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First published online 10 May 2006
doi: 10.1242/dev.02396

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Specification of ectoderm restricts the size of the animal plate and patterns neurogenesis in sea urchin embryos

Departments of Biology and Biochemistry/Microbiology, University of Victoria, POB 3020, STN CSC, Victoria, BC, V8W 3N5, Canada.

View larger version (87K): [in a new window]   Fig. 1. Blocking vegetal signaling produces permanent blastulae with an expanded animal plate that differentiates as unpatterned neural tissue. (A-D,F) Embryos injected with -cadherin RNA. (A) DIC image of embryo (72 hour) injected with -cadherin RNA. (B) Anti-serotonin immunoreactive cells in a -cadherin-injected embryo. Confocal stack of the animal pole. (C) Anti-synaptotagmin immunoreactivity (SpSyn B) is principally in neurites of the serotonergic cells. (D) Merged image of B,C. (E) Merged image of control embryo (72 hours); serotonin (green) and synB (magenta) co-localize in apical organ neurons and synB identifies ciliary band neurons. (F) Detail of serotonergic and non-serotonergic neurons of the animal plate of a -cadherin RNA injected embryo (120 hours). (G) Anti-serotonin immunoreactive cells in embryo expressing Gsk. Inset is DIC image. (H) Permanent blastula from animal cap blastomeres with abundant unpatterned anti-serotonin immunoreactive cells. Inset is DIC image. (I) Uninjected embryo (72 hours) expressing SpHnf6 (green) in ciliary band and SpNk2.1 protein (magenta). Only the animal plate co-expresses these transcription factors. (J) A single confocal, optical section at the midline showing a lateral view of the region co-expressing SpHnf6 and SpNk2.1 (bracket). Enlargement of the region shown with asterisk in inset. o, oral ectoderm; ab, aboral ectoderm. (K-M) Recombined confocal image of -cadherin RNA injected embryo from animal pole. All of the thickened epithelial cells express SpHnf6 (K) and SpNk2.1 (L). (M) Merged image of (K,L). (N) Lateral view of a -cadherin RNA-injected embryo labeled with in situ RNA hybridization of SpHnf6 and immunolocalization of SpNk2.1. Arrowheads indicate the border between thick and thin epithelium. (O) Single confocal optical section of -cadherin RNA-injected embryo. Serotonergic cells (green) express SpNk2.1 (magenta), but SpNk2.1 is cytoplasmic rather than nuclear in these cells (arrows). Scale bar: 20 µm.

View larger version (79K): [in a new window]   Fig. 2. Neurons do not differentiate in exogastrulated embryos injected with RNA encoding stabilized ß-catenin (XBC69) or a dominant-negative Gsk3ß (dnGsk). (A) Embryo (72 hours) injected with stabilized ß-catenin RNA. (B,C) Epifluorescent images of embryos labeled with anti-serotonin (B) and anti-synaptotagmin (SpSynB) (C). Neither neural marker is detected in exogastrulae. (D) Embryo injected with RNA encoding dnGsk. Neither serotonin (E) nor synaptotagmin (SpSynB) (F) can be detected in exogastrulae expressing dnGsk. (G-I) Exogastrula prepared by compressing embryos under a coverslip. (G) Exogastrula. (H,I) Epifluorescent images of G. Although the archenteron is not internalized, normal serotonergic neurons form in the apical organ (H, white arrowhead) and anti-synaptotagmin immunoreactive cells (I, yellow arrowhead) in mechanical exogastrula (72 hours). (J) Glycerol injected, control prism at 72 hours. (K,L) Epifluorescent images of J. (K) Serotonergic cells are immunochemically detected at the apical organ (white arrowhead). (L) Anti-synaptotagmin (Sp SynB) immunoreactive cells in the ciliary band (yellow arrowhead). (M-O) SpNk2.1 expression in embryos injected with RNA encoding stabilized ß-catenin. (M) The ectoderm is on the left of image. Neither anti-serotonin immunoreactive cells (N) nor anti-SpNk2.1 immunoreactive cells (O) are detected. SpNk2.1 localizes to the tip of the archenteron (O, asterisk). Scale bars: 20 µm.

View larger version (71K): [in a new window]   Fig. 3. Chimeric embryos and single blastomere injections indicate that signaling restricting the size of the animal plate is indirectly dependent on vegetal signaling. (A) The scheme for chimeras with animal half derived from a -cadherin RNA-injected embryo and vegetal half uninjected. (B) Scheme for octomere injections. (C-E) Epifluorescence of chimera labeled with anti-serotonin and anti-myc. (C) A normal number and pattern of serotonergic neurons form in the chimera. Inset is DIC of chimera (72 hours) the animal plate is marked with an asterisk. Normal archenteron forms in chimeras (arrowhead). (D) The recombinant embryo has co-injected myc protein lineage marker in ectoderm. Arrowhead indicates the position of endoderm and the asterisk is beside the animal plate. (E) Merged image of C,D. (F-H) Recombined confocal stack of an embryo in which one cell of eight-cell embryo was co-injected with RNA encoding stabilized ß-catenin and myc as a lineage marker. (F) Normally patterned apical organ with four serotonergic neurons differentiates in an octomere injected chimera. Inset is DIC, an asterisk marks the animal plate. (G) The lineage marker Myc and -cadherin is in ectoderm from injected octomere. (H) Merge of F,G. (I-K) Epifluorescent images of octomere-injected embryo. (I) SpNk2.1 protein is like uninjected embryos, indicating no direct effect of -cadherin. Inset is DIC image, asterisk indicates the animal plate. (J) Myc immunoreactive cells. (K) Merge of I,J. Scale bar: 20 µm.

View larger version (85K): [in a new window]   Fig. 4. Effects of treatments that alter ectoderm specification on formation and patterning of neurons in the animal plate. (A-D) Antivin-injected embryos (72 hours). (A) SpNk2.1 proteins in the animal plate of injected embryos. Inset is transmitted light image. (B) Ciliary band marker, SpHnf6, is expressed throughout the thickened ectoderm. (C) Serotonergic neurons are detected at the animal plate, but they are not normally patterned. (D) Ciliary band neurons (SpSynB) are scattered throughout lateral ectoderm. (E) Embryos treated with kinase inhibitor SB203580 have supernumerary, unpatterned serotonergic neurons. (F) Embryos treated with the TGFß receptor inhibitor, SB431542, express SpNk2.1 in the animal plate. Inset shows transmitted light image. (G) SB431542-treated embryos have extra serotonergic neurons lacking normal symmetry. (H-J) BMP-injected embryos (72 hours). (H) SpNk2.1 is restricted to the thickened ectoderm at the animal pole. (I) SpHnf6 is also restricted to the animal pole. (J) Serotonergic neurons in the animal plate lack normal patterning. (K-M) Nodal-injected embryos. (K) Distinctive bell-shaped embryos express SpNk2.1 throughout the dome. Inset shows transmitted light image. (L) SpHnf6 is expressed in the central part of the SpNk2.1 domain; thus, the animal plate is restricted to the thickened ectoderm at the pole. Inset shows entire SpHnf6 expression, including the postoral transverse ciliary band. (M) Serotonergic neurons are rarely detected in Nodal-injected embryos. (N) SpNk2.1 expression in Noggin-injected embryo. Inset shows transmitted light image. (O) In Noggin-injected embryos, few serotonergic neurons form in the animal plate. Scale bar: 20 µm.

View larger version (75K): [in a new window]   Fig. 5. Alterations of ectoderm specification in embryos and chimeras in which vegetal signaling is blocked. (A) Co-injection of -cadherin and Antivin results in permanent blastulae with numerous unpatterned serotonergic neurons in an enlarged animal plate. Inset shows transmitted light image. (B,C) Embryos co-injected with -cadherin and BMP. (B) SpNk2.1 is expressed throughout. Inset shows transmitted light image. (C) Numerous serotonergic neurons are scattered in thickened epithelium. (D-F) Embryos co-injected with -cadherin and Nodal. SpNk2.1 (D) and SpHnf6 (E) expression is identical to -cadherin alone. However, fewer serotonergic neurons form (F). Inset in S shows transmitted light image. (G,H) Chimeras in which the entire embryo expresses -cadherin and half co-expresses Antivin (G) and myc as a lineage tracer or BMP (H) and myc. Serotonergic neurons form in similar numbers as embryos injected with -cadherin. (I) Chimeras expressing -cadherin throughout and Nodal in one half have fewer serotonergic neurons. The serotonergic neurons that form in the half expressing -cadherin alone are several cell diameters from the interface with Nodal-expressing cells (arrowhead). (J-L) SpGsc-injected embryos. (J) SpNk2.1 protein is in all cells of embryos expressing SpGsc. Inset shows transmitted light image. Neither SpHnf6 (K) nor serotonin (L) are detected. (M-O) Embryos co-injected with -cadherin and SpGsc. (M) SpNk2.1 is expressed uniformly throughout the embryo. Inset shows transmitted light image. Neither SpHnf6 (N) nor serotonergic neurons (O) are detected. (P) In chimeras expressing -cadherin throughout, one half co-expresses SpGsc (myc-positive). Serotonergic neurons differentiate in the half of the embryo expressing only -cadherin. The serotonergic neurons form at the interface with cells expressing SpGsc (arrowhead). (Q) SB431542-treated -cadherin-injected embryos have a similar number of unpatterned serotonergic neurons as untreated embryos expressing -cadherin. (R) Embryos co-expressing -cadherin and Nodal, then treated with SB431542 form serotonergic neurons. Blocking Nodal receptors rescues the formation of serotonergic neurons. Scale bar: 20 µm.

View larger version (17K): [in a new window]   Fig. 6. Graphs showing the mean and standard error of the number of serotonergic cells in the animal plate of treated embryos. (A) Controls and treatments that block or enhance vegetal signaling. (B) Treatments that enhance the specification of ciliary band or aboral ectoderm. (C) Treatments that enhance the specification of oral ectoderm (D) Treatments of embryos in which vegetal signaling is blocked to eliminate endogenous specification of ectoderm. The control bars in A-C are the same data. Stars indicate treatments that produce changes that are significantly different from controls (P<0.01).

View larger version (21K): [in a new window]   Fig. 7. Model of restriction of size and patterning of animal plate. (A) The egg has a neuralizing potential in the animal half. The size and patterning of the neural tissue depends on the ß-catenin-mediated specification of endomesoderm. (B) During cleavage, specification of endomesoderm in vegetal blastomeres also specifies ectoderm as ciliary band, which restricts neurogenic ectoderm to a small animal plate. (C) As the ectodermal domain is specified as ciliary band, oral and aboral ectoderm, neuron formation is patterned in the animal plate. Nodal acts as a paracrine repressor of serotonergic neuron formation where oral ectoderm is adjacent to the animal plate. Serotonergic neurons form where the animal plate is adjacent to ciliary band and aboral ectoderm. Red, animal plate; blue, endomesoderm; purple, ciliary band; yellow, oral ectoderm; light green, aboral ectoderm; green cells, serotonergic neurons.

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