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First published online March 6, 2009
doi: 10.1242/10.1242/dev.032300

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The sea urchin animal pole domain is a Six3-dependent neurogenic patterning center

Zheng Wei, Junko Yaguchi^*, Shunsuke Yaguchi^*, Robert C. Angerer and Lynne M. Angerer

National Institute for Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

View larger version (54K): [in this window] [in a new window]   Fig. 1. cadherin-misexpressing embryos consist of animal pole domain (APD) tissues defined by expression of foxq2 and hbn. Whole-mount in situ hybridizations to embryos at mesenchyme blastula (A-D) and gastrulae (E-H) stages. (E,F) Glycerol-injected control. (G,H) cadherin-mRNA injected. (A,E,G) DIC; (B-D,F,H) Two-color fluorescence, hbn (green) and foxq2 (magenta) RNAs. Scale bar: 20 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 2. Temporal expression profiles of genes in the provisional early APD set. Profiling methods were as described by Wei et al. (Wei et al., 2006). Values at different hours post-fertilization from 2 to 72 are shown, as a percentage of the maximum signal intensity, for each gene at 2-cell (maternal RNA), 15-hour early blastula (EB), 30-hour late mesenchyme blastula (LMB), 48-hour late gastrula (LG) and 72-hour pluteus larva (PL). Profiles are grouped according to time of earliest detectable expression: (A) maternal; (B,C) early blastula; (D) early blastula to mesenchyme blastula. The position of the dashed line represents the time of assay in Six3 morphants. Data for the six3 gene are highlighted with a red arrow.

View larger version (83K): [in this window] [in a new window]   Fig. 3. Whole-mount in situ hybridization for six3 mRNA during development. Times as hours post-fertilization are shown in the upper right corner of each image. (A) Very early blastula. (B) Hatching blastula. (C-H) Mesenchyme blastula. (I,J) Late gastrula. (K) Pluteus. All embryos are shown in lateral view, except in G and H, which illustrate vegetal pole (vv), and animal pole view (apv), respectively. Arrows in I and K mark positions of secondary mesenchyme cells and cells flanking the mouth, respectively. Scale bar: 20 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 4. Loss of Six3 results in loss of neurons and the thickened epithelium characteristic of the APD. (A,B) Three-day embryos injected with Six3-MO2 at the one-cell stage, which are typical of stronger and weaker phenotypes, respectively. (C) Normal 3-day embryo. APD in A-C indicated by brackets; 1e11 (pan-neural, magenta), serotonin (green), DAPI (nuclei, blue). (D) Numbers of embryos containing either 0, 1, 2 or 3 serotonergic neuron per embryo in Six3 morphants. At this stage, normal embryos have from 3 to 5 serotonergic neurons. (E,F) hbn mRNA in normal mesenchyme blastulae (E) or in Six3 morphants (F). Scale bar: 20 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 5. The sensitivity of early APD regulatory genes to loss of Six3. (A) Three-day embryos injected with cadherin mRNA (top) or with cadherin mRNA and Six3-MO (bottom). The arrow indicates the orientation of the animal-vegetal axis. Embryos are immunostained with anti-serotonin and 1e11, a pan-neural marker. (B) QPCR cycle changes (blue and red bars) or log2 signal intensity differences (yellow bars) (y-axis, left) or fold changes (y-axis, right) in the levels of individual mRNAs in two batches (red and blue bars) of Six3 morphant and control 27-hour embryos, both containing cadherin. Genes whose expression is changed at least 3-fold are to the left of the green line.

View larger version (74K): [in this window] [in a new window]   Fig. 6. Misexpression of Six3 converts the embryo to an expanded APD. All embryos are 3 days old. (A) Normal embryo, DIC; blastopore view, oral up. (B,C) six3 mRNA-misexpressing embryos, DIC; (B) oral view, (C) lateral view; arrows in B indicate the border between the expanded animal plate and the more vegetal thin epithelium. (D,E) Serotonergic (sero, green) and all (1e11, magenta) neurons in normal embryos. (F-H) DIC and immunostains, as in D and E of six3 mRNA-misexpressing embryos; oral view, animal pole up. (I,I') Immunostains of normal 3-day embryos for NK2.1 (green) and Gsc (magenta); (I) lateral view; cells in the APD to the right of the white dashed line; (I') oral view, cells in the APD are between the dashed white lines. NK2.1-positive cells marked with arrowheads are in the blastocoel and not part of the APD; m, mouth. (J-T) six3-mRNA-misexpressing embryos. (J,K) NK2.1 (green); 1e11 (magenta). (L-N) NK2.1 (green); Gsc (magenta). (O-Q) DIC images of hbn whole-mount in situ hybridizations to control (O) and six3 mRNA-injected embryos (P,Q); animal pole up; white circle in O marks the mouth. (R-T) A through-focal series of an embryo stained with DAPI, and for 1e11 (green) and Spec1 (magenta) epitopes; Spec1 labels the thin, expanded aboral epidermis which collapses and wrinkles in preparation. (U) Diagram illustrating distribution of APD cell types in normal and Six3-mRNA-misexpressing embryos. The colored dots show the distribution of cells expressing the indicated proteins or mRNAs and black and purple ovals indicate serotonergic (Sn) and non-serotonergic neurons (n-Sn), respectively. Green and blue shadings identify facial epithelium and ciliated band, respectively. Six3 mRNA injection (arrow) results in a spherical 3-day embryo (right; see also B,C) consisting largely of the region outlined in blue in the normal embryo (left). In the Six3-mis-expressing embryo, the number of neurons and NK2.1/gsc-positive (yellow; supraoral) cells greatly expands and the hbn-positive cells (blue), which normally flank the animal plate on the oral side at this stage, are found at the vegetal pole. The arrows indicate the positions of the oral-aboral and animal-vegetal axes in both normal and Six3-misexpressing embryos. Scale bars: 20 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 7. Canonical Wnt-dependent APD restriction does not depend on Nodal and can be overridden by Six3 misexpression. (A,B,E,F) Nodal-MO only. (C,D) Nodal-MO and Six3 mRNA. (G,H) Nodal-MO and Six3-MO. (A,C,E,G) DIC; (B,D,F,H) immunostaining for serotonergic neurons (green) or all neurons (1e11, red). An, animal pole; Veg, vegetal pole. Scale bar: 20 µm.

View larger version (99K): [in this window] [in a new window]   Fig. 8. Misexpression of Six3 suppresses expression of genes encoding Wnt ligands and Nodal, but loss of Six3 is not sufficient to allow their expression in the APD. Whole-mount in situ hybridizations detecting mRNAs encoding Wnt1, Wnt8, Wnt16 and Nodal in 26-hour embryos injected with either Six3-MO or Six3 mRNA. Embryos in the top row were photographed using DIC illumination on a Zeiss Axiomat microscope; all others were photographed using phase contrast illumination on a Leica microscope. Scale bar: 20 µm

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