; Millimakiet al., 2007).pax2a mRNA expression appeared

in fewer cells in the mutant otic vesi-cle at 24 hr (Fig. 6I,J), consistent withits morphology and dlx3b expressionat that time. Nevertheless, early de-velopment of the otic placode was sur-prisingly normal in harpy mutants, asshown by Pax2-staining at 12 and 14hr (Fig. 6K–N). Moreover, despite theabsence of cell division, the number ofPax2-positive cells nearly doubledbetween 12 and 14 hr in mutants(19.2 6 3.8 cells at 12 hr, n ¼ 6; 37.26 4.4 cells at 14 hr, n ¼ 8), which issimilar to the fold-increase seen inwild-type embryos (82.9 6 7.9 at 12hr, n ¼ 8; 130.2 6 7.7 at 14 hr, n ¼ 5)This suggests that early expansion ofthe otic territory occurs by ongoingotic induction or recruitment, not celldivision.Lastly, ncad mRNA is expressed in

many regions of the embryo; here weuse it to visualize the lateral line

Fig. 5. Neural patterning defects in harpy mutants. Panels show wild-type control embryos andharpy mutants. A,B: Expression of deltaA (dlA) at 24 hr. Dorsal view of hindbrain. Note the nor-mal intensity of expression in mutant cells. C,D: Expression of notch1b at 24 hr. Dorsal view ofhead; insets show lateral view. Note expression in mutants is almost absent. E,F: Expression offgf8 at 24 hr. Dorsal view of hindbrain. Note ectopic staining in the fourth rhombomere of themutant. G,H: Anti-Pax7 staining at 24 hr. Arrows indicate neural crest cells migrating overthe eye and pharyngeal arches; while these cells are present in the mutant, they are not seen onthe migration pathway. I,J: Dorsal view of 3A10-stained Mauthner neurons at 30 hr, displayingabnormal pathfinding and absence of axons in the mutants. K,L: Anti-Grasp staining at 30 hr;dorsal view of the hindbrain. Arrows indicate commissural neurons that in the mutant are cha-otic. M,N: Anti-acetylated tubulin (AT) staining at 24 hr; dorsal view of the trunk. Although axonsare present in mutants, they do not express acetylated tubulin except in the tail region. mhb,midbrain–hindbrain border; mth, Mauthner neurons; ov, otic vesicle; r4, fourth rhombomere.Scale bar ¼ 50 mm in A–D, 20 mm in F,G, 100 mm in E–N.

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primordia. This group of cells movesdown the lateral sides of the embryoand leaves small rosettes of cells thatdevelop in the sensory structures ofthe lateral line (Fig. 6O). In the mu-

tant, this structure was small and of-ten difficult to find; when we did findit (Fig. 6P), it was hidden in variouslocations, somewhat randomly, on thesides of the embryo or the yolk sac.

Relationship Between

Endoreduplication and

Differentiation

During normal development, cell divi-sions become asynchronous andslower as cells begin to differentiate(Kane et al., 1992; Kane and Kimmel,1993; Kimmel et al., 1994; Zamir et al.,1997). We hypothesized that the slow-ing of endoreduplication might berelated to the initiation of cytodifferen-tiation that occurs beginning in thesegmentation stages of development.This must occur in only a fraction ofthe total number of cells because mostcells at these stages continue to showS-phase in both mutant and wild-typeembryos (Fig. 7A,B). To test this, weexamined patterns of BrdU incorpora-tion in embryos stained with markersof various specified and differentiatedcell types. We used three antibodiesknown to label early neuronal andmesodermal cell types: Anti-Islet1/2antibody stains Rohon-Beard sensoryneurons, primary motoneurons andtrigeminal ganglion neurons by 10 hr(Inoue et al., 1994). Anti-Engrailedantibody labels the midbrain–hind-brain border and muscle pioneer cells(Hatta et al., 1991). Anti-Pax2 anti-body, which primarily binds Pax2aprotein (Riley et al., 1999), labels cellsin the midbrain–hindbrain border, oticplacode, pronephros, reticulospinalneurons in the hindbrain, and CoSAspinal interneurons (Krauss et al.,1991b; Mikkola et al., 1992). 3A10antibody labels Mauthner neurons(Metcalfe et al., 1990).To assess whether DNA synthesis

occurred in these cells during the seg-mentation stages, BrdU was injectedat 12 hr and embryos were fixed andco-stained at 19 hr or later for BrdUand the individual cell type markers.Many of the specific cell typesexpressing the above markers did notshow BrdU incorporation (Table 2;Fig. 7D–I), though BrdU was detectedbroadly in surrounding cells. In par-ticular, BrdU incorporation was notseen in muscle pioneers, Rohon-Beardsensory neurons, primary motoneur-ons, trigeminal cranial ganglia,Mauthner, reticulospinal, and spinalinterneurons, all cell types that areknown to have early terminal mitoses(Mendelson, 1986; Myers et al., 1986;Mikkola et al., 1992; Hirsinger et al.,

Fig. 6. Placodal patterning defects in harpy mutants. Panels show wild-type referenceembryos and harpy mutants. A,B: Expression of dlx3b at 24 hr, showing reduced numbers ofpositive-expressing cells in the mutant. C,D: Optic vesicles in live embryos at 26 hr, showingextruded lens in the mutant. E,F: Otic vesicle in live embryos at 26 hr. Arrows indicate hair cells.Note the absence of layers and miniaturized structures in the mutant. G,H: Expression (arrows)of fgf8 at 24 hr in the otic vesicle. Note that expression is almost absent in the mutant vesicle(demarcated by oval). I–N: Expression of pax2a in the otic tissue. I,J: pax2a mRNA at 24 hr inthe otic vesicle. Note that expression reduced in the mutant vesicle (demarcated by oval).K–N: Staining with anti-PAX2 in the otic placodes, showing expansion of the PAX2 territorybetween 12 and 14 hr in the mutant. O,P: Expression of N-cadherin (ncad) at 24 hr, showing arosette of the lateral line (arrow head) and the lateral line primordia (arrow). In many mutants,the primordia could not be found. ls, lens; hc, hair cells; np, nose placode; oc, otocyst; op, oticplacode; ov, otic vesicle; px, pharynx. Scale bar ¼ 100 mm in A,B, 50 mm in C,D, 10 mm in E,F,20 mm in G–P.

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2004; Won et al., 2006; Caron et al.,2008; Rossi et al., 2009).

In contrast, BrdU was detected in asmall fraction of Pax2-staining cellsin the developing pronephric duct,within the otic vesicle and in the mid-brain–hindbrain border (Table 2; Fig.7C and data not shown). Each of thesedomains generate multiple diversecell types many of which do not differ-entiate until later. Together, thesefindings are consistent with the hy-pothesis that the process of endoredu-plication does not continue after cellshave begun terminal differentiation,but may still occur during earlierstages of organogenesis.

harpy Acts Both Cell-

Autonomously and Cell

Non-autonomously

To ask if the division characteristicsand the cell morphology of the harpymutant were cell autonomous, wetransplanted mixtures of wild-typeand mutant cells into wild-type ormutant host embryos, as describedpreviously (Ho and Kane, 1990). Inbrief, cells from two donors, one la-beled with rhodamine–dextran andone labeled with fluorescein–dextran,were transplanted into one host;donors were genotyped based on their24 hr phenotypes. We recorded thephenotypes of the individual donorcells as well as the number of cellsthat were produced by cell division. Ingeneral, we found that most of the di-vision and phenotypic characteristicsthat we identified in our lineage workabove were autonomous to the geno-type of the transplanted cells. Figure8 shows examples of transplanted

wild-type and mutant cells placed ineither wild-type or mutant hosts.Notably, harpy mutant cells thatbecame neurons were larger thantheir wild-type counterparts, regard-less of the genotype of the host. Mus-cle cells, as noted above, were almostnormal in size. However we did notesome nonautonomous effects: Nor-mally, harpy mutant neurons tend notto sprout axons. We found that wild-type cells placed in the CNS of harpymutants also tended not to sproutaxons (Fig. 8B,D), whereas harpycells in wild-type CNS sometimes did(Fig. 8C). We interpret this to meanthat some developmental environ-ments in the mutant are not normal.This is an interesting result becauseRca1 (in Drosophila) might havesome role in axogenesis (Dong et al.,1997). At least in vertebrates, thismay be a minor effect compared withnonautonomous effects in the embry-onic CNS.

Normally, cells that enter the neu-ral keel have a tendency to divide atthe midline with the daughter sibs of-ten moving to opposite sides of theforming neural tube. We noted thatwhen mutant cells were transplantedinto the CNS of wild-type or mutantembryos, the mutant cells tended tostay on one side of the neural keel(Fig. 8E,F) even when their wild-typeco-transplanted cells moved to bothsides.

We also counted the number of do-nor cells at 30 hr in our host embryos.For each chimera, 2 to 3 cells from twoseparate individuals were placed intoa host embryo in the late blastula. Thecell cycle number at this time would beapproximately cycle 12 or 13. Immedi-

ately after the transplant, each chi-meric embryo was recorded. Then at 6hr, just before gastrulation, the num-ber of cells in the chimera was re-inspected and recorded; similarly at 30hr. Regardless of host genotype, harpymutant cells do not tend to proliferateafter 6 hr whereas wild-type cells do(Fig. 8G).To test whether the mitotic block in

harpy mutants is cell autonomous, weproduced mosaic embryos by trans-planting cells from biotin–dextran la-beled donor embryos to unlabeledhost embryos during blastula stage,fixed the embryos at 24 hr, and doublestained them with a horseradish per-oxidase reaction product to detect do-nor cells and anti-phospho histone H3to detect mitotic cells. When we trans-planted cells from a mutant into awild-type embryo, we found that mu-tant cells never expressed phospho-histone H3, indicating that they donot enter mitosis (Fig. 8H). Labeledwild-type controls divided normally,even when transplanted into a mu-tant host (Fig. 8I). These data confirmthat disruption of harpy functionblocks mitosis cell autonomously.

DISCUSSION

We have shown that harpy and Rca1/emi1 mutations are allelic and havefurther characterized the cell cycledefect in the mutant as well as its de-velopmental consequences. Mutantsshow a global block to mitosis begin-ning early in gastrulation, after whichsome cells continue to grow in sizeas they undergo endoreduplication.Despite this cell cycle defect, mostaspects of global patterning occur

TABLE 2. BrdU Incorporation in Cells of harpy Mutants Expressing Markers of Specification and Differentiation

Marker Cell type No. cells BrdU positive No. cells counted No. embryos

Engrailed Muscle pioneers 0 124 7Islet1/2 Rohon-Beard sensory neurons 0 412 29

Primary motoneurons 0 408 29Trigeminal ganglion 0 243 29

Pax2 Reticulospinal primary neurons 0 94 26Spinal interneurons 0 272 22Otic Vesicle 26 451 11Pronephros 14 293 11Midbrain-Hindbrain Border 24 290 9

aBrdU, bromodeoxyuridine.

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relatively normally, though there arealso numerous local defects that arelikely attributable to loss of prolifera-tion or abnormal cell size.

The relatively normal global pat-terning seen in harpymutants is remi-niscent of previous studies in Xenopusshowing that early patterning and cell

type differentiation are surprisinglynormal in embryos blocked in DNAsynthesis by exposure to hydroxyureaand aphidocolin (Harris and Harten-stein, 1991; Rollins and Andrews,1991). In earlier studies on the analy-sis of early blood specification, we havedocumented that in harpymutants dif-ferentiation occurs on time for threemajor early blood histotypes although

Fig. 8.

Fig. 7.

Fig. 7. Patterns of bromodeoxyuridine (BrdU)incorporation. A–I: Images show lateral viewswith anterior to the left (A,B,E,F,H) or dorsalviews with anterior to the top (C,I) or to theleft (D,G). A,B: Head region of a wild-typeembryo and harpy mutant incubated withBrdU from 18 to 24 hr. Note that the majorityof cells incorporate BrdU in both mutant andwild-type embryos. C–I: harpy mutantsinjected with BrdU at 12 hr and fixed later at19 hr. Note that in all the cases shown, cellsthat express the indicated marker of differen-tiation (black) never incorporate BrdU (green).C: Anti-Engrailed stained midbrain–hindbrainborder. D,E: Anti-Isl1/2 staining showingRohon-Beard sensory neurons and trigeminalganglia. F: Anti-Engrailed stained muscle pio-neers. G: Anti-Pax2 stained spinal interneur-ons. H: Anti-Pax2 stained pronephric cells. I:3A10 antibody-stained Mauthner neurons;these were fixed at 27 hr. mhb, midbrain–hindbrain border; mp, muscle pioneers; Mth,Mauthner neurons; prt, pronephros; sin, spinalinterneurons; rb, Rohon-Beard sensory neu-rons; tg, trigeminal ganglia. Scale bar ¼ 100mm in A,B, 75 mm in C, 30 mm in D–G,I, 15mm in H.

Fig. 8. harpy acts both cell autonomouslyand non-autonomously. A–F: Examples oftransplanted wild-type (green) and mutant(red) cells placed in either a wild-type(A,C,E,F) or mutant (B,D) host. A,B: Compos-ite images of a neural and muscle chimera. C:Ultraviolet (UV) image showing a magnifiedview of neural and muscle chimera in A. Insetshows single harpy interneuron which pro-jected an axon. D: magnified views of neuralchimera in B. Note that the wild-type cells didnot project axons. E,F: Facial view of telence-phalic region (E) and dorsal view of brain (F)neural clones. Note that wild-type cells fre-quently cross the midline; mutant cells tend tonot. G: Comparison of number of cells inclones. For each chimera, two to three cellsfrom two separate individuals were placedinto a host embryo at 5 hr, and recorded im-mediately afterward (txp), then at 6 hr andagain at 30 hr. H,I: Mosaic embryos doublestained with HRP to detect donor cells(brown/black) and anti-phospho histone H3 todetect mitotic cells (green). H,H0: Labeled mu-tant cells transferred to an wild-type host; mu-tant cells are not dividing. I,I0: Labeled wild-type cells transferred to an unlabeled mutanthost. A dividing wild-type cell is indicated(arrow). Scale bar ¼ 200 mm in A,B,E,F, 50mm in C,D, 100 mm in F,I.

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the numbers of cells are reduced(Warga et al., 2009). These findingsunderscore the remarkable regulativecapacity of vertebrate embryos andfurther indicate that cell fate is influ-enced primarily by cell–cell interac-tions rather than lineage-restrictedcell fate determinants.

The Basis for Local

Developmental Defects

Development with relatively few cellsof large size would likely impose signif-icant spatial and geometrical con-straints on the morphogenesis of somestructures. For example, formation ofthin elongate structures such as thefloor plate and hypochord requiresfiner precision than can be accommo-dated in harpy mutants, possiblyaccounting for the spatial gaps notedin these structures (Fig. 4O). Addition-ally, complex organ systems requiringassembly of large numbers of cells isoften compromised in harpy mutants,as seen in the severely dysmorphic oticvesicle (Fig. 6F) and extrusion of thelens from the eye (Fig. 6D). Reducednumbers of cells would also tend tolimit cell type diversity, thereby dis-rupting essential local signaling inter-actions. This could account for abnor-mal migration of neuronal growthcones and neural crest cells (Fig.5H,J,L). Indeed, mutant neuronsdeveloping within a wild-type host of-ten showed improved axonogenesis,reflecting some degree of rescue fromnon-autonomous functions within thewild-type environment (Fig. 8C).

Large size alone probably perturbscertain cellular process required forproper development. For example,increased cell size could adverselyaffect cell signaling by altering rates ofdiffusion of secreted factors or by ham-pering the efficiency of signal trans-duction due to reduced surface:volumeratio. In some cases, defects in migra-tion could also arise from mechanicalchanges in membrane/cytoskeletal dy-namics of enlarged cells. Thus, thedelay in convergence-extension move-ments seen in harpy mutants (Fig.1B,E) could reflect sluggish cell move-ment or weakened signaling requiredfor planar cell polarity.

The lack of cell division itself alsolikely perturbs some aspects of pat-

terning and morphogenesis. In wild-type embryos, for example, cell divi-sions are oriented nonrandomlyduring late gastrulation and may con-tribute somewhat to convergence andextension (Kane et al., 1992; Kaneand Kimmel, 1993; Kimmel et al.,1994; Concha and Adams, 1998); suchforces would be absent in the mutant.Cell division in the wild-type neuraltube often separates daughter cellsinto right and left hemisegmentswhere they enter equivalent cohortsof cells and often adopt similar fates(Kimmel et al., 1994; Geldmacher-Voss et al., 2003). Disruption of thisprocess in harpy mutants possiblyaccounts for the asymmetric deficien-cies in Rohon-Beard sensory neuronsand primary motoneurons (Fig. 4I–K).This phenomenon is not limited to pri-mary neural cell types, as there was astrong general tendency of mutantclones in wild-type hosts to remainisolated on one side of the neural tube(Fig. 8E,F). Development of manyorgan systems relies on maintaining apool of stem cells or cycling progeni-tors. In the nervous system, for exam-ple, ongoing division of progenitorsgenerates successive layers with dis-tinct cell fates. The loss of prolifera-tive centers in the brain of harpymutants is reflected by the near ab-sence of notch1b expression (Fig. 5D)and is likely responsible for a severedeficiency of many neural cell types(Figs. 4D, 5L,N). Similarly, the pan-creas normally undergoes dramaticmitotic expansion but in the mutant itremains very small (Fig. 4H).

The Relationship Between

Endoreduplication and

Differentiation

BrdU incorporation shows that inmutants endoreduplication continuesin a large fraction of cells past 24 hrbut gradually ceases by 48 hr or soonthereafter. A recent study by Rhodeset al. (2009) confirms that cells inharpy hypomorphic mutants also con-tinue to undergo eudoreduplication.Similar results have been obtainedwith cultured mammalian cells, inwhich depletion of Rca1/emi1 leadsto limited endoreduplication withaccumulation of greater than 4NDNA content (Di Fiore and Pines,

2007; Machida and Dutta, 2007).Endoreduplication eventually stops incultured cells due to accumulation ofDNA damage, which activates check-point kinases that halt further DNAsynthesis (Verschuren et al., 2007). Asimilar process of checkpoint-medi-ated senescence could account for ces-sation of DNA synthesis in at leastsome cells in harpy mutants. How-ever, the complex pattern of BrdUincorporation revealed dramatic vari-ation in the time when different cellscease endoreduplication, suggestingthat cells are not regulated in a uni-form manner and that some otherprocess influences endoreduplication.Indeed, our findings suggest that dif-ferentiation accounts for much of thisvariation, allowing some cells to with-draw early from endoreduplication.Specifically, analysis of early differen-tiating cell types in harpy mutantsshowed that these cells no longerincorporate BrdU after 12 hr (Fig. 7;Table 2), similar to their wild-typecounterparts. Conversely, cell typesthat proliferate extensively in wild-type embryos, such as blood cells andperiderm, grow to an especially largesize in harpymutants (Fig. 3H,I), sug-gesting they undergo additionalrounds of endoreduplication in lieu ofcell division. Other developing tissuesthat continued to show sporadicBrdU-incorporation include the pro-nephros, otic placodes, and the mid-brain–hindbrain border. In each ofthese cases, expression of pax2a wasused to mark early stages in the de-velopment of these organ primordiarather than formation of terminallydifferentiated cell types. Thus, escapefrom endoreduplication in harpy mu-tant cells correlates with terminal dif-ferentiation, but not earlier steps incell fate specification of lineagerestriction.The precise relationship between

differentiation and cell division is onlypartially understood. The maternal-zygotic transition in zebrafish beginsat the tenth division, after which cellcycles lengthen and become asynchro-nous as cells begin to take on differentfates (Kane et al., 1992; Kane andKimmel, 1993). Cells fated to becomeprimary neurons become postmitoticas early as cycle 16 whereas secondaryneurons are born in cycle 17 or later(Kimmel et al., 1994). Differentiating

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cells can modulate the rate of cell divi-sion by changing expression levels ofcell cycle inhibitors, such as Rb andCip1/Kip1/p21, which repress E2F-dependent expression of Cyclins(reviewed by Robinson et al., 2002; DeFalco et al., 2006; Bicknell et al.,2007; Besson et al., 2008). Thisincludes Cyclin E, which is requiredfor entry into S-phase as well asendoreduplication (Knoblich et al.,1994; Geng et al., 2003; Parisi et al.,2003). Hence, the process of differen-tiation could block endoreduplicationin harpymutants by p21/Rb-mediatedrepression of Cyclin E.

Consequences of Elevated

Anaphase Promoting

Complex/Cyclosome

(APC/C) Activity in

harpy Mutants

APC/C is an E3 ubiquitin ligase thatnormally targets mitotic Cyclins andGeminin for destruction, cyclical regu-lation of which is necessary for coordi-nating DNA synthesis and cell-cycleprogression (Reimann et al., 2001a;Miller et al., 2006; Di Fiore and Pines,2007; Machida and Dutta, 2007). Weinfer that APC/C remains constitu-tively active in harpy mutants, as hasbeen shown in Drosophila, Xenopus,and mammalian tissue culture afterdisruption of Rca1/emi1 (Reimannet al., 2001a; Grosskortenhaus andSprenger, 2002; Machida and Dutta,2007). This model accounts for the cell-autonomous block to mitosis in harpymutants (Fig. 8H) as well as ongoingendoreduplication. This model alsoaccounts for the high frequency of cellsthat escape the mitotic block in thehypomorphic allele of harpy in thestudy by Rhodes et al. (2009).

Constitutive APC/C activity couldexplain some of the developmentaldefects seen in these mutants. APC/Cis known to target regulatory proteinsrequired for axon growth and synap-tic function (Juo and Kaplan, 2004;Konishi et al., 2004; van Roesselet al., 2004; Stegmuller et al., 2006).Although some aspects of axonogene-sis in mutant neurons were rescuedby development within a wild-typehost (Fig. 8C), rescue was neither uni-form nor complete, indicating that atleast some cell-autonomous defects

contribute to axonal defects. APC/Calso targets Id-related helix–loop–he-lix (HLH) proteins, which normallyinhibit differentiation by antagoniz-ing neurogenic and myogenic basicHLH (bHLH) proteins (Lasorellaet al., 2006). While in principle rapidclearing of Id proteins in harpymutants could accelerate differentia-tion, we detected no instances of pre-cocious expression of cell typemarkers. On the other hand, we didobserve that an early pattern ofexpression of fgf8 in the hindbrain isabnormally retained through at least24 hr in harpy mutants (Fig. 5F), pos-sibly reflecting failure in the normalprogression of interactions betweendiverse cell types.

Our short pulse BrdU incorporationstudies (Fig. 2I–N) suggested thatendoreduplication is occurring inharpy mutants. What with the stronglabeling of a fraction of the cells withonly few intermediately labeled cells,cells seem to be either in S-phase ornot, much like S-phase in cells inwild-type embryos. Nevertheless, ourwork only partially clarifies the dy-namics of S-phase in the mutants:although we refer to multiple roundsof DNA synthesis as endoreduplica-tion, this term is often used to suggestthat cells undergo definite S-phasesseparated by a G-phase. In principle,we do not know if there is a G-phasebecause we have not measured the S-phases delimiting this hypotheticalG-phase. Second, when Rca1/emi1 isablated, the APC/C could target gemi-nin for degradation and it wouldbecome possible to allow re-initiationof DNA synthesis, termed re-replica-tion (Sivaprasad et al., 2007; Porter,2008; van Leuken et al., 2008).Because we see only slightly highernumbers of positive nuclei with longpulses, it is possible that a large sub-set of the cell population may be in re-replication. Clearly, further experi-ments will be needed to answer thesequestions.

EXPERIMENTAL

PROCEDURES

Fish Strains, Staging,

and Genotyping

Wild-type zebrafish strains were de-rived from the AB line (Eugene, OR) or

the tupLongfin line (Tubingen, Ger-many). Embryos were developed infish water containing methylene blueat 28.5�C and staged according tostandard protocols (Kimmel et al.,1995). hrpti245 was recovered in alarge-scale ENU-mutagenesis screen(Haffter et al., 1996). hrpx1 wasinduced by postmeiotic ENU-muta-genesis (Riley and Grunwald, 1995)and recovered in a small-scale screen.

Lineage Tracing and Time

Lapse Analysis

Cell labeling was adapted from proto-cols previously described in Wargaand Nusslein-Volhard (1999) and inWarga et al. (2009). Briefly, an indi-vidual blastomere was labeled at the1K-cell stages with a 5% solution ofrhodamine–dextran (10,000 MW;Invitrogen, formerly MolecularProbes). Immediately after labeling,embryos were mounted in a 3% solu-tion of methylcellulose (Sigma) andrecorded in multiplane as describedin Warga and Kane (2003). Generallybetween 8 and 10 embryos wererecorded simultaneously for up to 12hr. Afterward, the recordings wereanalyzed using Cytos Software, asdescribed in Kane et al. (2005) and inMcFarland et al. (2005).

Cell Transplantation

Transplantation was carried outbetween doming and 30% epibolystage as described in Ho and Kane(1990). In some of our experiments,we included the fixable tracer biotin-ylated-Dextran (Invitrogen). In suchexperiments, hosts were fixed in 4%paraformaldehyde, and the clone wasvisualized as described in Warga andKane (2007) with the following modi-fication: Diaminobenzidine (Sigma)was used as the enzyme substratefollowing protocols previously des-cribed in Warga and Nusslein-Volhard(1999).

Analysis of Gene Expression

Whole-mount in situ hybridizationwas carried out as described previ-ously in Thisse et al., 1993) andPhillips et al. (2006). Whole-mountimmunostaining was performed asdescribed in Riley et al. (1999) and

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Phillips et al. (2006) using antibodiespurchased from the DevelopmentalStudies Hybridoma Bank directedagainst Grasp (zn8,1:100), Islet1/2(39.4D5 1:100), Pax7 (1:150), 3A10(1:20), or anti-HuC (ZIRC, EugeneOR, 1:100), or commercial antibodiesagainst Pax2 (Covance, 1:100), phos-pho-histone H3 (Upstate 06-570,1:350), or acetylated tubulin (Sigma,1:2,00).

BrdU Incorporation

BrdU pulse labeling was performedas described by Phillips et al. (2006)using anti-BrdU (Beckton-Dickinson,1:250).

DAPI Staining

Embryos were fixed and stainedsimultaneously for 15–20 min in a 4%paraformaldehyde solution in phos-phate buffered saline containing0.01% DAPI and either photographedimmediately or disassociated bygently crushing between coverslipsbefore photographing.

Dissociation of Embryos

To obtain cell counts, dechorionatedembryos were incubated for 5–10 minat 37�C in modified Holtfretter’sbuffer (60 mM NaCl, 0.6 mM KCl, 0.9mM CaCl, 5 mM HEPES pH 7.4) con-taining collagenase (Sigma C9891) ata concentration of 20 mg/ml (embryosat 12 hr or older) or 7 mg/ml (embryosat 6 hr). Embryos were monitoredwhile gently triturating with a glasspipette until dissociation was com-plete. Dissociated cells were sus-pended in Hotfretter’s buffer andcounted with a hemocytometer.

ACKNOWLEDGMENTSWe thank Sumi Himangshu and AprilKraus for spirited help on the harpyproject through the years, and RobertHo, Yi-Lin Yan, Benjamin Geiger, Yun-Jin Jiang, and Sharon Amacher forprobes and advice. B.B.R. was fundedby a National Institutes of Healthgrant and D.A.K. received a PewFellowship.

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