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Seminars in Cell & Developmental Biology 20 (2009) 942–946

Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology

journa l homepage: www.e lsev ier .com/ locate /semcdb

eview

ive imaging of development in fish embryos

on Clarke ∗

RC Centre for Developmental Neurobiology, King’s College London, New Hunt’s House, 4th Floor, Guy’s Hospital Campus, London SE1 1UL, United Kingdom

r t i c l e i n f o

rticle history:vailable online 12 August 2009

a b s t r a c t

Understanding the cellular and molecular mechanisms that drive the development of embryos requires a

eywords:ebrafishmbryosorphogenesis

detailed knowledge of the way cells divide, move, change shape, interact with one another and die duringembryogenesis. Ideally this should be analysed in intact embryos using minimally invasive techniques.Because of their easy accessibility, external development and excellent transparency the teleost embryohas emerged as probably the premier vertebrate model for this type of study. This review will discusssome of the recent advances in this field including attempts to image every cell and their movementsduring the first 24 h of development as well as other studies that focus on the development of specific

ime-lapsen vivo imaging organs or high resolution analyses of the behaviour of individual cells.

© 2009 Elsevier Ltd. All rights reserved.

ontents

1. Some technical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9421.1. Embryo stabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9421.2. Mosaic or ubiquitous labelling? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 943

2. Capturing every cell movement and division in the embryo: generating the digital embryo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9433. In toto imaging or more cell specific imaging? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9444. Subcellular resolution in vivo? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9445. Seeing the unexpected . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9446. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945

If you want to understand how a house is built a reasonablepproach would be to actually sit and watch a team of buildersonstructing a house from bricks and mortar. It may not tell youho is directing the builders or how they receive their instruc-

ions, but it will tell you where the bricks come from, how they areoved around and how they combine together to create different

hapes. If you want a detailed knowledge of this process you willeed to view the construction from more than one viewpoint andome parts of the build will only be visible from inside the housetself. To understand how an embryo is constructed needs a similarpproach. If we want a deep understanding of the mechanisms that

ing in their normal environment, but for many vertebrate embryosthat develop either within the reproductive tract of the mother orwithin protective, opaque shells this is simply not possible. Teleostembryos however have long been recognised for their transparencyand accessibility for time-lapse microscopy [1] and have emergedas the premier vertebrate model for in vivo imaging studies of earlydevelopment. This article will focus on recent progress made study-ing zebrafish embryos, but other teleost species are being used togood effect too (e.g. references [2,3]).

1. Some technical considerations

ontrol the development of embryos, we need to understand howells and tissues behave (move, interact, change shape, divide, die)uring the processes that shape an embryo. Ideally our analysesf these behaviours should be made in intact embryos develop-

∗ Tel.: +44 20 7848 6463.E-mail address: jon.clarke@kcl.ac.uk.

084-9521/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcdb.2009.08.003

1.1. Embryo stabilization

A small number of variations on a theme are being used to sta-

bilize the position of embryos as they grow. Most include the useof a non-toxic, highly transparent, low melting point agarose gel inwhich the embryo is embedded. Others use a viscous solution ofmethyl cellulose. The methyl cellulose approach has the advantage

J. Clarke / Seminars in Cell & Developmental Biology 20 (2009) 942–946 943

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ig. 1. Watching organogenesis—building the zebrafish neural tube. Mosaic fluoresy 8 h of confocal time-lapse acquisition reveals many neural plate cells cross the midline crossing starts in frame F and is completed by frame K. This midline cross

mportant step in morphogenesis of the lumen of the fish neural tube (ref. [4]). Fram

hat it easily accommodates the changing morphology of thembryo (for example the outgrowth of the tail), but the overall sta-ility of the embryo is consequently lower than that of low meltgarose and therefore comes with the risk of regions of interestoving out of the field of view. Agarose gel will constrain the mor-

hological changes of the embryo, but stability is high. The tails the most problematic region for growth, but careful cutting ofhe agarose gel away from the tail can allow for growth of thisegion while maintaining good stability of the rest of the embryo.t stages beyond 24 h of development muscular contractions canlso lead to problems of stability for imaging. This can be overcomey anaesthetising with tricaine or paralysing with neuromuscularlockers.

Imaging one embryo at a time is a rather slow way to collectata, but this is now efficiently overcome by the use of a motorizedY specimen stage coupled to software with a point finding func-

ion. These allow rapid and accurate sequential imaging of severalmbryos throughout a single time-lapse period.

.2. Mosaic or ubiquitous labelling?

If you need a detailed analysis of the behaviour of individualells then this is most easily obtained by visualizing a relativelymall number of fluorescently labelled cells scattered throughout aissue of unlabelled cells. If the cells are labelled with a fluorescent

embrane protein this affords the best resolution of the outline ofhe cell and will include the filopodial and lamelipodial extensionsn the cell surface. The inclusion of a nuclear signal (for exam-le H2B-RFP) helps give visual weight to the cell soma as well asbviously being essential for the examination of nuclear dynamicsnd accurate monitoring of cleavage at mitoses. Obtaining mosaicabelling in the zebrafish embryo is simply achieved by injecting

RNAs for the fluorescent proteins into only one cell at the earlyleavage stages of the embryo. In our lab we reliably inject intone cell at the 64 or 128 cell stage [4,5]. This results in fluorescentells scattered throughout the tissues and investigators then sim-ly screen through their injected embryos to find labelled cells inhe particular location of interest (Fig. 1).

If on the other hand you need to understand how a populationf cells moves to generate a particular organ or region then it isrobably necessary to analyse the movements of all the cells inhat population. This can be achieved in the zebrafish embryo bynjecting the mRNAs of fluorescent proteins into the 1 cell stage ofevelopment. Actually this often results in rather uneven labelling

f cells as the RNA appears to be often unevenly distributed throughhe early cleavages. Nonetheless this can be a very informativepproach (e.g. [6,7]). Alternatively there are many excellent trans-enic lines available expressing for example H2A-GFP in every cellucleus [8] or the actin-GFP line that outlines the cortex of every

belling of cells in the left hand side of the zebrafish embryo’s neural plate followedof the neural primordium (arrowed) to contribute to both sides of the neural tube.ent is dependent on cell division and further analyses have shown that this is anwere selected from 8 h of time-lapse acquisition that captured images every 5 min.

cell, and these lines have excellent even labelling of all cells. Thesecell-ubiquitous lines of course do not identify particular popula-tions of cells and tissue specific lines can be helpful if appropriatedrivers are available. A good example here is the CldnB::lynGFP linewhich allows excellent visualization of the lateral line primordia(e.g. [9]).

2. Capturing every cell movement and division in theembryo: generating the digital embryo

The ambition to capture essentially all the movements andall the divisions of every cell during the first day of zebrafishdevelopment has amazingly recently been achieved [7]. This team,comprising physicists and developmental biologists, developed anew mode of microscopy that they called digital scanned laser lightsheet fluorescence microscopy or DSLM for short. DSLM delivers asheet of laser light to the specimen rather than a single beam. Thissheet of light is moved rapidly through the embryo (in reality itis the embryo that is moved) and the fluorescence emitted fromthe specimen is detected through an objective that lies orthogonalto the light sheet. Use of a sensitive EMCCD camera for widefielddetection captures all the fluorescence emitted from the specimenat once; detection is therefore quick. Although most of the cells ofthe embryo have a high transparency they lie on the more opaqueyolk cell mass and if this is in the light path it will degrade the signalresolution. To overcome this the authors captured simultaneousimages from two orthogonal light paths (horizontally and verti-cally through the embryo). The data obtained from the two pointsof view are first processed separately before being recombined togenerate a single complete dataset. The advantages reported by theauthors for DSLM are a highly efficient and uniform illuminationintensity at all levels through the embryo, high image quality and avery low excitation energy that results in very low photobleachingand very low phototoxicity. The physics and measurements thatsupport these claims are beyond the scope of this overview butare available in detail with the original article. The datasets cap-tured in this study are staggering. For each embryo they captured1.5 billion voxels of information per minute over a 24-h recordingperiod, which generated 55 million nucleus positions in the com-plete dataset gathered from 5 wild-type embryos and one mutant.Digital reconstruction of the data then allows automated trackingof all nuclei movements and divisions and the generation of “dig-ital embryos”. Viewing this amount of data and making sense ofit is a challenge for our brains, but the simple steps of colour cod-

ing the directionality of movements and divisions helps the viewerinterpret the mass of dynamic information. Since the informationis digitized it can be quantified and compared both between indi-vidual embryos and between normal and mutant behaviours. As asimple example of how this technology leads to quantification of

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44 J. Clarke / Seminars in Cell & Deve

orphogenetic information the authors compared data collectedrom wild-type embryos and a mutant embryo that lacks the abil-ty to respond to secreted Nodal signalling. This loss of signalling isnown to result in a loss of anterior mesendoderm and now by usingSLM this can be quantified to reveal that whereas in wild-typembryos this anterior tissue is derived from approximately 1550ells that undergo an internalization step during epiboly, only about0 cells are capable of a similar morphogenetic movement in theutant. All of the wild-type and mutant data compiled in this study

re publicly available (www.embl-heidelberg.de/digitalembryo) sothers can mine this data for their own research.

. In toto imaging or more cell specific imaging?

While the DSLM study is astonishing it “only” tells us abouthe position, movements and divisions of nuclei. It does not tells how cells are packed, organised and reorganised to drive theynamic tissue movements of embryogenesis. For that you alsoeed to visualize a membrane label to see the outline of cells andheir processes. The ambition to capture this level of behaviourf all cells (termed in toto imaging) in the developing zebrafishmbryo was first expressed by Megason and Fraser [10]. Theirpproach is to label every cell in the embryo at both the membranemembrane-GFP) and the nucleus (H2B-RFP) and capture com-rehensive z-stacks of information over time using multiphotonicroscopy. The membrane label not only gives a good represen-

ation of the overall shape of cells and the way they are packed inissues, but the authors also cleverly use it as a method to moreompletely separate the nuclear labelling in neighbouring cells. Inensely packed tissues the nuclei of adjacent cells are often so closeogether they cannot be resolved by software as separate objects.ut by including a differently coloured membrane label which will

ie between the adjacent nuclei this membrane signal can be sub-racted from the nuclear signal and thus increase the separationf adjacent nuclear signals. This is an important step to aid auto-ated recognition of individual nuclei and automated tracking to

ollow their movements and fates. These authors are also develop-ng their own custom 4D cell tracking software called “GoFigure”hat will hopefully go some way to handling the huge datasets gen-rated by in toto imaging. The ambition of in toto imaging of theegason and Fraser paper has yet to be fully realised in the form

f either a reconstructed digital embryo or a reconstruction of aarticular tissue or organ but information about this project andhe continuing development of the GoFigure software is availablet https://wiki.med.harvard.edu/SysBio/Megason/GoFigure. Theseuthors also have provided some excellent papers that consider theechnical challenges of in vivo imaging and data handling [11,12]nd serious students of in vivo imaging are recommended to readhis work.

While attempting to analyse all cells and tissues in the embryot this level of detail remains a long-term goal, much importantnformation is likely to be obtained by concentrating only on imag-ng specific tissues or cells through limited periods of development.ome very informative high resolution in vivo time-lapse analysesf organ development are being obtained and perhaps the mostmpressive are those concerning the development of the lateral linerimordium in the zebrafish. This is a relatively small, very discreteopulation of cells that migrates from a position just behind theeveloping ear all the way down the lateral flank of the embryo.s it migrates it organises the cells towards the rear of the pri-

ordia into epithelial rosettes and these rosettes are deposited

t regular intervals along the path of migration. Once depositedhese rosettes develop into the sensory neuromast structures thatetect water movements. This behaviour and the complex cellularearrangements required for directed migration and rosette for-

ntal Biology 20 (2009) 942–946

mation, deposition and replenishment all occur very convenientlyvery superficial to the embryos surface. This is perfect for excellentoptics and the movies and level of analyses they allow are amaz-ing (e.g. [9,13–16]). Another good example of analyses that focuson the dynamics of a single event is the development of axonalarbor morphology and synapse formation in the developing tec-tum [17]. Rather than looking at the collective behaviour of smallnumbers of cells this study concentrates on only a small part of asingle cell and is able to resolve how the morphology of the axonterminal branches is determined by the stabilization of synapticconnections.

4. Subcellular resolution in vivo?

Understanding many aspects of dynamic cell behaviour dur-ing development requires more than just being able to watch cellmovements. We need to be able to visualize the subcellular dis-tribution of proteins and how this may change over time duringfor example cell migration or the development of cell polarity.This is again relatively easily achieved in the zebrafish embryo asRNA injections into the early blastomeres allow us to express pro-teins fused to fluorescent reporters. Recent examples explore thedynamics of Golgi apparatus, centrosomes during in vivo migra-tion, differentiation and cell polarization [4,18,19]. Others combinethis approach with a deletion strategy to determine which pro-tein domains are required for correct subcellular localization in vivo[20,21].

This simple approach may be limiting for some studies becauseof the worry that these observations depend on an over-expressionof the protein of interest and thus may not precisely recapitulate theendogenous protein levels or distribution. This should be overcomein the future by generating stable transgenics that drive fusion pro-tein expression from endogenous control elements.

5. Seeing the unexpected

While it is good to have a very precise aim when making invivo time-lapse analyses, sometimes the data obtained reveal inter-esting and unexpected findings that have not previously beendetected or even predicted from analyses of static images. Thismay be because some cellular behaviours are simply too quick tobe frequently picked up in static images or because reconstruct-ing the sequence of dynamic behaviours is not easy from staticimages. Other behaviours are simply counterintuitive and thus invivo time-lapse can reveal cellular behaviours not predicted fromfixed specimens or from watching cells in culture. An exampleof this comes from our own and others recent work imaging thebehaviour of neural tube progenitors as they divide in the earlyneural tube. Neural progenitors in the vertebrate retina and neuraltube are elongated, highly polarized cells that stretch from the api-cal (ventricular) surface of the neuroepithelium to the basal (pial)surface. Analyses of static images had previously suggested thatwhen these cells divide they detach and retract their long basalprocess, round up and undergo mitoses at the apical side of theepithelium [22–24]. In vivo time-lapse imaging of zebrafish retinalprogenitors as well as time-lapse imaging of mammalian and avianneuroepithelia in slice cultures have however demonstrated thatin fact the basal process is maintained during what is in fact a mor-phologically very asymmetric mitosis [25–29]. More recent higherresolution time-lapse of zebrafish neural tube progenitors in vivo

and mammalian and avian progenitors in slice cultures demon-strates the details of this process are even more unexpected [5]. Inthe early neural tube the cytokinetic event is actually initiated quitedistant from the apically located cell body at the basal extremity ofmany of the progenitors, and the thin basal process itself is split as

J. Clarke / Seminars in Cell & Developmental Biology 20 (2009) 942–946 945

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ig. 2. Seeing the unexpected. Eight frames selected from a time-lapse movie of and cytoplasm are labelled with GFP and nucleus is labelled with histone-RFP. Neuraterial remains close to the apical surface of the neuroepithelium (shown arrowe

a)) and this precedes segregation of the daughter chromosomes (first seen in (f)) b

leavage appears to “unzip” the process from basal to apical (Fig. 2).leavage of the basal cell process occurs approximately 15 min indvance of the anaphase events in the apical cell body [5]. Divisionf neuroepithelial cells is thus rather unusual in two respects: firsthe spindle does not lie along the long axis of the cell and seconddistinct cleavage event is initiated before anaphase in contrast

o many cells in which cleavage is only initiated after the sisterhromatids have segregated. Location of the cleavage furrow is gen-rally thought to be established in reference to the microtubuleased mitotic apparatus in the cell body [30]—this co-ordinatespindle orientation and sister chromatid separation with contrac-ile activities at the cell cortex. How the initiation of cleavage ofhe basal process of neuroepithelial cells is regulated so far distantrom the cell body and so far in advance of chromatid separation isn intriguing problem.

. Conclusion

It seems likely that the teleost embryo will remain the premierertebrate system for in vivo imaging of development. In additiono its accessibility and optical transparency there are now manyhousands of mutant and transgenic lines that when coupled toive-imaging studies will help drive our understanding of the cel-ular and molecular processes that build the vertebrate embryo.ollaborations between biologists, physicists, optical engineers andata handlers will undoubtedly move the technology forwards soe can obtain, analyse and understand ever more sophisticated

evels of optical information. It is an exciting time to put an embryonder the microscope.

cknowledgements

I would like to thank all past and present members of my labora-ory who have helped me try to understand zebrafish development

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itor in the neural tube of a zebrafish embryo undergoing division. Cell membraneenitors are elongated highly polarized epithelial cells. During M-phase the nuclear

a)). Cytokinesis is unexpectedly initiated at the basal end of the cell (asterisked inroximately 15 min. Total time from frame (a) to frame (h) is 44 min.

and I would like to apologize to the many labs whose work I have notbeen able to mention in this short article. The time-lapse sequencein Fig. 1 was captured by Dave Lyons and the sequence in Fig. 2 byPaula Alexandre.

References

[1] Trinkhaus JP. Surface activity and locomotion of Fundulus deep cells duringblastula and gastrula stages. Dev Biol 1973;30:68–103.

[2] Hirose Y, Varga ZM, Kondoh H, Furutani-Seiki M. Single cell lineage andregionalization of cell populations during Medaka neurulation. Development2004;131:2553–63.

[3] Rembold M, Loosli F, Adams RJ, Wittbrodt J. Individual cell migration serves asthe driving force for optic vesicle evagination. Science 2006;313:1130–4.

[4] Tawk M, Araya C, Lyons DA, Reugels AM, Girdler GC, Bayley PR, et al. Amirror-symmetric cell division that orchestrates neuroepithelial morphogen-esis. Nature 2007;446:797–800.

[5] Kosodo Y, Toida K, Dubreuil V, Alexandre P, Schenk J, Kiyokage E, et al. Cytoki-nesis of neuroepithelial cells can divide their basal process before anaphase.EMBO J 2008;27:3151–63.

[6] England SJ, Blanchard GB, Mahadevan L, Adams RJ. A dynamic fate map of theforebrain shows how vertebrate eyes form and explains two causes of cyclopia.Development 2006;133:4613–7.

[7] Keller PJ, Schmidt AD, Wittbrodt J, Stelzer EH. Reconstruction of zebrafishearly embryonic development by scanned light sheet microscopy. Science2008;322:1065–9.

[8] Pauls S, Geldmacher-Voss B, Campos-Ortega JA. A zebrafish histone variant H2AF/Z and a transgenic H2A. F/Z:GFP fusion protein for in vivo studies of embryonicdevelopment. Dev Genes Evol 2001;211:603–10.

[9] Valentin G, Haas P, Gilmour D. The chemokine SDF1a coordinates tissue migra-tion through the spatially restricted activation of Cxcr7 and Cxcr4b. Curr Biol2007;17:1026–31.

10] Megason SG, Fraser SE. Digitizing life at the level of the cell: high-performancelaser-scanning microscopy and image analysis for in toto imaging of develop-ment. Mech Dev 2003;120:1407–20.

11] Megason SG, Fraser SE. Imaging in systems biology. Cell 2007;130:784–95.

12] Megason SG. In toto imaging of embryogenesis with confocal time-lapse

microscopy. Methods Mol Biol 2009;546:p.317–32.13] Gilmour D, Knaut H, Maischein HM, Nüsslein-Volhard C. Towing of sensory

axons by their migrating target cells in vivo. Nat Neurosci 2004;7:491–2.14] Haas P, Gilmour D. Chemokine signaling mediates self-organizing tissue migra-

tion in the zebrafish lateral line. Dev Cell 2006;10:673–80.

9 lopme

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46 J. Clarke / Seminars in Cell & Deve

15] Lecaudey V, Cakan-Akdogan G, Norton WH, Gilmour D. Dynamic Fgf signalingcouples morphogenesis and migration in the zebrafish lateral line primordium.Development 2008;135:2695–705.

16] Nechiporuk A, Raible DW. FGF-dependent mechanosensory organ patterningin zebrafish. Science 2008;320:1774–7.

17] Meyer MP. Smith SJ Evidence from in vivo imaging that synaptogenesis guidesthe growth and branching of axonal arbors by two distinct mechanisms. J Neu-rosci 2006;26:3604–14.

18] Zolessi FR, Poggi L, Wilkinson CJ, Chien CB, Harris WA. Polarization and orien-tation of retinal ganglion cells in vivo. Neural Dev 2006;1:p2.

19] Pouthas F, Girard P, Lecaudey V, Ly TB, Gilmour D, Boulin C, et al. In migrat-ing cells, the Golgi complex and the position of the centrosome depend ongeometrical constraints of the substratum. J Cell Sci 2008;121:2406–14.

20] Reugels AM, Boggetti B, Scheer N, Campos-Ortega JA. Asymmetric localizationof Numb:EGFP in dividing neuroepithelial cells during neurulation in Danio

rerio. Dev Dyn 2006;235:934–48.

21] von Trotha JW, Campos-Ortega JA, Reugels AM. Apical localization of ASIP/PAR-3:EGFP in zebrafish neuroepithelial cells involves the oligomerization domainCR1, the PDZ domains, and the C-terminal portion of the protein. Dev Dyn2006;235:967–77.

22] Sauer FC. Mitosis in the neural tube. J Comp Neurol 1935;62:377–405.

[

[

ntal Biology 20 (2009) 942–946

23] Sauer FC. The interkinetic migration of embryonic epithelial nuclei. J Morphol1936;60:1–11.

24] Seymour RM, Berry M. Scanning and transmission electron microscope studiesof interkinetic nuclear migration in the cerebral vesicles of the rat. J CompNeurol 1975;160:105–25.

25] Das T, Payer B, Cayouette M, Harris WA. In vivo time-lapse imaging of celldivisions during neurogenesis in the developing zebrafish retina. Neuron2003;37:597–609.

26] Miyata T, Kawaguchi A, Okano H, Ogawa M. Asymmetric inheritance of radialglial fibers by cortical neurons. Neuron 2001;31:727–41.

27] Miyata T, Kawaguchi A, Saito K, Kawano M, Muto T, Ogawa M. Asymmetric pro-duction of surface-dividing and non-surface-dividing cortical progenitor cells.Development 2004;131:3133–45.

28] Noctor SC, Martínez-Cerdeno V, Ivic L, Kriegstein AR. Cortical neurons arise insymmetric and asymmetric division zones and migrate through specific phases.

Nat Neurosci 2004;7:136–44.

29] Wilcock AC, Swedlow JR, Storey KG. Mitotic spindle orientation distinguishesstem cell and terminal modes of neuron production in the early spinal cord.Development 2007;134:1943–54.

30] von Dassow G. Concurrent cues for cytokinetic furrow induction in animal cells.Trends Cell Biol 2009;19:165–73.