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Home , Nephrozoa, Orthonectida, Planulozoa, Platynereis dumerilii, Scalidophora

OEB51 Lecture 9 Mollusk Embryology Porifera Parahoxozoa Echinodermata Hemichordata Deuterostomia Cephalochordata Chordata Urochordata Craniata Cycliophora Annelida Brachiopoda Phoronida Gastrotricha Protostomia Platyhelminthes Gnathostomulida Micrognathozoa Rotifera Nucleariida Fungi Opisthokonta Filasterea Ichthosporea Holozoa Animals Choanoflagellata Nematoda Tardigrada Arthropoda Spiral cleavage general properes II • Four cell stage: – May be equal sized blastomeres or unequal • (at 2 and 4 cell stages) Some Spiralia show Polar Lobe formaon Polar Lobe Formaon. Example 1.

Acila castrensis

Remember the polar lobe is not the same as the polar body! You can see both of these “polar things” in this movie. Make sure you know which is which! Polar Lobe Formaon. Example 2.

Pulsellum sp. Mollusca Snails, bivalves, squid

• direct and indirect development • vast majority of classes show spiral cleavage • e.g. Ilyanassa obsoleta (snail) • emerging system for molecular embryology • classes have very modified cleavage (and modified body plans) • e.g. Loligo pealei (squid): no spiral cleavage Spiral cleavage can be sinistral (to the le) or dextral (to the right)

Lymnaea stagnalis Shibazaki et al. (2010) Curr Biol Microtubules (red) and microfilaments (green) during third cleavage:

Sinistral Dextral Turns aer Turns before cleavage cleavage

The spiral arrangement can be achieved by different mechanisms Lymnaea stagnalis Shibazaki et al. (2010) Curr Biol The roles of microtubules (red) and microfilaments (green) during third cleavage: Turns aer Turns before cleavage cleavage Nocodazole inhibits Latrunculin A inhibits microtubule polymerizaon acn polymerizaon

Microtubules needed for Polymerized acn needed for Dextral cleavage Sinistral cleavage Cytoskeletal elements play different roles in dextral and sinistral embryos Lymnaea stagnalis Shibazaki et al. (2010) Curr Biol NATURE | Vol 462 | 10 December 2009 LETTERS

Sinistral or dextral arrangement of cleavage can be changed manually Lucifer Yellow

Sinistralization of Dextralization of dextral embryo sinistral embryo a d g k

+ Trace b e h l o 1Q

c f i m p 2q 2Q 1q

j n q Lymnaea stagnalis 1 Kuroda et al. (2009) Nature 1q 1q2

Figure 1 | Reversal of the third cleavage directions by micromanipulation chirality by manipulating as in d–f and culturing them. The resultant and the resultant 8-, 12- and 16-cell stage embryos. Dextral embryos at the dextral-typeeight-cellstage sinistralembryo (g, k) was compacted(h, l, o) and metaphase-anaphase (a) and sinistral embryos at the telophase (d) of the then cleaved into 12- (i, m, p) and 16-cell (j, n, q) embryos, which arose from third cleavage were manipulated. The first quartet of micromeres getting the typical non-synchronous division of macromeres (1Q) and micromeres generated was continuously pushed towards the direction opposite to (1q). Each blastomere of 1Q (o) and 1q (p) divided in the dextral-type normal by glass rods (sinistrally for the dextral embryo (b) and dextrally for anticlockwise direction and produced their descendants 2q-2Q (p) and 1q1- the sinistral embryo (e)), which resulted in chirality-inverted sinistral-type 1q2(q), respectively. a–j, Bright field image; k–q, fluorescence image with (c) and dextral-type (f) eight-cell embryos, respectively. Fluorescence- outline of blastomeres (o–q). Arrows (o, p) indicate the spindle orientation. imaged cell-lineage tracing was carried out by injecting Lucifer Yellow dye Scale bar, 100 mm. into one quadrant of the four-cell stage sinistral embryo, then reversing the Fig. 2p, v) and their internal organ asymmetry was examined in detail. do not show them20 (see below). SD is a helical deformation of the Fully grown ‘sinistralized’ and ‘dextralized’ snails had pulmonary sac, blastomeres at the metaphase–anaphase, and SI is a spiral orientation anus, male and genital pores open at the left or right side of the of the four spindles, as a consequence of SD, before the cleavage 20 body (Fig. 2e, k), just like the normal sinistral (Fig. 2q) and the dextral furrow ingression . We have succeeded in making F7 congenic ani- (Fig. 2w) snails, respectively, and internal organs, such as heart, mals, which inherit 99.2% of sinistral strain-derived and 0.8% of the stomach, liver coiling and gut looping, with the shape and positions dextral strain-derived . Remarkably, SD and SI were observed (Fig. 2f, l) just like the normal sinistral (Fig. 2r) and dextral (Fig. 2x) in all the dextral embryos oviposited by F7 animals that inherit the snails, respectively. Thus, the chirality-reversed embryos at the eight- dextrality gene(s), but not in any of the sinistral embryos oviposited cell stage developed to situs inversus. We did not observe situs solitus by F7 snails devoid of the dextrality gene(s). Thus, the organismal or situs ambiguus. The reversed-coiled snails were fertile, and pro- handedness-determining gene(s) is strongly linked to, or is, the gene duced sinistral or dextral progenies dictated by their genotype and that induces or activates SD and/or SI. We made dextral snails by not the reversed body handedness (Supplementary Table 1). pushing the micromeres of sinistral embryos from the telophase Although chirality is the most prominent at the third cleavage, it without SD. can be traced back to the first and second cleavages21. We altered, by These results suggest that chiral blastomere configuration is the key manipulation, the directions of blastomere rotations of both the factor in handedness determination, which is achieved by SD and SI sinistral and dextral embryos at the first or the second cleavage to genetically in the wild, and by micromanipulation in our experiments. produce reversed blastomere configuration at the four-cell stage. The epigenetic manipulation reprograms the left–right asymmetry However, the manipulated embryos all reverted to the original-type determination most probably by altering blastomere arrangement third cleavage (Supplementary Fig. 2). We also observed that sinistral around the 3D organizer which is specified at the 24-cell stage23. In embryos occasionally showed dextral-type blastomere arrangement the case of C. elegans, it has been reported13 that mechanical treatment at the four-cell stage even in the egg capsules, but they all showed at the six-cell stage produced chirality-reversed animals, similar to the normal anticlockwise cleavage at the third division. Thus, macro- case of L. stagnalis. Although spindle orientation is important in both mere–micromere cell contacts at the eight-cell stage embryo appear species, L. stagnalis appears to adopt a different chirality determining to be the first determining step for asymmetric development of snails. pathway (see below). We have studied the orthologues of Ga and We have previously reported that dextral and sinistral snail several cell polarity-related proteins (for example, Par6, atypical embryos are not mirror images of each other at the third cleavage PKC) for the sinistral and the dextral L. stagnalis, but no chirality- (refs 20, 22). The dominant dextral snails exhibit spiral deformation dependent difference was observed in their expression (T. Homma, (SD) and spindle inclination (SI), while the recessive sinistral snails M.S. and R.K., unpublished results). 791 ©2009 Macmillan Publishers Limited. All rights reserved Ilyanassa obsoleta – development to veliger Ilyanassa obsoleta – micromere fate maps

8 cell stage

First quartet micromeres: 1a, 1b, 1c, 1d 1a and 1c make normally

When 1a and 1c are removed, eyes are missing Ilyanassa obsoleta – micromere potenals

Cytoplasmic determinants are not the whole story

Put 1a into the posion of 1b: 1a no longer makes eyes!

What prevents 1b and 1d from making eyes? Ilyanassa obsoleta – micromere potenals

Experiment: FIRST Remove the polar lobe contents at first cleavage THEN Put 1d into the posion of 1a

1d can make eyes!

The polar lobe contents give 1D the power to repress 1d’s making potenal Ilyanassa obsoleta – recall that micromeres have different potenals

What is special about the polar lobe (and the D lineage) that makes it able to repress potenals of neighbouring cells? Ilyanassa obsoleta – one molecular mechanism for segregang cytoplasmic determinants The D lineage inherits specific mRNAs encoding for signaling molecules This enables 1D to signal to neighboring cells like 1d

mRNA tubulin centrosome DNA mRNAs segregate to specific cell lineages Loligo pealeii – these mollusc embryos look nothing like spiralian embryos Cephalopod hatching

hps://www.youtube.com/watch?v=r6geyV0i3QI RESEARCH | REPORTS

ORIGIN OF NOTOCHORD by double WMISH (Fig. 2, F to L). Although none of the genes were exclusively expressed in the mesodermal midline, their combined Development of the annelid coexpression was unique to these cells (implying that mesodermal midline in and chor- damesoderm in are more similar to axochord: Insights into each other than to any other tissue). It is unlikely that the molecular similarity between annelid notochord evolution and mesodermal midline is due to in- dependent co-option of a conserved gene cas- Antonella Lauri,1*† Thibaut Brunet,1* Mette Handberg-Thorsager,1,2‡ sette, because this would require either that this Antje H.L. Fischer,1§ Oleg Simakov,1 Patrick R. H. Steinmetz,1‖ Raju Tomer,1,2¶ cassette was active elsewhere in the body (which is not the case) or that multiple identical inde- Philipp J. Keller,2 Detlev Arendt1,3# pendent events of co-option occurred (which is The origin of has been debated for more than a century, with one key issue being unparsimonious). As in vertebrates, the meso- the emergence of the notochord. In vertebrates, the notochord develops by convergence dermal midline resembles the neuroectodermal and extension of the chordamesoderm, a population of midline cells of unique molecular midline, which expresses foxD, foxA, netrin, slit, identity. We identify a population of mesodermal cells in a developing , the marine and noggin (figs. S6 and S7) but not brachyury or annelid dumerilii,thatconvergesandextendstowardthemidlineandexpressesa twist.However,unlikeinchicken(10), the an- notochord-specific combinationofgenes.Thesecellsdifferentiateintoalongitudinalmuscle, nelid mesodermal and ectodermal midline pop- the axochord, that is positioned between central nervous system and axial blood vessel and ulations are not directly related by lineage (fig. S2). secretes a strong collagenous extracellular matrix. Ancestral state reconstruction suggests that Last, the Platynereis mesodermal midline is contractile mesodermal midline cells existed in bilaterian ancestors. We propose that these devoid of paraxis,whichisexclusivelyexpressed cells, via vacuolization and stiffening, gave rise to the notochord. in laterally adjacent mesoderm (fig. S8), in line with its vertebrate ortholog demarcating par- axial mesoderm (11). In vertebrates, this segre- efining the propertiesFischer et al. andFrontiers characteristics in 2010, 7:31bone morphogenetic protein (BMP) body side (5) gation depends on canonical Wnt signaling, with of the last commonhttp://www.frontiersinzoology.com/content/7/1/31 ancestor of bilaterian that stood out by early and continuous expression b-catenin–positive cells preferentially adopting animals, Urbilateria, is a key question of of colA1,encodingcollagentypeA(Fig.2,AtoD). aparaxialfateandposition(12). Consistently, we the evolution and development field (1). SiMView light sheet microscopy (6)revealedthat observed nuclear localization of b-catenin in the In an attempt to infer a possible urbila- these cells moved underneath the neuroectoderm more-lateral mesoderm only, and b-catenin over-

Dterian precursor for the chordate notochord (2, 3), toward the midline until they contacted their bi- activation converted the mesodermal midline on February 4, 2016 RESEARCH Open Access we reasoned that this structure should occupy lateral counterpart (movie S1 and Fig. 2E). Sub- toward a more lateral fate and position (fig. S8). asimilarpositionwithregardtooverallmor- sequently, these cells narrowed and elongated We next compared the developmental fate of phology and molecular topography during de- without a net increase in cell surface (fig. S1), annelid and vertebrate mesodermal midline cells. velopment and in theThe adult body normal plan of living developmentand additional adaxial mesodermal of Platynereis cells were Phalloidin dumerilii staining and expression analysis of descendants (Fig. 1, A and B); that it should observed to intercalate between the elongating muscle markers (fig. S9) revealed that, after express, during its development,(, a suite of genes Annelida)pairs (Fig. 2E), reminiscent of the processes by elongation, the Platynereis mesodermal midline that have proven specificAntje HL and Fischer indispensable1*, Thorsten for Henrichwhich1,2, Detlev the chordamesoderm Arendt1* converges and ex- cells differentiate into the previously described

notochord formation in the chordates; and that tends (table S1) (7). Lineage tracking by targeted “medial ventral longitudinal muscle” (13)(Fig.3A). Downloaded from it should be of widespread occurrence in bilate- photoconversion of the fluorescent protein kikGR Given the ropelike appearance and axial position rian body plans (Fig. 1C). We focused our search confirmed the origin of these cells from the meso- of this muscle, we propose to call it “axochord.” A Abstract on the model annelid Platynereis dumerilii,which dermal bands (fig. S2). muscular nature of a putative invertebrate count- is amenable to molecularBackground: studies andThe has retained annelidThePlatynereis unique location, dumerilii is large an emerging size, and model specific organismerpart for of the the study chordate of notochord is consistent with more ancestral featuresmolecular than Drosophila developmental melano- processes,arrangement evolution, of neurobiology the Platynereis andmesodermal marine biology. mid- Annelidsthe observationbelong to the that in the most basal chordate, gaster or CaenorhabditisLophotrochozoa, elegans (4). By looking the so far for understudiedline cells third allowed major their branch unambiguous of bilaterian animals identifi- besidesamphioxus, the notochord and is composed of spe- cell populations thatecdysozoans. would resembleP. dumerilii the verte-has provencation highly after relevant whole-mount to explore in ancient situ hybridization bilaterian conditionscialized via muscle comparison cells to(14 the)andexpressesthesame brate chordamesodermdeuterostomes, (a population because of mesoder- it has accumulated(WMISH) and less thus evolutionary expression change profiling than byconventional con- muscle ecdysozoan markers models. (15). We further observed segmen- mal midline cells thatPrevious converge staging medially was tomainly give referringfocal to imaging. hours post To fertilization test a possible but did homology not allow of matchingtal sets stages of transverse between studies muscles connecting to the rise to the notochord;performed red in Fig. at1A), (even we identified slightly) differentthese temperatures. cells with the To chordamesoderm, overcome this, and we to chose provide aaxochord first comprehensive (“ventral oblique muscles”)(13)(Fig.3,A segmental pairs of mesodermaldescription cells of P. on dumerilii the nonnormal– a development, chordamesoderm-specific a temperature-independent gene set accord- staging systemand B, is needed.and fig. S3). Scanning electron microscopy Results: Platynereis dumerilii normaling development to the following is subdivided criteria: (i) into specificity 16 stages,—their startingrevealed with the zygotethat, in and adult ending worms, the axochord is with the death of the mature wormscombined after delivering expression their uniquely gametes. defines The stages the chor- describeddeeply can be embedded easily identified in the fibrous sheath of the 1Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), D-69117by Heidelberg. conventional2Janelia light Farm microscopydamesoderm; or even by dissecting (ii) conservation scope. Developmental—their chorda- landmarksventral such nerve as the cord beginning (16)andremainsconnectedto of Research Campus, 19700 Helixphototaxis, Drive, Ashburn, the visibility VA 20147, of the stomodealmesoderm opening expression and is of conserved the chaetae, in at the least first three occurrencethe transverse of the ciliary muscles bands, (Fig. the 3, C and D). Immu- USA. 3Centre for Organismalformation Studies, University of the parapodia, of the extensionof four vertebrate of antennae species; and cirri, and the (iii) onset function of feeding— andnostainings other characteristics confirmed its are axial position between Heidelberg, Heidelberg, Germany.used to define different developmentalthey have stages. proven The essential morphology for chordamesodermof all larval stages as wellneuropil as of andjuveniles blood and vessel (fig. S12; similar to the *These authors contributed equallyadults to is this documented work. †Present address: by light microscopy. We also provide an overview of important steps in the development of Institute for Biological and Medical Imaging and Institute of development or signaling. We thus investigated notochord; Fig. 1, A and B). Axochord contractility Developmental Genetics, Helmholtzthe nervous Zentrum systemMünchen, and of the musculature,expression of using seven fluorescent transcription labeling factors techniques(brachyury, andwas confocal evident laser-scanning from live imaging (fig. S9, E to G, and Ingolstädter Landstrasse 1, D-85764microscopy. Neuherberg, Timing Germany. of each developmentalfoxA, foxD, stagetwist, not refers, soxD to,and hourssoxE post), fertilization the signal- at 18movie ± 0.1°C. S2) For and comparison, occurred in alternation with the ‡Present address: Max Planckwe Institute determined of Molecular the Cell pace Biology of developmenting molecules of larvaenoggin raisedand at 14°C,hedgehog 16°C, 20°C,[chordin 25°C, 28°Ctransverse and 30°C. muscles A staging (movie S3). Electron micro- and Genetics, Pfotenhauerstrasseontology 108, 01307 representing Dresden, Germany the comprehensive list of developmental stages of P. dumerilii is available online. §Present address: Department for Molecular and Cell Biology, appears absent from annelid (8)], and graphs confirmed the muscular nature of axochor- Harvard University, 16 DivinityConclusions: Ave, Cambridge,Our MA 02138, atlas USA. of Platynereisthe guidancedumerilii normal factors developmentnetrin and representsslit (table an S2 importantdal cells resource and revealed for the a tight physical connection to ||Present address: Departmentgrowing for MolecularPlatynereis Evolutioncommunity and andfor references).can also be applied Transcripts to other for nereidid all but one annelids. [the transverse muscles (Fig. 3, E to I). Laser ablation of Development, University of Vienna, Althanstrasse 14, A-1090 Wien, not gene (2)] were detected (figs. S3 to S5) in the axochord impaired crawling (fig. S10 and Austria. ¶Present address: Howard Hughes Medical Institute, accordance with previously reported brachyury movie S4) and confirmed anchoring of the trans- Stanford University, Stanford,Background CA 94305, USA. #Corresponding in P. dumerilii and vertebrates [8,9]. author. E-mail: [email protected] expression (9), and their coexpression confirmed verse musculature. Additionally, we found that the In the past decades, the annelid Platynereis dumerilii during development of the two-celled larval eye may has been established as a marine model for reflect the bilaterian ground pattern [10]. SCIENCE sciencemag.orgdevelopmental, evolutionary and neurobiological Bilaterian animals comprise12 three SEPTEMBER main taxa: 2014 deuteros-• VOL 345 ISSUE 6202 1365 research as well as for ecology and toxicology [1-6]. It is tomes (e.g. chordates, , ), especially suitable for comparative studies because sev- ecdysozoans (e.g. , ), and lophotro- eral lines of evidence indicate that its evolutionary line- chozoans (mollusks, annelids and other marine inverte- age has been slow-evolving. For example, P. dumerilii brates). “Classical”,well-establishedanimalmodels has a highly conserved gene structure [7] and genes belong to the ecdysozoans (fruit fly, C. elegans)ordeu- involved in the development of the central nervous sys- terostomes (mouse, chicken, fish). Lophotrochozoans tem are expressed in a conserved molecular topography are still largely under-represented despite their obvious relevance to comparative approaches that seek to unra-

* Correspondence: [email protected]; [email protected] vel the ground pattern of all bilaterians. 1Developmental Biology Unit, European Molecular Biology Laboratory, D- P. dumerilii, which has been kept in laboratory culture 69117 Heidelberg, Germany since 1953, easily breeds in captivity where it produces Full list of author information is available at the end of the article

© 2010 Fischer et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Fischer et al. Frontiers in Zoology 2010, 7:31 Page 3 of 39 http://www.frontiersinzoology.com/content/7/1/31

1D 1c 1d 1C 1b 1a 1A 1B

ld ld 1C 1D 1B 1A

pt ld ld mm mm

apt le pt ld ld sf mm Figure 1 Schemes of developmental stages of P. dumerilii: zygote, cleaving embryo, stereoblastula - stereogastrula, protrochophore and early . Left: The scheme indicates the key characteristics of each developmental stage. Right: Next to the scheme a brief summary of the key features for the stage is given. The time points indicated with a star mark the end of each stage and are excluded from this stage. Bold: The name of the stage and in brackets the beginning and end of each stage is given. Italic: The key characteristics, which can be used to determine the beginning of each stage. Normal: Additional features of each stage are given, including some landmarks, which can be seen in the developing nervous system and musculature. From top to bottom: zygote (apical view), cleaving embryo (apical view), stereoblastula - stereogastrula (ventral view, apical up), protrochophore (ventral view, apical up) and early trochophore (ventral view, apical up). Abbreviations see abbreviations list. Fischer et al. Frontiers in Zoology 2010, 7:31 Page 32 of 39 http://www.frontiersinzoology.com/content/7/1/31

Figure 28 Series of conventional light microscopy images of P. dumerilii, small atokous worm and heteronereis. All images: Dorsal view, anterior side left. A: Overview, small atokous worm with 48 chaetigerous segments. B: Head of the same specimen as shown in A, at a higher magnification. The dorsal and ventral branch of the anterior and posterior cirri are visible, as well as the palpi (pl), antennae (ant) and the well developed jaws (j). C: Posterior end of the same specimen at higher magnification, posterior to the last chaetigerous segment is the growth zone (gz) located where new segments are formed. D: Overview, male and female may be similarly sized. Female (♀), top, appears yellowish, due to the oocytes in the coelomic cavity. Male (♂) bottom, appears white due to spermatozoans in the anterior part and red due to dense accessory blood vessels in the posterior part. The parapodia (pp) in the posterior body part are flattened with paddle-like chaetae. E: Detail of the male and female body at a higher magnification. Note the numerous visible oocytes (ooc) inside the female’s body and the different shape of the parapodia (pp) in the anterior and posterior body part. F: Anterior part of a mature male. Note the enlarged adult eyes (ae) in comparison to B. The dorsal cirri of the parapodia (dcpp) in segment three to nine are clearly club-shaped. D: Anterior part of a mature female, note also here the enlarged adult eyes (ae) and the oocytes inside the coelomic cavity and the parapodia. The dorsal cirri of the parapodia (dcpp) in segment three to seven are slightly club-shaped. Scale bar in A: 500 μm. Scale bar in B and C: 100 μm. Scale bar in D, E, F and G: 2 mm. Further abbreviations see abbreviations list. Fischer et al. Frontiers in Zoology 2010, 7:31 Page 34 of 39 http://www.frontiersinzoology.com/content/7/1/31

192 3-segmented errant juvenile 180 168

156 “7 days”

°C 144 at 18 f 132 “6 days” late nectochaete 120 108 “5 days” 96 84 mid-nectochaete the developmental stage in hp f 72 }early nectochaete late metatrochophore 60 } }mid-metatrochophore }early metatrochophore

equivalent o 48 }late trochophore 36 }mid-trochophore 24 }early trochophore }protrochophore 12 }stereoblastula/ stereogastrula }cleaving embryo 0 }zygote 1 12 24 36 48 60 72 hpf 14°C 16°C 18°C 20°C 25°C 28°C 30°C Figure 29 Influence of temperature on developmental speed in P. dumerilii. Diagram, which shows the developmental stage reach 12 h, 24 h, 36 h, 48 h, 60 h and 72 h post fertilization, growing at 14°C, 16°C, 18°C, 20°C, 25°C, 28°C and 30°C. Sampling was performed every 12 h for 72 h. The error bars indicate the range within the stage, at which the embryos or larvae were found. Within this range it was not possible to determine the stage more precisely. The gradient in the chart background indicates the decreasing synchrony from the late nectochaete stage onwards. The transversal lines indicate the end of one stage and the beginning of the following one, The duration of the late nectochaete stage and the beginning of the following stage vary, indicated by the bold blurred line.

The developmental stages defined here are based on (with a narrow band of long cilia - the prototroch) 3) existing terminology used for P. dumerilii and for other metatrochophore I (simplest form of a segmented larva) [6,36,37,62]. Häcker [36] distinguished five 4) metatrochophore II (parapodia appear but are not yet stages of polychaete development: 1) protrochophore used for locomotion) and 5) nectochaete (parapodia are (with a broad preoral ciliated band), 2) trochophore the main swimming apparatus). Although the meaning RESEARCH ARTICLE Mesoteloblast‐Like Mesodermal Stem Cells in the Polychaete Annelid Platynereis dumerilii (Nereididae) ANTJE H.L. FISCHER* AND DETLEV ARENDT* Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

ABSTRACT Spiral cleavage is observed in animals that belong to the , a large group of marine . As characteristic for spiral cleavage, the bulk of mesoderm forms from one cell, the “4d blastomere.” This process has not yet been followed in cellular detail in annelids except in the leech, where “mesoteloblasts,” a pair of mesodermal stem cells, generate two bands of mesoderm precursor cells in an iterative fashion. It is so far unknown whether such stem cell‐like lineage is a general property of 4d‐derived mesoderm in spiralian larvae. To address this, we have analyzed the cell lineage of the 4d blastomere in the polychaete annelid Platynereis dumerilii, an emerging model for lophotrochozoan and spiralian embryology (Fischer et al., 2010), by 4D microscopy, a semi‐automated cell tracking technique based on differential interference contrast serial imaging (Schnabel et al. '97). Our data reveal that the two daughter cells of the 4d cell undergo seven consecutive rounds of unequal cell divisions. They bud off smaller cells in ventral‐vegetal orientation and thus show mesoteloblast‐ and stem cell‐like behavior. Based on these findings, we suggest that mesoteloblast‐like mesodermal stem cells that form continuous mesodermal bands are part of the Errantia Sedentaria ground pattern. In the course of annelid evolution, the number consecutive divisionsþ of these cells would have been low initially with <10 division cycles, giving rise to larval segments only, and then increased up to 35 as observed in clitellates. J. Exp. Zool. (Mol. Dev. Evol.) 320B:94–104, 2013. © 2013 Wiley Periodicals, Inc. ‐ J. Exp. Zool. How to cite this article: Fischer AHL, Arendt D. 2013. Mesoteloblast like mesodermal stem cells (Mol. Dev. Evol.) in the polychaete annelid Platynereis dumerilii (Nereididae) J. Exp. Zool. (Mol. Dev. Evol.) 320B:94–104, 2013 320B:94–104.

Annelids (including sipunculids echiurans and pogonophorans), Additional supporting information may be found in the online version of molluscs, dicymemid mesozoans (obligate parasites living in the this article. Competing interests: The authors declare that they have no competing renal appendages of , consisting of around 30 cells), interests. nemerteans, , and free‐living platyhelminthes, The present address of Antje H.L. Fischer is Eugene Bell Center for share a highly specific cleavage pattern called spiralian cleavage Regenerative Biology and Tissue Engineering, Marine Biology Laboratory, (Henry and Martindale, '99). Recent phylogenetic analyses group Woods Hole, MA. animals with spiralian cleavage within the lophotrochozoans (e.g., Grant sponsor: Marie Curie Research Training Network Zoonet; Grant number: MRTN‐CT‐2004‐005624. Dunn et al., 2008; Giribet, 2008; Paps et al., 2009a,b). *Correspondence to: Antje H.L. Fischer, Eugene Bell Center for Spiralian cleavage is a stereotypic, holoblastic cleavage program Regenerative Biology and Tissue Engineering, Marine Biology Laboratory, that offers the unique opportunity to identify individual blastomeres Woods Hole, MA. E‐mail: [email protected] among several major animal taxa, analyze their cell lineages and *Correspondence to: Detlev Arendt, Developmental Biology Unit, compare their cell fates on a single cell level (Meyer et al., 2010). European Molecular Biology Laboratory, Meyerhofstraße 1, 69012 Heidelberg, Germany. E-mail: detlev.arendt@embl‐heidelberg.de Spiral cleavage is characterized by a series of divisions with an Received 5 April 2012; Revised 12 October 2012; Accepted 17 December oblique spindle orientation with respect to the animal–vegetal axis 2012 of the developing embryo (Henry and Martindale, '99). The first two Published online 13 February 2013 in Wiley Online Library (wileyonline cleavages are meridional and can be equal or unequal, depending on library.com). the species. The result of the first cleavage are the blastomeres AB DOI: 10.1002/jez.b.22486

© 2013 WILEY PERIODICALS, INC. MESODERMAL STEM CELLS IN THE POLYCHAETE PLATYNEREIS 97

Figure 1. Continued.

J. Exp. Zool. (Mol. Dev. Evol.) MESODERMAL STEM CELLS IN THE POLYCHAETE PLATYNEREIS 101

Figure 3. Overview of the cell divisions during the proliferation phase. (A) Cell genealogy of 4d descendants. The color code is the same as that used in Figures 1 and 2. The offspring of m6 outlined with a red square give rise to the first differentiating muscle cells. The dotted lines indicate cell divisions that could not be observed in all individuals. (B,C) The representation of the nuclei of each cell of the M‐blastomere lineage at 23.5 hpf in the SIMI BioCell software in different orientations. The offspring of m6 that give rise to the first differentiating muscle cells are outlined by red dotted lines in B and C. The white dotted circle indicates the outline of the embryo. B: Anterior view, dorsal side up. C: Ventral view, anterior side up. (D) Fluorescent nuclei staining with DAPI in a 24 hpf trochophore larva, ventral view, anterior side up. Z‐projection of confocal images, the ventral‐most and dorsal most parts of the animal are not shown. The cells that will differentiate into the first muscle cells, located at the anterior end of the mesodermal bands, are indicated by the red dotted line. The mesodermal bands are outlined with white dotted lines. development, the primordial germ cells migrate anteriorly into the et al., 2005). There is no evidence that 4d‐descendants contribute “primary gonads” and later to the base of the parapodia (Rebscher to any “non‐mesodermal” structure in P. dumerilii. et al., 2007, 2012). Lineage tracing experiments using fluorescent It will be very interesting to investigate whether individual cells dextran injections, have shown that 4d‐descendants also give rise of the 4d‐cell lineage give rise to specific segmental structures. to the entire body musculature in P. dumerilii (Ackermann This cannot be concluded from the present dataset. The spatial

J. Exp. Zool. (Mol. Dev. Evol.)