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Research Article

Cryptic organisation within an apparently irregular rostrocaudaldistribution of interneurons in the embryonic zebrafishspinal cord

Simon Wellsa,d,⁎, John G. Conranb, Richard Tammea,1, Arnaud Gaudinc,Jonathan Webba,2, Michael Lardellia,d

aDiscipline of Genetics, School of Molecular and Biomedical Sciences, University of Adelaide, Adelaide, South Australia 5005, AustraliabEcology and Evolutionary Biology, School of Earth and Environmental Sciences, University of Adelaide, Adelaide,South Australia 5005, AustraliacSchool of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, AustraliadThe Special Research Centre for the Molecular Genetics of Development, University of Adelaide, Adelaide, South Australia 5005, Australia

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. School of MolecularFax: +61 08 8303 7534.

E-mail addresses: simon.wells@adelaide.edua.gaudin@uq.edu.au (A. Gaudin), jonathan.webb

Abbreviations: BCIP, 5-bromo-4-chloro-3′-inCNS, central nervous system; DIC, differential inDNA, deoxyribonucleic acid; DNP, dinitrophenoGABA, gamma-aminobutyric acid; HCL, hydrochtetrazolium chloride; ntl, no tail; PBS, phosphreaction; RA, retinoic acid; RNA, ribonucleic aci1 Current address: Department of Gene Techn2 Current address: Worcester College, Univers

0014-4827/$ – see front matter © 2010 Elseviedoi:10.1016/j.yexcr.2010.06.020

A B S T R A C T

Article Chronology:

Received 20 April 2010Revised version received 31 May 2010Accepted 23 June 2010

Available online 1 July 2010

The molecules and mechanisms involved in patterning the dorsoventral axis of the developingvertebrate spinal cord have been investigated extensively and many are well known. Conversely,knowledge ofmechanisms patterning cellular distributions along the rostrocaudal axis is relativelymore restricted. Much is known about the rostrocaudal distribution of motoneurons and spinal

cord cells derived from neural crest but there is little known about the rostrocaudal patterning ofmost of the other spinal cord neurons. Here we report data from our analyses of the distribution ofdorsal longitudinal ascending (DoLA) interneurons in the developing zebrafish spinal cord. Weshow that, although apparently distributed irregularly, these cells have cryptic organisation. Wepresent a novel cell-labelling technique that reveals that DoLA interneurons migrate rostrallyalong the dorsal longitudinal fasciculus of the spinal cord during development. This cell-labellingstrategy may be useful for in vivo analysis of factors controlling neuron migration in the centralnervous system. Additionally, we show that DoLA interneurons persist in the developing spinalcord for longer than previously reported. These findings illustrate the need to investigate factorsand mechanisms that determine “irregular” patterns of cell distribution, particularly in the centralnervous system but also in other tissues of developing embryos.

© 2010 Elsevier Inc. All rights reserved.

Keywords:

Dorsal longitudinal ascendinginterneuronCryptic organisationNeuronal migrationRostrocaudal patterningSpinal cordtbx16

and Biomedical Sciences, The University of Adelaide, Adelaide, South Australia 5005, Australia.

.au (S. Wells), john.conran@adelaide.edu.au (J.G. Conran), rtamme@ttu.ee (R. Tamme),@worc.ox.ac.uk (J. Webb), michael.lardelli@adelaide.edu.au (M. Lardelli).dolyphosphate p-toluidine salt; BMP, bone morphogenetic protein; cDNA, complementary DNA;terference contrast; DLF, dorsal longitudinal fasciculus; DMNB, 2, 3-dimethyl-2, 3-dinitrobutane;l; DoLA, Dorsal Longitudinal Ascending; Dpf, days post-fertilisation; FGF, fibroblast growth factor;loric acid; Hpf, hours post-fertilisation; isl, islet; MO, morpholino oligonucleotide; NBT, nitroblueate-buffered saline; PBT, PBS+0.1% Tween-20; PBTI, PBT+0.3% IGEPAL; PCR, polymerase chaind; SDS, sodium dodecyl sulfate; Shh, sonic hedgehog; UTR, untranslated region; UV, ultravioletology, Tallinn University of Technology, Tallinn 19086, Estonia.ity of Oxford, Oxford OX1 2JD, UK.

r Inc. All rights reserved.

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Introduction

The rostral part of the vertebrate central nervous system (CNS)exhibits extensive genetic and morphological regionalisation. Indeveloping zebrafish, this regionalisation appears to begin as earlyas gastrulation [1–3]. The rostrocaudal axis of the developing CNSis sequentially subdivided into broad regions that will become theforebrain and midbrain; the hindbrain and spinal cord; and themidbrain-hindbrain boundary. The hindbrain is further parti-tioned into rhombomeres that achieve different identities and thusdevelop different neuronal cell types through the action of variousconserved transcription factors including the hox genes [4–6].

A battery of signalling molecules network in complex interac-tions to initiate dorsoventral patterning of the developingvertebrate neural tube. Fibroblast growth factors (FGFs) producedby the caudal mesoderm must be down-regulated by retinoic acid(RA) released by the paraxial mesoderm before neural differen-tiation can occur. Sonic hedgehog (Shh) protein released by thenotochord and floor plate has been shown to act in a concentra-tion-dependent manner to induce ventral neurons. Similarly, bonemorphogenetic proteins (BMPs), produced by the roof plate, act ina concentration-dependent manner to induce dorsal neurons. Theoverlapping gradients of BMPs and Shh interact with each other ina repressive manner and the resultant gradients are interpreted byan array of interacting homeodomain proteins, the result beinggenetic partitioning of the dorsoventral axis [7–9], leading tospecific cell fates [10].

In the developing vertebrate spinal cord, a dorsal roof plate andventral floor plate that extend along the rostrocaudal axis areseparated by densely packed neural progenitors and glia. Duringdevelopment, neuronal cells must differentiate in the correctpositions to ensure that they can form an interconnecting patternof enormous complexity. Neuronal cell fate in the embryonicspinal cord is determined by interpretation of patterning signalson the rostrocaudal and dorsoventral axes. Our understanding ofthe molecules involved in patterning the dorsoventral axis of thevertebrate spinal cord is quite advanced [7–9].

In invertebrates, the rostrocaudal axis of the CNS often displaysa segmented pattern. For example, the nerve cord of Drosophilamelanogaster is genetically and morphologically partitioned intosegments. Initial segmentation occurs by interaction betweensegment polarity genes and columnar genes resulting in asegmented ectoderm fromwhich the ventral nerve cord is derived[11]. The CNS of the mud leech, Haemopis marmorata, contains aline of segmentally derived ganglia that display differentialneuronal cell content [12]. Various neurons in this organismhave been identified that are distributed in multisegment patterns,suggesting the existence of supersegmental counting mechanisms[12].

In contrast, the vertebrate spinal cord, although geneticallysegmented by hox genes [3,13,14] does not display obviousmorphological segmentation. Additionally, the majority of neuro-nal cell types do not appear to vary at different regions along therostrocaudal axis of the spinal cord [15,16], although experimentsby Hale et al. have shown rostrocaudal changes in cell morphologyin zebrafish that are likely to be genetically determined [16].Exceptions to this observation include somemotoneuron subtypes[17] and the poorly characterised IC interneurons that are locatedin the developing hindbrain and only the rostral region of the

spinal cord [18]. Recent work shows that the genetic hox networkof the spinal cord is involved in determining the target-muscleconnectivity of motoneurons [14]. The overlying lateral somiticmesoderm appears to impart sectional segmentation guidance tosome cells of the spinal cord. Lewis and Eisen have shown thatprimary motoneuron identity and cell body positioning, andprobably secondary motoneuron positioning are influenced byoverlying somites [19]. Additionally, the pattern of motoneuronand sensory neuron axonal projections from the spinal corddisplays a morphological metamerism guided by flanking somiticmesoderm [20,21].

In embryonic zebrafish both metameric and non-metamericpatterns of spinal neuron distributions can be observed. Primarymotoneurons are patterned such that there are three cells per spinalcord hemisegment [19,22,23]. (A hemisegment is that area of oneside of the spinal cord defined with reference to adjacent somitemetamerism.) In this paper we describe this as a “regular”(metameric) distribution (in population dispersion parlance itwould be known as a “uniform” dispersion). Mutation of the genetbx16 (previously known as spadetail (spt)) causes morphologicaldefects in somite formation that affect motoneuron distributionindicating that the regular distribution of motoneurons is controlledby signals from the flanking somitic mesoderm [19,24]. In contrast,the Rohon–Beard dorsal sensory neurons do not show any obviousmetameric distribution. These occur at a frequency exceeding onecell per flanking somite in what we define as a “frequent irregular”distribution. (We cannot describe this dispersion as “random” sincethis has not yet been demonstrated analytically.) The number ofthese cells does not appear to be affected by the tbx16mutation [24]suggesting that there is no control by signals from somitic tissue.However, mutations in Notch signalling pathway members canaffect the number/distribution of Rohon–Beard neurons [25–27]since the “frequent irregular” distribution of these cells probablyarises as a consequence of Notch signalling, acting somewhatstochastically within an equivalence field of precursor cells [25] togenerate different cell fates.

A third rostrocaudal cell distribution may be found in the spinalcord,withwhatwe define as an “infrequent irregular”pattern of lessthan one cell per flanking somite. For example, dorsal longitudinalascending (DoLA) interneurons are found at a frequency of 0.06 cellsper flanking somite for the 5th to 8th formed somite at 18 h post-fertilisation (hpf) [28]. The majority of identified embryonic spinalcord neurons have such infrequent irregular distributions, [28]. Thishighlights the importance of exploring the mechanisms thatdetermine cell position for this class of patterning.

The genetic pathways underlying infrequent irregular patternsof neurons are particularly difficult to dissect. Mutation studiesand analyses in developmental biology typically involve disruptionof normal, regular developmental patterns. However, any disrup-tion to an infrequent irregular pattern would most likely cause asimilar, apparently stochastic arrangement. Despite their appar-ently irregular distribution, the number of DoLA neurons thatdevelop in the embryonic spinal cord is tightly regulated(21.4±3.4 cells at 24 hpf and 22.7±2.9 cells at 30 hpf [29])implying that strong constraints can exist on cell numbers in suchinfrequent irregular cell distributions. These constraints and theirmolecular foundations are completely unknown.

We have previously reported that tbx16-expressing cells in thedeveloping dorsal embryonic spinal cord are most likely to be DoLAinterneurons exclusively [29]. This is based on their coexpression of

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neuronal marker genes islet1 (isl), islet2a, and islet2b [30–32], huC[33], their dorsoventral position, and their rostrocaudal distributionand the time at which they are first observed [28,34]. Wedemonstrateda juxtapositionof tbx16-expression in thedorsocaudalregion of newly formed somites and in the spinal cord at the samedorsoventral level. However, tbx16-expressing neurons do not formadjacent to every somite suggesting the existence of an “inefficient”patterning stimulus either originating from the somites, or from asource signalling to both the tbx16-expressing somite and neuronalcells. Higher numbers of tbx16-expressing neurons are foundrostrally compared to caudally in the embryonic spinal cord andthe rostral-most DoLA neuron position alters over time. This can befound juxtaposed to the 10th formed somite pair at 16 hpf but isfrequently juxtaposed to the 6th formed somite pair at 24 hpfsuggesting thepossibility that themost rostral cellsmaydifferentiatelater or that DoLAs may migrate rostrally after birth [29].

In the work described in this paper we set out to dissect theapparently infrequent irregular distribution of DoLA interneuronsand the constraints upon this distribution. We show that there iscryptic organisation in the rostrocaudal and left-right distributionsof these cells. However, we show that there is no statisticalrelationship between the position of DoLA neurons and tbx16-expression in the dorsal caudal regions of newly formed somitesand that presomiticmesoderm is not required for differentiation toa DoLA fate. Using an UV-activatable dye we have developed alabelling procedure to show that DoLA neurons migrate rostrallyafter birth. They are the only cells that we have detected migratingrostrally in the developing caudal spinal cord prior to 24 hpf. Thisprocedure may be useful for analysis of factors affecting neuronalcell migration in vivo.

Materials and methods

Embryos and staging

Embryos were collected and allowed to develop at 28.5 °C.Morphological features of embryos were consistent with thezebrafish staging guide [35]. All animal procedures were carriedout under the auspices of the Animal Ethics Committee and theInstitutional Biosafety Committee of the University of Adelaide.

Whole-mount in situ transcript hybridisation

A cDNA clone, 26M, corresponding to transcription from tbx16wasisolated in a whole-mount in situ hybridisation screen of zebrafishembryos [29] and was subsequently cloned into the pGEMT vector(Promega Corporation, Madison, WI, USA). The clone for produc-tion of probe against islet1 transcripts was obtained from HitoshiOkamoto [30]. The inserts of these clones were amplified by PCRwith M13 primers and then transcribed with T3 or T7 RNApolymerases to produce unlabelled or digoxigenin-labelled anti-sense probes. Unlabelled RNA was post-labelled with dinitrophe-nyl (DNP) using the HybQUEST™ Label IT® (DNP) Kit (MIR 3825,Mirus Bio LLC, Madison, WI, USA).

Whole-mount in situ hybridisation was performed essentiallyas described [36] but the double staining reaction was undertakenwith BCIP/NBT, and subsequently, the second staining reactionused the Vector Red Alkaline Phosphatase Substrate Kit I (SK-5100,Vector Laboratories Inc., Burlingame, CA, USA). Inactivation of the

first alkaline phosphatase reaction was achieved by heating to65 °C for 1 h in PBS.

Morpholino knockdowns

Antisense morpholino oligonucleotides (MOs) were designed tobind over the start codon or the 5′ UTR regions of zebrafish genesand synthesised by Gene Tools (Gene Tools LLC, Philomath, OR,USA). We used a previously described MO against no tail (ntl) aswell as the standard negative control MO (CON-MO) supplied byGene Tools. Zebrafish tbx16 cDNA (AF077225) was used to designtbx16-MO: 5′-GCTTGAGGTCTCTGATAGCCTGCAT-3′. The under-lined region denotes sequence complementary to the ATG startcodon. MOs were diluted to 0.5–1 mM in deionised water andinjected into developing embryos at the one-cell stage beforeincubation at 28.5 °C until the desired developmental stage.

Dye uncaging

All activation and imaging was undertaken with a Bio-Rad MRC-1000UV confocal laser scanning microscope system (Bio-RadLaboratories Inc., Hercules, CA, USA) and a Nikon Diaphot 300inverted microscope (Nikon Instech Co., Ltd., Kawasaki, Kangawa,Japan). We diluted DMNB-caged fluorescein dextran (Invitrogen)to 2 mM in deionised water and injected through the yolk into thedeveloping animal at the 1-cell stage with or without tbx16-MO.Embryos developed at 25 °C until 13 h post-fertilisation (hpf) indarkness to prevent premature dye activation. Embryos wereanaesthetised in 0.02% tricaine/embryo medium (Sigma-Aldrich,Germany). The dye was activated in the unsegmented tail tip byexposure to 351 nm light generated by an UV-argon laser focusedby a 40× objective lens. Dye activation was confirmed by visualinspection of fluorescein. Embryos were allowed to develop at28.5 °C until reaching the appropriate stage and examined formigrant cells by imaging fluorescein distribution. To identifymigrant cells, embryos positive for migrant cells in the spinal cordwere immediately fixed in 4% formaldehyde in PBT and subse-quently processed by whole-mount in situ transcript hybridisationwith an antisense RNA probe against tbx16.

Time-lapse observations

All photoconversion and observations were undertaken using anOlympus IX81 inverted confocal microscope. We used thetransgenic line Tg(elav3:Kaede)rw0130a (also known as HuC:Kaede)which expresses Kaede under the control of the zebrafish HuCpromoter (kindly provided by Hitoshi Okamoto) [38]. At 16 hpfembryos were anaesthetised in 0.02% tricaine/embryo mediumprior to being embedded in 0.02% tricaine immersed 0.5% agarose.The caudal-most Kaede positive cells in the spinal cord werephotoconverted by a short UV laser pulse focussed by a 40× waterimmersion lens. We immediately began imaging for both greenand red Kaede emission at 5 min intervals for 7 h. At every timepoint we imaged a z-stack of 2 μm steps through one side of thespinal cord. Using ImageJ (National Institutes of Health, USA) theimage stack was flattened along the z-axis using a maximumintensity projection protocol and in a similar way along the x-axisafter a 90° rotation around the rostrocaudal axis. Both orientationsweremontaged together and annotationswere added using AdobePhotoshop (Adobe Systems Incorporated, USA).

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Embryo observations

Wild-type or MO-injected embryos were collected and fixed in 4%formaldehyde in PBT before carrying out whole-mount in situtranscript hybridisation with an antisense RNA probe against tbx16.Light field observations were conducted under a Zeiss Axiophot™microscope (Carl Zeiss Jena GmbH, Jena, Germany) with a 20xobjective using differential interference contrast (DIC) optics.

The analysis of the rostrocaudal distribution of tbx16 positiveCNS cells in relation to each other was undertaken as previouslydescribed [29]. The final number of somite pairs that develop in azebrafish embryo can vary somewhat. Also, it can be difficult toidentify with certainty the rostral-most somite pair in fixedembryos. Therefore, in order for data on DoLA position to becomparable between embryos we used the somite directly dorsalto the caudal tip of the yolk extension as our positional reference.Later, when data from all embryos had been collected, this somitewas designated to be somite 15 counting from the tail end since noembryo we observed had more than 14 somites caudal to thissomite. This allowed all DoLA positional data to have positivevalues that assisted statistical analysis.

For comparison of the somitic and neural tbx16 expression inthe region of the most recently formed somites, cells on the left orright side of the embryo were considered separately and theirpositions were examined relative to the somites on the same side.The four most recently formed somites (on each side) with caudalborders were numbered and divided into rostral, medial andcaudal regions. tbx16 positive cells in the CNS were scoredaccording to their position relative to these subdivisions. tbx16CNS cells that were found corresponding to the border of twosomites or that were located caudal to the most recently formedsomite were not considered in this study.

Confocal imaging of embryos was conducted on a Bio-RadMRC-1000 UV confocal laser scanning microscope system (Bio-Rad) using a Nikon Diaphot 300 inverted microscope (NikonInstech). Fluorescence was observed using a krypton/argon laserwith excitation at 488/10 nm and emission at 522/35 nm forAlexa 488 and with excitation at 568/10 nm and emission at 605/32 nm for Vector Red. Data were processed with Amira Visagesoftware package (Visage Imaging Inc., Andover, MA, USA).

Statistical calculations

To determinewhether therewas a relationship between the left andright sides in terms of cells expressing tbx16, the totalled tbx16expression frequencies in the developing spinal cord juxtaposed toeach somite on the two sideswere L vs. R cross-correlated after log10transformation using SYSTAT® 10 [39]. This shows if the two seriesbeing tested behave in a similar manner to each other. To examinemore closely the nature of any such relationship, the data weredifferenced to remove common trends to see if there were anysignificantly related points in the series. In this way, effects of theexistence of tbx16-expressing cells on one side influencing theexistence of tbx16-expressing cells on the other might be detected[39].

The log10 transformed total frequency data (L+R for eachsomite) were also subjected to autocorrelation plot analysis inSYSTAT® using increasing numbers of size classes (from two toeight) to see if there was any pattern in the positioning of tbx16-expressing cells along the rostrocaudal axis whichmight be related

to the existence of tbx16-expressing cells at other positions on theaxis. Autocorrelation plots allow investigation of periodicity—i.e. isthe expression of the gene at time/position X significantlycorrelated with later expression at position Y. In this way, ifthere is a more or less regular distance between regions where thegene is expressed, a pattern will emerge. For analysis of spatialautocorrelation we used Moran's index I [40]. To further filter thenoise caused by intervening positions, the datawere also subjectedto partial autocorrelation, where the influence of interveningpoints is partialed out for the specified lag or interval space [39].This is equivalent in spatial autocorrelation to changing thenumber of distance classes, although large numbers of classesare generally not recommended because of the loss of power withreduced sample size per class [41,42].

Western blotting

Standard control MO and tbx16-MO-injected embryos werecollected at 12 hpf. Deyolked embryos were placed in samplebuffer [2% sodium dodecyl sulfate (SDS), 5% β-mercaptoethanol,25% v/v glycerol, 62.5 mM Tris–HCl (pH 6.8), and bromophenol-blue], heated immediately at 100 °C for 2 min, and then storedat −20 °C prior to separation on a 12% SDS-polyacrylamide gel.Proteins were transferred to nitrocellulose membranes using asemi-dry electrotransfer system. For Western blotting with anti-Tbx16 antibodies (kindly provided by Charles B. Kimmel), themembrane was blocked with 5% horse serum in phosphate-buffered saline containing 0.1% Tween-20 (PBT) for 4 h at roomtemperature and incubated with a monoclonal mouse anti-Tbx16antibody (1/100 in PBT containing 2% horse serum) overnight at4 °C. The filter was washed four times each for 15 min in PBT andincubated for 1 h in a 1/20,000 of donkey anti-mouse IgG coupledto horseradish peroxidase (Jackson ImmunoResearch Laboratories,Inc., Baltimore, PA, USA) in PBT containing 2% horse serum. ForWestern blotting with anti-β-tubulin antibodies (Antibody E7,Developmental Studies Hybridoma Bank, The University of Iowa,IA, USA) we used the same procedure except that the primaryantibodies were diluted 1/200 and donkey anti-mouse IgGsecondary antibodies (Jackson ImmunoResearch) were diluted1/3,000. After incubation with secondary antibodies, the mem-brane was washed four times in PBT and visualized with luminalreagents (Sigma-Aldrich) by exposure to X-ray films (X-Omat-AR,Kodak Ltd., London, UK).

Results

There is underlying pattern in the apparently irregulardistribution of DoLA interneurons

DoLAneurons in the developing spinal cord are apparently distributedin an infrequent irregular pattern (Fig. 1A and B; see the Introductionfor discussion of distribution types). We have shown previously thatthere is a tendency for higher numbers of these cells at rostral somitelevels compared to caudal somite levels [29]. To determine if there isany regularity in the positioning between individual cells along therostrocaudal axis we made a detailed analysis of the position of thesecells in20size-matchedembryosat22–24 hpf thathadbeensubjectedto in situ transcript hybridisation against tbx16 transcripts.We plottedthe positions of these cells using the flanking somite boundaries as

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coordinates and we measured the distance between cells in terms ofthese “somite units” (hemisegments). In fixed embryos subjected to insitu transcript hybridisation it can be difficult to identifywith certaintythe rostral-most somite. Therefore, to ensure the comparability of theplotted cell positions between embryos we used the somite pair

immediately overlying the end of the yolk extension as a commonreference position upon which to align position data from individualembryos and we counted somites from the most recently formedsomite at the caudal end of the embryo (Fig. 1C; for more details onsomite designation see Materials and methods). This method was

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deemed more accurate than measurement of the physical distancebetween tbx16-expressing cells as it should be insensitive to slightvariations in embryo size.

As previously observed [29], the frequency of DoLA neurons(tbx16-expressing cells per flanking somite) was higher rostrallythan caudally (Fig. 1D). The highest frequency of DoLAs appears tooccur in the spinal cord region juxtaposed to somites 21–24 (whencounting from themost recently formed somite)which correspondsto somites 9–12 (when counting from the rostral-most somite).When position data from all embryos was pooled, the frequencydistribution of cells at any particular positionwas not randomundera Poisson model (Fig. 1E) and a Fourier analysis did not showperiodicity. Therefore, DoLA neurons may possibly show what istermed a “clumped” or “contagious” distribution (in populationdispersionparlance) ipsilaterally on the rostrocaudal axis (since theydo not show a metameric/uniform or random distribution).

The non-random, non-metameric and possibly clumped distri-bution of DoLA neurons on the rostrocaudal axis may be requiredfor their normal function and suggests the possibility of interactionbetween these cells. Therefore, we examined the position of eachDoLA relative to others ipsilaterally using autocorrelation analysis(see Fig. 1F). This indicated that the existence of a DoLA neuron atany one position may raise the possibility of finding another DoLAipsilaterally within the next 4 somite units (i.e. clumps may span 5somite units or less, see asterisks in Fig. 1F that indicatesignificance at p<0.05). Conversely, at a distance of 13 somiteunits or greater there may be a reduced probability of finding aDoLA neuron (i.e. clumps are separated by 12 somite units orgreater). This analysis supports the possibility of a clumped natureof the distribution. It also supports that the distribution of DoLAneurons is similar on the left and right sides of the spinal cord (seeFig. 1F). However, according to the Nyquist–Shannon samplingtheorem the results of our analysis can be nomore than suggestivesince the length of embryos is not sufficient to make an accurateprediction of the proposed pattern (i.e. the length in somite units isnot at least twice the “bandwidth” of 12+5 somite units) [43].

DoLA neurons are first observed in the spinal cord at thedorsoventral level of the future dorsal longitudinal fasciculus(DLF) and their cell bodies become wrapped around this axonbundle [29]. Initially, their single axon extends along the DLF andthey have no other observable neurites. We would not expectDoLAs to interact across the midline of the developing spinal cord.However, upon examining the contralateral correlation betweenDoLA neurons we were surprised to observe a very significant(though not absolute) association (Fig. 1G).

Fig. 1 – DoLA neurons display a rostral clumping distribution. (A anddistribution of tbx16-expressing cells (DoLA interneurons) from dorsaup in B (scale bar: 250 μm). (C–G) Statistical examination of DoLA neu(C) Somite unit coordinate system for plotting the position of tbx16-eembryoswithdiffering total numbers of somites. The somite locatedabThis was found to be somite number 15 counting from the caudal endfrequency of tbx16-expressing cells per flanking somite in the somite csimilar [29]. (E) Frequency histogram of the number of tbx16-expressdeparture from random distribution under a Poisson model). (F) Autbetween tbx16-expressing cells. Data is from 20 embryos combined. Veaxis shows distances between cells on the left, “L,” right, “R” and wheautocorrelations). (G) Cross-correlation plot of logn transformed left aposition showingapositive correlationbetween somiteson the left andis a greater likelihood of a cell on the other side at the same somite co

Somitic mesoderm is unnecessary for DoLA neuron birth

The segmental distribution and identity of primary motoneurons isdetermined by signals from adjacent somitic mesoderm. In ouranalysis of rostrocaudal DoLA distribution above we did not seeevidence for an influence from somitic mesoderm although it isremarkable that tbx16 expression is seen at the same dorsoventrallevel in both the developing spinal cord and newly formed somites[29]. Indeed, in our previous publication we show that cells in thedorsocaudal extremity of newly formed somites express tbx16 athigher levels, and appear to be frequently juxtaposed to the tbx16 cellsin thedeveloping spinal cord (see Fig. 3 in Tammeet al. 29). To analysethis possibility in more detail we examined this region in thedeveloping spinal cords of 60 embryos by dividing the dorsalextremity of each somite adjacent to tbx16 expressing cells intothree virtual partitions: rostral, medial, and caudal, each spanning onethird of the rostrocaudal width of a somite. We then assessed thejuxtaposition of any tbx16 expressing CNS cells to these regions of thesomite. We did not find any significant bias for the juxtaposition ofthese cells to anyparticular rostrocaudal regionof a somite (Fig. 2BandC), which does not support a rostrocaudal positioning influence frommesoderm.

It is conceivable that inductive signals from presomitic mesodermmight be important in the differentiation of DoLAs from precursorcells. To exclude a role for paraxialmesoderm inDoLA birthwe soughtto remove this tissue from developing embryos bymanipulating geneactivity. Amacher et al. have demonstrated complete loss of caudalmesoderm in embryos lacking activity of both the tbx16 and no tail(ntl) genes [44]. We attempted to phenocopy this loss of gene activityby simultaneous injection into developing embryos of MOs blockingtranslation of tbx16 and ntl. Inhibition of ntl activity by MO injectionhaspreviouslybeendemonstrated [37]. This causesmorphological andbiochemical defects indistinguishable from the ntlmutant phenotype[45,46]. However, inhibition of tbx16 activity by MO injection had notpreviously been demonstrated so we first validated the inhibitoryactivity of our tbx16-MO by Western blot analysis of tbx16-MO-injected embryos. This revealed severe (although incomplete)reduction of the tbx16 protein expression (Fig. 2A). Injection oftbx16-MO(Fig. 2F)phenocopies themorphological defectsobserved inthe homozygous tbx16mutant line [47] (Fig. 2E). The injection of ntl-MO causes loss of posterior somitic mesoderm (Fig. 3G) and thesimultaneous injection of tbx16- and ntl-MOs causes the complete lossof somitic mesoderm (Fig. 2H and I). tbx16 mRNA remains in a smallnumber of cells in these embryos in the vestigial tail (Fig. 2I). Toconfirm that all paraxial mesoderm had been removed and that the

B) Trunk and tail region of a 30 hpf embryo stained to show thel (A) and lateral (B) perspectives. Rostral is to the left and dorsal isron distribution (data represent values from 20 embryos).xpressing cells and to allow comparison of these values betweenove the endof the yolkextension is usedas a referencepoint (“0”).in the embryo with the greatest number of somites. (D) Mean

oordinate system. Left and right sideswere found tobe statisticallying cells occurring at any position (χ2 value indicates significantocorrelation analysis of the relative positions (in somite units)rtical axis is Moran's I spatial autocorrelation statistic. Horizontaln position data are combined, “total” (* indicates significantnd right side tbx16-expressing cell frequency data for each somiteright sides of theembryo (i.e. if a cell exists onone side, then there

ordinate position.) Threshold lines indicate 95% confidence limits.

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remaining cells transcribing tbx16were DoLA neurons we performeddouble in situ transcript hybridisation againstmRNAs of tbx16 and isl1.All cells containing tbx16 mRNA also transcribed isl1 indicating thatthese cells are neuronal and not of mesodermal origin (Fig. 2I). Thisconfirmed that paraxial mesoderm is not required for the differenti-ation of DoLA neurons. It also indicated that tbx16 activity, while amarker of DoLA neurons, does not appear to be required for their

differentiation. Indeed, reduction of tbx16 activity may increase DoLAdifferentiation (see below).

DoLA interneurons migrate rostrally after birth

We have previously observed tbx16-expressing neuronal cells withsingle, rostrally extending processes that are reminiscent of either

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axonal growth cones or migratory processes. These are juxtaposed to,approximately, the 7th to 14th most recently formed somites in 22 hpfembryos [29]. Interestingly, these processes can apparently beobserved by whole-mount in situ transcript hybridisation since theycontain tbx16mRNA [29]. Migration of cells can typically be observedafter dye-labelling of cells in living embryos. However, the infrequentirregular distribution of DoLA interneurons makes the directedlabelling of individual cells very difficult since their positions cannotbe predicted. Therefore, we used an ultraviolet (UV) activatable dye(“dye uncaging”) to develop a strategy for labelling a region of thedeveloping spinal cord in the expectation that cellsmigrating rostrallywould be seen to leave this region. Briefly, we injected embryos at theone-cell stage with DMNB-caged fluorescein dextran. The dye wasactivated in the unsegmented tail region at 13 hpf with an UV laser.Embryoswere thenallowed todevelop for varying timeperiods beforeexamination microscopically for fluorescence. In most cases, at leastone cell could be observed in the developing spinal cord rostral to theregion that had been uncaged. In many cases several cells could beobservedwithunique, irregulardistributions(Fig. 3A). Todemonstratethat these rostrally migrating cells are DoLA neurons we tookindividual labelled embryos immediately after observation and fixedthem for in situ hybridisation against tbx16. In every case (n=10) theirregular pattern of distribution of tbx16-expressing cells in theseembryosmatched that previously observed for the fluorescing cells intheCNS rostral to theuncagedzone (Fig. 3B shows the sameembryoasin Fig. 3A) indicating that the rostrally migrating cells are DoLAinterneurons (for additional embryo alignments see supplementaryfile S1). No other cell types in the caudal spinal cord were observed tomigrate rostrally at this time. To support these findings further, weundertook time-lapse analysis utilising a HuC-Kaede transgeniczebrafish line to highlight neurons in the dorsal spinal cord.Fluorescent protein in neurons expressing the transgene was photo-convertedat16 hpf andembryosobserved for7 h.During this time thetail is extending in the caudal direction while a limited number ofneurons arenoted tobemigrating in the rostral directionalong theDLF(Fig. 3C and D and Supplementary Fig. S2). Given the position of thecells on the DLF and the developmental time at which they areobserved we conclude that these migratory cells are the same as weobserve by the dye uncaging protocol and are DoLA interneurons.

Tbx16 protein is not necessary for DoLA migration

T-box transcription factors have been implicated in a number ofmorphological cell movements in the developing embryo includingbrachyury duringmouse gastrulation [48,49], tbx5 duringmigration oflimb precursor cells [50], and tbx16 during migration of trunkmesoderm [47]. Since the differentiation of DoLA interneurons does

Fig. 2 – Influenceofoverlying somiticmesodermonDoLA interneuronzebrafish lysates demonstrating efficient inhibition of Tbx16 proteinembryos; right lane, lysates from tbx16-MO-injected embryos. (B) Diaformed somites into rostral (R),medial (M), and caudal (C) regions forneurons in the spinal cordwhencompared toadjacent recently formedembryos at 24 hpf. Rostral is to the left anddorsal is up. (D) Standard co(G) ntl-MO injected. (H) Dual tbx16- and ntl-MO injected. (I) Dual tbx1(blue)and tbx16 transcript (red)expression.White arrowheads indicatorganised somites. Asterisk indicates notochord (D) or a lack of notocdue to incorrect cellular movement during tail extension. Scale bars i

not require Tbx16 activity (see above) and DoLA neurons migraterostrally, we asked whether Tbx16 might be required for migration ofDoLAs. By co-injecting embryoswith tbx16-MOand theUV-activatabledye we were able to use our dye-labelling strategy to determinewhether tbx16 activity is required for rostralwardsmigration of DoLAs.We observed that cells possessing tbx16 mRNA but with greatlydecreased Tbx16 levels (Fig. 2A) could still migrate rostrally from thezone of activated dye (Fig. 3E and F). The pattern of migration wassomewhat disturbed but thismaybe due to the disruption of overlyingparaxial mesoderm through loss of tbx16 activity. Our results implythat tbx16 is not required for DoLA migration although we cannotexclude that tbx16 is required for correctmigratory pathfinding or thatthe tbx16-MO still permits low levels of Tbx16 protein synthesis thatare sufficient for DoLA migratory behaviour. The majority of tbx16-expressing cells (97%, n=121) inwild type embryosmigrate 5 somiteunits or less (Fig. 4), consistent with the idea that the first DoLA cellsmay differentiate at the 10-somite stage, as we discuss below.

Interestingly, during the course of our experiments we notedthat injection of tbx16-MO into embryos led to an increase in theobservable numbers of DoLA neurons. This was an increase from20.5±1.27 (SEM) per embryo at 24 hpf in wild type to 32.0±3.72(SEM) per embryo (P>0.001, n=85, data not shown). Thus,apparently tbx16 expression suppresses the formation of DoLAneurons, although we cannot exclude an indirect effect caused bydisruption of trunk paraxial mesoderm.

DoLA neurons persist after hatching

Previous analyses of DoLA neurons have presented conflicting resultsregarding the persistence of DoLA neurons after embryogenesis.Bernhardt et al. (1990) suggested that DoLAs may die before larvalstages while Higashijima et al. (2004) identified a small number ofGABA-positive, retrograde labelled interneurons with DoLA-likemorphologies at 4 days post-fertilisation (dpf). We have been unableto detect tbx16-expressing cells in the spinal cord after 3 dpfsupporting the idea that DoLAs either do not survive to this stageor that they repress tbx16 transcription after this time. Developmentof a strategy to label easily DoLA neurons provided us with theopportunity to test these possibilities. Four embryos with dyeactivated at 13 hpf were observed at 2, 4 and 8 dpf and showed thepersistence of labelled cell bodies until at least 8 dpf in the expectedposition on the dorsoventral axis of the spinal cord (Fig. 5). Thisconfirms that DoLAs persist after hatching but down regulate tbx16expression. The background fluorescence generated by the dyeuncaging technique increases with time and did not permitobservation of neurites to assess the arbour morphology of thesecells.

originandpositioning. (A)Western immunoblot analysisof 12 hpftranslation. Left lane, lysates from standard control MO-injectedgram displaying the strategy used to divide the most recentlyDoLA position analysis. (C) There is no preferred position for DoLAsomiticmesoderm. (D–I) Lateral viewsofmutantandMO-injectedntrol (CON)-MO injected. (E) tbx16mutant. (F) tbx16-MO injected.6-MO and ntl-MO-injected embryo stained to show isl1 transcriptedual-stainedcells. Brackets identify regionsofmesodermlackinghord (G). Black arrowheads show accumulation of cells in the tailndicate 100 μm.

Fig. 3 – DoLA interneurons migrate rostrally after birth. (A) 17 hpf embryo 4 h post-dye uncaging/activation reveal migrant cells onthe dorsal longitudinal fasciculus in the developing spinal cord rostral of the region activated (white arrowheads). (B) The sameembryo from (A) stained by in situ transcript hybridisation to show tbx16 transcripts. Black arrowheads indicate neurons in thespinal cord that appear to have identical spatial patterning and positions to cells that have migrated rostrally. See supplementaryfile S1 for additional alignments. (C and D) Time-lapse analysis of migratory neural cells in the spinal cord of HuC-Kaede transgeniczebrafish. Arrowheads (a and b) indicate the same non-migratory cells in C and D for positional reference. Arrows (1, 2, and 3)indicate individual cells migrating rostrally (towards the left) at 105 min (C) and 370 min (D) post-Kaede activation. Cell 2 can beseen in both C and D. See Supplementary Fig. S2 for the time-lapse movie from which these still images are taken. (E) A 17 hpfembryo co-injected with tbx16-MO and uncaging dye exhibits migrant cells (white arrowheads) in the spinal cord rostral to theactivated region. (F) The same embryo as in (E) stained by in situ transcript hybridisation against tbx16 transcript displaying tbx16-expressing cells with an identical spatial pattern to the migrant cells (black arrowheads). Rostral is to the left and dorsal is up inall images. Size bars indicate 100 μm.

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Discussion

The need to analyse irregular cell distributions in developingembryos

Most experimental analyses in developmental biology are based ondisrupting the mechanisms that control a process of interest. Theresultant abnormal embryos are then used as assays for investigatingthesemechanisms. A classic example of this is usingmodel organisms

to screen for mutations affecting embryo development. When amutation is found that disrupts a developmental process of interest,themutatedgene canbe identified. Analysis of thegene thenacts as anentry point into understanding themolecularmechanisms underlyingthat developmental process. This strategy has been remarkablysuccessful leading to a significant expansion in our knowledge ofdevelopmental control mechanisms over recent decades. However,this strategy is critically reliant on a researcher's ability to identifydisruptions to a normal pattern. As a result it is strongly biased toanalysis of regular patterns. Mechanisms controlling apparently

Fig. 4 – Analysis of the distancemigrated by DoLA neurons rostralto the area of uncaged dye and relative to adjacent somitic tissue.

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irregular patterns of cell distribution have been largely ignored.However, apparently irregular distributions are common in embryodevelopment, especially in the developing CNS where the majority ofneurons show these distributions.

In previous work in zebrafish we found that the gene tbx16 isuniquely useful as a marker of a single neuronal cell type—theDoLA neurons that develop in the embryonic spinal cord. In thisstudywe attempted to gain insight into the factors that control andconstrain the apparently irregular distribution of DoLA neurons.

There is a cryptic organisation in the apparently irregulardistribution of DoLA interneurons

DoLA neurons, like most of the identified interneuron cell types inthe embryonic spinal cord of zebrafish, are arranged along therostrocaudal axis with an infrequent irregular distribution of less

Fig. 5 – DoLA interneurons persist for longer than previouslyreported. (A) Lateral view of a 4 dpf embryo that has undergonethe uncaging procedure showing 2 dye-labelled DoLAinterneurons (white arrowheads) in the developing spinal cordrostral to uncaged tissue (demarcated by dashed white lines).(B) A dorso-lateral viewof the same embryo at 8 dpf showing thesame dye-labelled cells (note that the different viewing angles inA and B make the width of the embryo appear different in eachimage). Rostral is to the left. Size bars indicate 100 μm.

than onecell per spinal hemisegment.When examinedmore closely,the distribution of DoLA neurons is found to show contralateralcorrelation, an increase in cell abundance rostrally aswell as possibleipsilateral clumping. One possible explanation for the significant butimperfect contralateral correlation of DoLA neurons could be thesimultaneous birth of contralateral cell pairs during trunk and tailformation followed by variability in their distance of rostralwardsmigration. Irregular death of DoLAs could also contribute to theapparent irregularity of the pattern but the rapid nature of apoptosisand the irregular distribution of DoLAs would make apoptosingDoLAs difficult to observe.

DoLAs appear to be the only cells showing rostralwardsmigration in the early caudal embryonic spinal cord

Single cell labelling of DoLA neurons, while possible, is haphazardsince their infrequent irregular distribution makes it difficult topredict their position on the rostrocaudal axis. We were able tospecifically label DoLAs using an activatable dye approach. Inconjunction with time-lapse observation we discovered that DoLAsare the only rostrallymigrating neurons thatwe could observe in thecaudal embryonic spinal cord prior to 24 hpf. Our data support thatthe majority of these cells migrate for distances to a maximum ofapproximately five somite units. We do not understand how thismigratory distance is controlled or its significance in DoLA function.We have shown that lateralmesoderm is not required for DoLA birthbut it remains possible that the mesoderm plays a role in organisingthe final, possibly clumped distribution of DoLAs.

DoLAs (or their precursors) probably arise with tailbudformation and persist after hatching

At 24 hpf the rostral-most tbx16-expressing neurons are foundneighbouring the 5th formed somite pair. Prior to this, at 16 hpf alltbx16-expressing neurons are separated from the head region by atleast 9 pairs of somites; that is, they are juxtaposed to the 10th formedsomite pair. This difference of 5 somite pairs correlates our uncagingdata that indicates that themajority of tbx16-expressing cellsmigratethe equivalent of 5 somite units. These data taken together suggestthat DoLA neurons or their precursors may develop in a position inthe spinal cord nomore rostral than the level of the 10th somite pair.Spatially, this region is close to the position where the tailbud firstdevelops at 11–12 hpf (3–6 somite pair stage) [51]. One possibility isthat DoLA cells (or their precursors) may differentiate as pairsprogressively in thenascent spinal cord of the extending tail from thetime the tailbud is formeduntil the tail has fully extended. Thecells ofeach pairmay thenmove to theDLF on opposite sides of the embryo.The first DoLAs begin to express tbx16 by approximately 16 hpf (the10 somite stage) and, sometime thereafter, migrate rostrally byapproximately 4–5 somite units to reach their maximum rostralextent adjacent to the fifth somite pair. This possible birth ofDoLAsortheir precursors in pairs in the nascent spinal cordmight explain thestrong cross-correlation in their final position. A dependence of DoLAdifferentiation on the start of tail formation is thus consistent withthe rostral limit of their distribution in the spinal cord.

DoLAs cease to express tbx16 after three days. Initially they areGABAergic cells [34,52,53],which is characteristic ofmany inhibitoryneurons, but they appear to down regulate GABA expression around4 dpf [53]. This and other data [28] suggested that these cells mightdie after hatching (2–3 dpf), which implied that DoLA activity was

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only needed prior to hatching. This combined with the projection ofDoLA axons into the hindbrain [54,55] and the close physical contactbetween DoLA cell bodies and the dorsal longitudinal fasciculus [29]might suggest that DoLAs have an inhibitory role on embryo trunkand tail movement before hatching.We have shown the presence ofDoLAneurons to8 dpf (5–6 daysposthatching) indicating that thesecells persist, and possibly continue to function after hatching.

A new in vivo system for studying factors controllingneuronal migration

In vivo labelling of individual DoLA cells is difficult since theirinfrequent irregular distribution means that we cannot anticipatetheir position. In order to observe whether DoLAsmigrate rostrallywe developed a novel labelling strategy using an UV-activatabledye to label the caudal extent of the developing spinal cord out ofwhich migrating cells then move. This procedure revealed thatDoLAs are, indeed, migratory and that they are the only spinal cordcells to move in this way from this area at the stages analysed.

Studies determining the role of particular genes and proteins inneuronal migration typically involve the use of explanted tissue orcultured cells placed on manipulated growth media under condi-tions drastically different from those occurring in the developingCNS. The ability to identify a unique migratory neuronal cell type inliving zebrafish embryos suggests that this will be a valuable systemfor studying factors controlling neuronal migration under in vivoconditions. The activity of a number of genes can be manipulatedsimultaneously in zebrafish embryos by injection of morpholinoantisense oligonucleotides and/or mRNAs to block gene activities ordrive gene expression respectively. Embryos can also be placeddirectly intodrug solutions to inhibit or activatemolecularprocesses.We injected a mixture of an MO blocking tbx16 translation togetherwith the UV-activatable dye to follow the effects of loss of tbx16activity on DoLA migration. This showed that loss of tbx16 does notappear to inhibit DoLA migration. We have also shown that tbx16 isnot required for DoLA differentiation. Rather, loss of tbx16 functionappears to increase the number of DoLAs that form. Thus, the uniquerole that tbx16 plays in the formation and/or function of DoLAneurons before hatching remains to be determined.

Conclusions

Careful statistical analysis of an apparently “irregular” (non-uniform) pattern of interneuron distribution in the embryonicspinal cord of zebrafish has revealed the existence of crypticorganisation. This supports that greater efforts should be made tounderstand the factors directing and constraining the formation ofirregular patterns of cell distribution in the CNS and other tissues ofdeveloping embryos. We have also used a novel cell-labellingstrategy to demonstrate that DoLA neurons migrate rostrally. Thisstrategy may be useful for in vivo analysis of factors controllingneuron migration.

Authors’ contributions

S.W. undertook the embryo injection, in situ transcript hybridisa-tion, uncaging technique, western blotting, and some of thestatistical calculation and drafted the manuscript.

J.C. contributed to the statistical analysis.R.T. performed some of the statistical analyses and the cell

counting.A.G. performed the time-lapse analysis of neuronal migration

in HuC-Kaede transgenic fish and produced associated media filesJ.W. analysed neural position in relation to neighbouring

somites.M.L. directed the research and edited the manuscript.

Acknowledgments

We thank Meredith Wallwork for assistance with confocal micros-copy and processing of images. We thank various people for gifts ofresources: the transgenic line Tg(elav3:Kaede)rw0130a and the clonefor production of the probe against isl1 from Hitoshi Okamoto, thetbx16 mutant zebrafish line from Heather Verkade, and the Tbx16antibody from Charles B Kimmel. This workwas supported by fundsfrom the Australian Research Council via its Special Research Centrefor the Molecular Genetics of Development (S00001531). The workwas carried out under the auspices of the Animal Ethics Committeeand the Institutional Biosafety Committee of the University ofAdelaide. We thank Judith Eisen, Patty Solomon, and Jarrod Johnsonfor critical comments on the manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.yexcr.2010.06.020.

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