OEB51 Lecture 9 Mollusk Embryology Ctenophora Animals Porifera Placozoa Cnidaria Xenacoelomorpha Parahoxozoa Ambulacraria Echinodermata Hemichordata Planulozoa Deuterostomia Cephalochordata Chordata Urochordata Bilateria Craniata Chaetognatha Bryozoa Entoprocta Cycliophora Nephrozoa Annelida Trochozoa Mollusca Nemertea Brachiopoda Phoronida Spiralia Gastrotricha Protostomia Platyhelminthes Gnathostomulida Micrognathozoa Gnathifera Rotifera Nucleariida Orthonectida Fungi Dicyemida Opisthokonta Filasterea Priapulida Ichthosporea Scalidophora Holozoa Animals Loricifera Choanoflagellata Kinorhyncha Nematoida Nematoda Ecdysozoa Nematomorpha Tardigrada Panarthropoda Onychophora Arthropoda Spiral cleavage general proper=es 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. Phylum 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 • cephalopod classes have very modified cleavage (and modified body plans) • e.g. Loligo pealei (squid): no spiral cleavage Spiral cleavage can be sinistral (to the leT) 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 ac=n 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 female 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 genome. 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 larva Ilyanassa obsoleta – micromere fate maps 8 cell stage First quartet micromeres: 1a, 1b, 1c, 1d 1a and 1c make eyes normally When 1a and 1c are removed, eyes are missing Ilyanassa obsoleta – micromere poten=als Cytoplasmic determinants are not the whole story Put 1a into the posi=on of 1b: 1a no longer makes eyes! What prevents 1b and 1d from making eyes? Ilyanassa obsoleta – micromere poten=als Experiment: FIRST Remove the polar lobe contents at first cleavage THEN Put 1d into the posi=on of 1a 1d can make eyes! The polar lobe contents give 1D the power to repress 1d’s eye making poten?al Ilyanassa obsoleta – recall that micromeres have different poten=als What is special about the polar lobe (and the D lineage) that makes it able to repress poten=als 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 annelid mesodermal midline, their combined Development of the annelid coexpression was unique to these cells (implying that mesodermal midline in annelids and chor- damesoderm in vertebrates 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 vertebrate 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,