PATTERNS & PHENOTYPES

Bone Growth in Zebrafish Fins Occurs viaMultiple Pulses of Cell ProliferationIsha Jain,1 Christine Stroka,1 Jianying Yan,2 Wei-Min Huang,2 and M. Kathryn Iovine1*

Fin length in the zebrafish is achieved by the distal addition of bony segments of the correct length. Geneticand molecular data provided evidence that segment growth uses a single pulse of growth, followed by aperiod of stasis. Examination of cell proliferation during segment growth was predicted to expose agraphical model consistent with a single burst of cell division (e.g., constant, parabolic, or exponentialdecay) during the lengthening of the distal-most segment. Cell proliferation was detected either by labelinganimals with bromodeoxyuridine (during S-phase) or monitoring histone3-phosphate (mitosis). Resultsfrom both methods revealed that the number of proliferating cells fluctuates in apparent pulses as asegment grows (i.e., during the growth phase). Thus, rather than segment size being the result of a singleburst of proliferation, it appears that segment growth is the result of several pulses of cell division thatoccur approximately every 60 microns (average segment length � 250 microns). These results indicatethat segment lengthening requires multiple pulses of cell proliferation. Developmental Dynamics 236:2668–2674, 2007. © 2007 Wiley-Liss, Inc.

Key words: ontogeny; saltations; bone growth; segment length; zebrafish

Accepted 22 June 2007

INTRODUCTION

The diversity of life forms is mostclearly manifested as differences inshape and structure. Variations inmorphology arise as organs and limbsgrow to different proportions with re-spect to each other and to the body.The final form is the result of thesedifferent growth rates. Understandingthe pattern of growth provides insightinto the underlying mechanisms of de-velopment. For example, the findingthat human bone growth occurs bysaltations (i.e., discrete pulses ofgrowth) rather than in a continuousprocess, suggests the existence of apreviously unknown hormonal syn-chronizing mechanism regulating

growth (Lampl et al., 1992). Such dis-coveries will lead to the identificationof the cellular and molecular mecha-nisms involved in size regulation,which remain largely unknown.

Short generation time, high fecun-dity, ease of maintenance, and a pre-dominantly sequenced genome (http://www.sanger.ac.uk/ ) make the zebrafisha popular model for evaluating underly-ing molecular pathways in vertebratesystems. More particular to the ques-tions of growth or size, the caudal fin iseasily measured and amputated for his-tochemical analyses. Furthermore, asthe fin is nonessential for viability (es-pecially in a laboratory setting) and hasa comparatively simple structure with

few cell types, it has been the focus ofmany studies (reviewed in Poss et al.,2003; Akimenko et al., 2003).

The caudal fin is bi-lobed and com-posed of 16–18 fin rays of varyinglength. There is a single shortest finray in the medial or central part of thefin, and symmetric lobes extendinglaterally in both dorsal and ventraldirections. Each fin ray grows by thedistal addition of bony segments (Gossand Stagg, 1957; Haas, 1962) addedsynchronously in the adult fin (Gold-smith et al., 2003, 2006). Fin rays arecomposed of two opposed concavehemirays of bone matrix, or lep-diotrichia, surrounding mesenchymalcells including undifferentiated fibro-

1Lehigh University, Department of Biological Sciences, Bethlehem, Pennsylvania2Lehigh University, Department of Mathematics, Bethlehem, PennsylvaniaGrant sponsor: the NIDCR; Grant number: DE014863; Grant sponsor: the NIH-NCRR; Grant number: P40 RR12546.*Correspondence to: M. Kathryn Iovine, Lehigh University, Department of Biological Sciences, 111 Research Drive, IacoccaB-217, Bethlehem, PA 18015. E-mail: mki3@lehigh.edu

DOI 10.1002/dvdy.21270Published online 5 August 2007 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 236:2668–2674, 2007

© 2007 Wiley-Liss, Inc.

blasts, nerves, and blood vessels (San-tamaria et al., 1992). Proliferatingcells in the distal mesenchymal com-partment contribute to outgrowth bycondensation along specialized collag-en-like fibers called actinotrichia, dif-ferentiation as bone-forming cells (os-teoblasts), and ultimately depositionof bone matrix by intramembranousossification (Landis and Geraudie,1990; Goldsmith et al., 2003).

Recent analyses revealed that onto-genetic fin growth is a saltatory pro-cess, and suggest that the bony seg-ment is the unit of growth. Thus, finlength is achieved by discrete pulsesof growth (when a segment is pro-duced) separated by phases of rest, orstasis. The occurrence of saltationswas revealed by the discovery that ananonymous growth marker fa93e10 (agene-based marker shown to correlatewith distal mesenchymal cell prolifer-ation) is expressed episodically andwith decreasing frequency as animalsgrow or age (Goldsmith et al., 2003).Indeed, 100% of young fins expressedfa93e10 (at the growing ends of all finrays), suggesting either that youngfins are always in a state of growth orthat rest phases are very short. Olderanimals, 24 weeks of age, expressedfa93e10 only 5% of the time (again,across all fin rays). Since fin growthoccurs by the synchronous addition ofnew segments, it has been suggestedthat one saltation is responsible forthe growth of one segment (Iovine andJohnson, 2000; Goldsmith et al.,2003).

Growth in vertebrates is regulatedprimarily by changes in cell number(Conlon and Raff, 1999), indicatingthat cell proliferation may be used togauge growth. Thus, the next level ofprocesses to consider during fingrowth is the pattern of cell divisionthat results in the formation of an in-dividual bone segment. This examina-tion was predicted to expose a graph-ical representation of proliferationconsistent with a single burst of celldivision (e.g., constant, parabolic, ex-ponential decay) during the lengthen-ing of the distal-most segment. To as-sess these possibilities, we monitoredcell proliferation using markers for re-cently dividing cells (BrdU) and mito-sis (H3P) and followed the growth ofthe ultimate segment in each the long-est and shortest fin rays in the caudal

fin. Interestingly, our data do not sup-port the initial hypothesis that seg-ment growth is the result of a singlesaltation. Instead, multiple apparentfluctuations of cell proliferation areidentified during the growth of an in-dividual segment, revealing multiplebursts of cell division. We find evi-dence for four pulses in the longestrays (average segment length �250microns) and three pulses in theshortest rays (average segment length�200 microns), suggesting that thenumber of saltations (but not the am-plitude) controls the length of seg-ments. One intriguing possibility forhow fin shape is regulated is that asystemic factor coordinates saltationsacross the fin, but individual fin raysrespond to that factor differentially.

RESULTS AND DISCUSSION

Cell Proliferation DuringSegment Growth Is Pulsatile

Recently, Goldsmith et al. (2006) iden-tified a physiological transition fromjuvenile (i.e., paddle-shaped caudalfin) to adult (i.e., bi-lobed caudal fin)form during ontogenetic fin growth.Segment addition was found to beasynchronous until after the bi-lobedshape is established, when segmentsare added concurrently across the fin.We chose to monitor cell proliferationafter the transition to adult growth,when segment addition is synchro-nous, and when fins are growing rap-idly (�12–14 weeks of age). It was pos-sible to confirm that fin growth hadtransitioned to adult growth by count-ing the number of segments from thelongest and shortest rays (i.e., at least12 segments in the longest rays and atleast 8 segments in the shortest ray).Furthermore, because segment lengthtends to be longer in the lobe fin raysthan in other regions of the fin (Iovineand Johnson, 2000), we completed ouranalyses in both the longest (i.e., “lat-eral”) and shortest (i.e., “medial”) finrays to provide insight into how differ-ences in segment length across the finare attained.

Vital markers for dividing cellshave not been developed, so we exam-ined cell proliferation from a popula-tion of 40 animals using histochemicaltechniques, and reasoned that grow-ing segments of varying lengths would

be identified. Cell proliferation duringgrowth of the ultimate fin ray segmentwas assessed by labeling animals withthe thymidine analog bromodeoxyuri-dine (BrdU), which incorporates intoDNA during the S-phase of the cellcycle and is detectable using a mono-clonal antibody. Initially, we evalu-ated the number of mesenchymalBrdU-labeled cells with the length ofthe ultimate (i.e., incomplete, or grow-ing) segment using Confocal micros-copy to identify labeled cells from theentire distal mesenchymal compart-ment (Fig. 1). Labeled cells werecounted at level with or distal to theultimate joint. Of interest, the patternof proliferating cells was not consis-tent with the predicted patterns for asingle pulse of dividing cells men-tioned above. Rather, we identifiedmultiple apparent pulses duringgrowth in both the longest and short-est fin rays (Fig. 1). Furthermore, theoverall slope of the data is positive,suggesting an increase in the numberof dividing cells per pulse as the seg-ment grows longer.

Cells Proximal to theGrowing Segment Contributeto Outgrowth

One explanation for the increasingnumber of BrdU-labeled cells with in-creasing segment length is that thenewly forming segment recruits an in-creasing number of dividing cells as itgrows. If true, this would suggest thatthe rate of segment growth increaseswith increasing length. Although thisremains a formal possibility, we do notfind a preponderance of fins with shortultimate segments (as would be pre-dicted if the segment grew slowly ini-tially). Alternatively, the positiveslope in Figure 1 may simply reflectthat, as a segment lengthens, wewould naturally identify BrdU-posi-tive cells from a larger area.

One way to correct for the latterpossibility is to count BrdU-positivecells over fixed distances (i.e., 60, 150,250 microns) from the distal end of thefin, even if this means counting cellsproximal to the most distal joint. In-deed for any fixed distance, the fluctu-ations in cell proliferation are still ap-parent (Fig. 2), but the overall slope ofthe data no longer appears positive. Ofinterest, the difference in labeled cells

CELL PROLIFERATION AND BONE GROWTH IN ZEBRAFISH FINS 2669

Fig. 1. Cell proliferation during segmentgrowth appears pulsatile. A: Overlayed imagesof ZNS5� cells (osteoblasts) in brown and bro-modeoxyuridine-positive (BrdU�) cells in red.Arrowheads indicate joints. BrdU� cells distalto the most distal joint were counted (boxedregion) from individual fin rays and plottedagainst the length of the ultimate segment fromthe same fin. B: Number of BrdU� cells in thelongest fin ray in the ventral lobe. C: Number ofBrdU� cells in the shortest fin ray in the fin.Each data point in B and C represents a singlefin ray. N � 28 rays.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 2. Cells proximal to the most distal jointappear to contribute to segment growth. A: Over-layed images of ZNS5� cells (osteoblasts) inbrown and bromodeoxyuridine-positive (BrdU�)cells in red. Arrowheads indicate joints. BrdU�cells were counted from three areas: 60 �m fromthe end of the fin (blue box), 150 �m from the endof the fin (black box), 250 �m from the end of thefin (red box); and plotted against the length of theultimate segment in B and C. B: BrdU� cellscounted in each of the three areas, from the long-est fin ray in the ventral lobe. C: BrdU� cellscounted in each of the three areas, from the short-est fin ray in the fin. Each data point in B and Crepresents a single fin ray. N � 28 rays.

Fig. 3. The number of mitotic cells during seg-ment growth appears pulsatile. A: Confocal im-age of a single section with ZNS5� cells in greenand H3P� cells in red. The yellow arrowheadindicates the joint; the blue arrowhead indicates asingle H3P� cell in this section (and in this fin ray).The boxed region represents an area 250 �mfrom the end of the fin. B: H3P � cells in thelongest fin ray in the ventral lobe. C: H3P� cellsfrom the shortest fin ray in the fin. Each data pointin B and C represents a single fin ray. N � 25 rays.

Fig. 4. Mathematical representation of cell pro-liferation during segment growth in the longest(top) and shortest fin rays (bottom). The dottedline represents the mean number of bromode-oxyuridine-positive (BrdU�) cells.

2670 JAIN ET AL.

COLOR

increases appreciably when measuredfrom 60 microns to 150 microns, indi-cating that many cells within thisarea likely contribute to outgrowth. Incontrast, there is not a notable differ-ence in the number of labeled cellsfrom 150 microns to 250 microns, sug-gesting that cells more proximal than250 microns do not add significantly tothe number of proliferating cells. Toaccount for all potential proliferatingcells involved with segment growth,we used data from the 250 micron pop-ulation for further analyses.

The distinction between countingcells over fixed or variable distancesmay seem arbitrary since pulses ofBrdU-positive cells are apparent us-ing either method of counting dividingcells. Therefore, the method for count-ing proliferating cells does not alterthis conclusion. The next question toconsider is whether or not it is reason-able to conclude that the labeled cellsproximal to the ultimate joint contrib-ute to the outgrowth of the ultimatesegment. Fin regeneration studieshave demonstrated that proliferatingmesenchymal cells one to two seg-ments proximal to growing segmentsin fact contribute to outgrowth (Poleoet al., 2001; Nechiporuk and Keating,2002). Our findings are consistentwith the recruitment of more proximalcells contributing to the growth ofmore distal segments, and furthersuggest that underlying mechanismsof cell proliferation are similar duringfin regeneration and ontogeny (Mari-Beffa et al., 1996).

We next used an independentmethod to detect and follow activelydividing cells during segment growth.Phosphorylation of serine 10 on his-tone H3 occurs only during mitosis(Hendzel et al., 1997), and the anti-body against this phosphorylated res-idue serves as a specific marker formitotic cells (H3P, Wei et al., 1999).We followed the number of H3P-posi-tive cells and found results consistentwith our analysis of BrdU-labeledcells. Specifically, the number of mi-totic cells fluctuates in apparentpulses as the ultimate segment in-creases in length (Fig. 3). Thus, prolif-erating cells detected by means ofBrdU or H3P reveals pulsatile pat-terns of cell division during segmentgrowth.

Mathematical RepresentationDescribing the Pattern ofCell Proliferation

To provide additional support for theperiodicity of cell proliferation, weconducted a statistical analysis to testour conclusions. Indeed, the apparentfluctuations in the number of BrdU-positive cells may represent actualfluctuations in cell proliferation, ormay simply represent normal varia-tion for a model of constant cell prolif-eration. Therefore, we tested the fit ofour data to a periodic curve (alternatehypothesis) and to a horizontal line(null hypothesis). We began with anequation representing the cosine func-tion, which is a periodic curve fluctu-ating in both the positive and negativequadrants. To account for both thediscontinuous and necessarily positivenature of the data, we used the abso-lute value cosine curve (i.e., choosingnot simply to shift the cosine curveinto the positive quadrant).

The equation for the alternate hy-pothesis is y � a�cos(bx-c)� � d � �; theequation for the null hypothesis is y �k. An F-ratio test statistic of both thealternate hypothesis and the null hy-pothesis resulted in a P value of 0.034for the longest fin rays and P � 0.035for the shortest fin rays, indicatingthat the alternate hypothesis (i.e., ab-solute value cosine curve) is a morevalid representation of the data than ahorizontal line (Fig. 4). The mostlikely source of error in our data col-lection is in distinguishing betweennewly forming segments and recentlycompleted segments. Therefore, we re-peated the test of variance omittingthe first interval for the lateral rays.In this case, the P value for the long-est fin rays was P � 0.000 (data notshown). The statistical significance isconsiderably stronger when usingonly the last three periods, providingincreased support for our derivedequation as representative of the pat-tern of cell proliferation during seg-ment growth.

As mentioned, more medial regionsof the caudal fin tend to have shortersegments than in the lobes. The cellu-lar basis for the differences in seg-ment length are likely reflected by thedata presented here. Of interest, theultimate segment in the shortest finray displays only three periods of

growth, whereas the ultimate seg-ment in the longest ray displays fourpulses (Fig. 4). In contrast, the meannumber of dividing cells in the longestrays (n � 26.7 � 12.6) was not signif-icantly different from the mean num-ber of dividing cells in the shortestrays (n � 21.8 � 12.0), suggesting thatthe overall amplitude of the periodsare similar across the fin. Thus, thefrequency of pulses is likely the pri-mary contributor for the regulation ofsegment length across the fin rays ofthe entire fin.

As a final test of our data, we com-pleted an additional statistical analy-sis to provide confidence that the areaover which BrdU-positive cells werecounted did not influence the overallpattern or mathematical model forboth the lateral and medial fin rays.Because it is apparent already thatincreasing the area containing BrdU-positive cells also results in an in-crease in the number of BrdU-positivecells, it did not make sense to comparethe amplitudes of the curves. Rather,it was important to demonstrate thatthe overall shape of the curves is sim-ilar.

We limited our analyses to the150-�m and 250-�m data sets becauseit is apparent that the number of cellsmeasured from 60 �m was insufficientfor identifying the number of dividingcells that reasonably contribute tonew segment growth. A z-test forequal slope parameters was com-pleted. When comparing the differ-ences in slope across the entire curve,we found that the slopes were not dif-ferent (i.e., we did not reject the nullhypothesis, that 150 �m � 250 �m)between the 150 �m and 250 �m datasets for either the lateral (P � 0.88) orthe medial (P � 0.78) fin rays. There-fore, the patterns of cell proliferationare similar among the 150 �m and 250�m data sets.

Updated Model of SegmentAddition During Fin Growth

Previous genetic and molecular anal-yses revealed the saltatory nature offin growth. This cellular analysis ofcell division confirms that growth oc-curs in pulses and more clearly de-fines a single saltation as the prolifer-ation of �40 cells (i.e., approximatenumber of BrdU-positive cells during

CELL PROLIFERATION AND BONE GROWTH IN ZEBRAFISH FINS 2671

the peak of the pulses). If the length ofthe cell cycle is less than 6 hours (thetime of exposure to BrdU), the numberof proliferative progenitors could belower than the estimated 40 cells de-scribed here (i.e., if the initial progen-itors divided more than once duringthe labeling period). Still, that doesnot preclude the conclusion that salta-tions occur as part of segment growth.Unexpected insights from this reportrevealed that multiple pulses of cellproliferation are required for segmentsize rather than a single saltation, aspreviously predicted (Iovine and John-son, 2000; Goldsmith et al., 2003).How do these saltations fit into earliermodels for fin growth?

Prior models of fin growth predictedthat a single growth phase/saltationwould account for the growth of a sin-gle segment and that rest phasesflank each segment addition. Molecu-lar evidence supporting this model re-vealed that expression of a gene-basedgrowth marker, fa93e10, occurred100% of the time in young, rapidlygrowing fins, and less frequently withaging, slowly growing fins. One possi-

bility is that the saltations that wedefine here represent the saltationsidentified using fa93e10, but the as-sumption that one saltation was equalto one segment is false (Fig. 5, ModelA). If correct, then growth and restsimply alternate as predicted previ-ously, and rapidly enough that it isnot possible to identify young fins inthe rest phase (i.e., fa93e10-negative).This is the simplest interpretation ofour data. Alternatively, the saltationswe define may represent minisalta-tions within a growth phase (Fig. 5,Model B). In this model, fa93e10would be positive throughout growthof a single segment. For example, finswould be fa93e10-positive at alltimes—in the “valleys” of less thanfive BrdU-positive cells, during the“peaks” of cell proliferation, and at allpoints in between. This may explainwhy it has not been possible to iden-tify young fins that are fa93e10-nega-tive even though we readily identifyyoung fins with a small number ofBrdU-positive cells. Indeed, finswould be fa93e10-negative only dur-ing a “true rest” phase occurring upon

segment completion. If correct, then arapidly growing fin is in growth phase(i.e., fa93e10-positive) until a segmentis complete and regardless of the num-ber of proliferating cells (i.e., �5–40cells for the longest fin rays). This in-terpretation suggests there are layersof control during segment growth, andpredicts that one can distinguish be-tween “valleys” in the growth phaseand a hypothetical “true rest” phaseby following both fa93e10 expressionand BrdU labeling.

To test this prediction, we labeled30 young fish with BrdU as before,and processed the caudal fins for bothfa93e10 expression and BrdU. Indeed,it was possible to identify fa93e10-pos-itive fin rays that contained only oneto five BrdU-positive cells (Fig. 6), in-dicating that fins with a small numberof BrdU-positive cells are in fact in thegrowth phase. This finding occurred�25% of the time (7/28), which is con-sistent with the frequency of “BrdUvalleys” from the initial profile of pro-liferating cells (i.e., �23% of fin raysin valleys in Fig. 2), indicating that asimilar population was sampled inboth experiments. These data providestrong evidence favoring Model B (Fig.5) and indicate that the valleys iden-tified in our cell proliferation profilesare part of the normal segment growthcycle and are distinct from a “true rest”phase (i.e., fa93e10-negative).

Conclusions

In this report, we identify saltationsduring fin growth, define a saltation asthe proliferation of up to 40 cells, andreveal that fin shape is partially regu-lated by the number of saltations occur-ring during new segment growth. Theconclusion that the growth of a singlesegment requires multiple saltations issupported by two independent methodsto identify dividing cells. Furthermore,mathematical modeling not only pro-vides an equation representing the pat-tern of cell proliferation, but also pro-vides statistical support for theexistence of saltations.

What is the purpose of saltations?In humans, it has been suggested thatgrowth and stasis reflect synchronizedcell cycle activity at the organismallevel and that long episodes of stasisare required for normal developmentto proceed (Lampl et al., 1992; Lampl

Fig. 5. Updated models for how regulation of growth (cell proliferation) and stasis contributes tosegment growth. Model A: Valleys during pulsatile growth represent the stasis period throughoutfin growth. Model B: Valleys during pulsatile growth are distinguishable from true stasis periods.

Fig. 6. Double labeling of young fins with the growth marker fa93e10, and with bromodeoxyuri-dine (BrdU). A: Fins positive for fa93e10 may have a low number of BrdU-positive cells (�5; n � 7)or a high number of BrdU-positive cells (� 5; n � 21). B: An example of a fa93e10-positive fin(bracket) with a high number of BrdU-positive cells (arrows, n � 13). C: An example of afa93e10-positive fin (bracket) with a low number of BrdU-positive cells (arrows, n�3). Brown cellsare melanocytes. N � 28 fins.

2672 JAIN ET AL.

and Johnson, 1993). However, the co-ordination of cellular processes hasnot been examined during bonegrowth in humans. In zebrafish, onepossibility is that regulation of salta-tions contributes to the maintenanceof fin shape in adult fins (recall thatthe establishment of fin shape occursduring juvenile fin growth; Goldsmithet al., 2006). Circulating systemic fac-tor(s) likely induce cell proliferation inthe distal population of undifferenti-ated mesenchymal cells during fingrowth, and individual fin rays mayrespond differentially, resulting inslight variations in segment lengthacross the fin. Environmental condi-tions, such as nutrition, may providethe systemic factor regulating cell di-vision. Indeed, it has recently beendemonstrated that inhibition of insu-lin/insulin-like growth factor signal-ing by means of rapamycin treatmentsignificantly reduces cell proliferationduring fin growth (Goldsmith et al.,2006). When nutrients are limiting,the fin may remain in stasis or initiatea single saltation as nutrients becomeavailable; when nutrients are plenti-ful saltations may occur more fre-quently. “Valleys” identified here mayprovide additional opportunities to en-ter stasis in the event that nutrientsare depleted during the growth of asegment (�5–7 days, Iovine and John-son, 2000). Similarly, as body growthand fin growth are coordinated (Iovineand Johnson, 2000), saltations may al-low for finer regulation of the couplingof growth processes and ensure ade-quate nutrient distribution. Such pos-sibilities are not mutually exclusive,and in fact likely describe differentaspects of the same problem, i.e., reg-ulation of the growth and morphogen-esis of the adult fin. Future aspects ofthis research will elucidate the molec-ular pathways regulating the dynam-ics of vertebrate bone growth.

EXPERIMENTALPROCEDURES

Stocks and Fish Rearing

The wild-type fish stocks were ob-tained from the C32 strain. Fish weremaintained at a constant temperatureof 28°C and exposed to a 14-hr light:10-hr dark photoperiod (Westerfield,1993).

Detection of BrdU-LabeledCells

Fish were allowed to swim in BrdU(50 mg/ml) in fish water for 6 hr (Ne-chiporuk and Keating, 2002). The cau-dal fins were amputated asymmetri-cally to preserve dorsal and ventralidentity and fixed in 4% paraformal-dehyde at 4°C overnight. After fixa-tion, fins were rinsed twice in metha-nol and stored at �20°C. They wererehydrated into PBTx (phosphatebuffered saline [PBS] with 0.3% Tri-ton X-100) and incubated in DnaseI(50 units/ml) for 45 min. Tissue wasincubated with PBTxB (PBTx with0.25% bovine serum albumin [BSA])for 45 min and then oscillated over-night at 4°C in mouse anti-BrdU an-tibody (1:50). After extensive washing,fins were incubated in anti-mouse Al-exa-546 at 1:200 overnight at 4°C. In-dividual fins were examined for BrdU-positive cells by confocal microscopy.

Confocal Microscopy

The results of the BrdU staining wereobserved using the Zeiss LSM 510Meta confocal microscope. To optimizethe visible range, optical z-sectionswere collected through the distal mosttips of the fin rays. Individual pictureswere transferred to Microsoft OfficePowerPoint and recompiled in az-stack. The longest and shortest finrays were examined from each fin.The longest fin ray in the ventral lobewas typically the third fin ray fromthe lateral side of the ventral lobe(identified by harvesting fin with anasymmetric cut), and is also referredto as the “lateral” ray. The shortest finray was identified as the most medialfin ray in the fin, and is also referredto as the “medial” ray.

ZNS5 Detection

After confocal microscopy, fins weretransferred to individual wells in a 48-well dish. Fins were treated with col-lagenase (1 mg/ml) in PBS for 45 minat room temperature, washed in ablocking reagent (2% BSA in PBS) andincubated at 4°C in mouse ZNS5 anti-body at 1:200 (Zebrafish internationalresource center, http://zebrafish.org/zirc/home/guide.php). After removal ofthe primary antibody, fins were

washed and exposed to an unlabeledgoat anti-mouse secondary antibodyfor 2 hr at room temperature. Subse-quently, samples were washed and in-cubated in mouse PAP (peroxidase an-tiperoxidase) overnight at 4°C. Lastly,fins were washed in PBS and rinsed indiaminobenzidine (DAB; 0.03% DABin 0.1 M PBS); 0.01% hydrogen perox-ide was added. Fins were mounted inglycerol and visualized using a NikonSMZ1500 microscope, and segmentlength was measured using ImageProsoftware.

Detection of Mitotic Cells

Phosphorylation of Ser10 on histoneH3 occurs specifically during mitosis(Hendzel et al., 1997). We examinedfins for both mitotic cells using ananti–H3-phospho histidine antibody(H3P, Upstate Biotechnology) and forZNS5 to follow the number of mitoticcells during segment growth. The H3Pantibody was used at 1:100 and wasdetected by using anti-rabbit Alexa-546 following the ZNS5 detection pro-tocol (i.e., double labeling along withZNS5/anti–mouse Alexa-488). Finswere analyzed by confocal microscopy.

Mathematical Modeling

To statistically confirm the periodicnature of cell division, a mathematicalmodel of the form y � a�cos(bx-c) � �d � � was proposed, where the numberof dividing cells was a function of dis-tal-most segment length. The period band shift c were approximated so wecould treat �cos(bx-c) � as a new predic-tor variable, say x, and the remainingfitting parameters a and d were foundusing simple linear regression methodand using the Minitab StatisticalPackage 14. We tested the normality,equal variance, and independence as-sumptions of the linear regressionmodels. We did not find significant vi-olation of these assumptions. This al-ternative hypothesis was testedagainst the null hypothesis y � k(mean number of dividing cells), usingthe F-ratio test. Equations were lin-early transformed, and correspondingP values were also found usingMinitab. Final equations were plottedusing Maple 9.5.

To determine whether the meannumber of dividing cells was statis-

CELL PROLIFERATION AND BONE GROWTH IN ZEBRAFISH FINS 2673

tically different from the lateral andmedial fin rays, we completed a two-sample t-test. To begin, we found thedata passed the three normalitytests in Minitab (i.e., P valuesgreater than significance level �0.05 for each test), and the datapassed the equal variance test for anormal distribution (P � 0.818).Completion of the two-sample equalvariance t-test revealed a P value of0.162, indicating that there was nostatistical difference in the meannumber of dividing cells from thetwo data sets.

Double-Labeling Withfa93e10 and BrdU

Fish (14 weeks and 12 lateral fin raysegments) were treated with BrdU for6 hr, immediately harvested, fixed,and stored in methanol as describedabove. In situ hybridization using an-tisense RNA probe (UTP-digoxigenin)was completed as described (Poss etal., 2000).

After manual color development,fins were processed for BrdU as de-scribed above. BrdU-positive, mesen-chymal cells were identified by confo-cal microscopy.

ACKNOWLEDGMENTSThe authors thank Jacob Fugazzottofor care and maintenance of the ze-brafish colony and Matthew Gold-smith for careful reading and discus-

sion of this manuscript. The ZebrafishInternational Resource Center is sup-ported by the NIH-NCRR. M.K.I. wasfunded by the NIDCR.

REFERENCES

Akimenko MA, Mari-Beffa M, Becerra J,Geraudie J. 2003. Old questions, newtools, and some answers to the mysteryof fin regeneration. Dev Dyn 226:190–201.

Conlon I, Raff M. 1999. Size control in an-imal development. Cell 96:235–244.

Goldsmith MI, Fisher S, Waterman R,Johnson SL. 2003. Saltatory control ofisometric growth in the zebrafish caudalfin is disrupted in long fin and rapunzelmutants. Dev Biol 259:303–317.

Goldsmith MI, Iovine MK, O’Reilly-Pol T,Johnson SL. 2006. A developmentaltransition in growth control during ze-brafish caudal fin development. Dev Biol296:450–457.

Goss RJ, Stagg MW. 1957. The regenera-tion of fins and fin rays in Fundulus het-eroclitus. J Exp Zool 136:487–507.

Haas HJ. 1962. Studies on mechanisms ofjoint and bone formation in the skeletonrays of fish fins. Dev Biol 5:1–34.

Hendzel MJ, Wei Y, Mancini MA, Van Hoo-ser A, Ranalli T, Brinkley BR, Bazett-Jones DP, Allis CD. 1997. Mitosis-spe-cific phosphorylation of histone H3initiates primarily within pericentro-meric heterochromatin during G2 andspreads in an ordered fashion coincidentwith mitotic chromosome condensation.Chromosoma 106:348–360.

Iovine MK, Johnson SL. 2000. Geneticanalysis of isometric growth controlmechanisms in the zebrafish caudal Fin.Genetics 155:1321–1329.

Lampl M, Johnson ML. 1993. A case studyof daily growth during adolescence: a

single spurt or changes in the dynamicsof saltatory growth? Ann Hum Biol 20:595–603.

Lampl M, Veldhuis JD, Johnson ML. 1992.Saltation and stasis: a model of humangrowth. Science 258:801–803.

Landis WJ, Geraudie J. 1990. Organiza-tion and development of the mineralphase during early ontogenesis of thebony fin rays of the trout Oncorhynchusmykiss. Anat Rec 228:383–391.

Mari-Beffa M, Mateos I, Palmqvist P,Becerra J. 1996. Cell to cell interactionsduring teleosts fin regeneration. Int JDev Biol Suppl 1:179S–180S.

Nechiporuk A, Keating MT. 2002. A prolif-eration gradient between proximal andmsxb-expressing distal blastema directszebrafish fin regeneration. Development129:2607–2617.

Poleo G, Brown CW, Laforest L, AkimenkoMA. 2001. Cell proliferation and move-ment during early fin regeneration in ze-brafish. Dev Dyn 221:380–390.

Poss KD, Shen J, Nechiporuk A, McMahonG, Thisse B, Thisse C, Keating MT. 2000.Roles for Fgf signaling during zebrafishfin regeneration. Dev Biol 222:347–358.

Poss KD, Keating MT, Nechiporuk A. 2003.Tales of regeneration in zebrafish. DevDyn 226:202–210.

Santamaria JA, Mari-Beffa M, Becerra J.1992. Interactions of the lepidotrichialmatrix components during tail fin regen-eration in teleosts. Differentiation 49:143–150.

Wei Y, Yu L, Bowen J, Gorovsky MA, AllisCD. 1999. Phosphorylation of histone H3is required for proper chromosome con-densation and segregation. Cell 97:99–109.

Westerfield M. 1993. The zebrafish book: aguide for the laboratory use of zebrafish(Brachydanio rerio). Eugene, OR: Uni-versity of Oregon Press.

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