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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02577

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Repression of mesodermal fate by foxa, a key endoderm regulator of the sea urchin embryo

¹ Division of Biology, 156-29, California Institute of Technology, Pasadena, CA 91125, USA.
² Department of Biology, Duke University, Durham, NC 27707, USA.

View larger version (113K): [in a new window]   Fig. 1. Spatial expression pattern of Spfoxa. (A-L) WMISH was performed at seven different developmental stages, as indicated in the upper right corner of each panel. Note that starting at 21 hours postfertilization, asymmetric expression of foxa is observed across the endoderm (C,H,L). This is not as apparent in D, which is purposely overdeveloped to reveal that only endoderm expresses foxa. The asterisks in E-G,K,L indicate the stomodeal region of the oral ectoderm. Arrows (I,J) indicate higher expression levels in foregut and hindgut relative to the midgut. LV, lateral view; VV, view from the vegetal plate; OV, view from the oral ectoderm.

View larger version (12K): [in a new window]   Fig. 2. Temporal expression of the Spfoxa gene in embryonic development. QPCR data obtained from different time points were converted to number of transcripts per embryo, by reference to a known standard (see Materials and methods). The transcript prevalence describes an oscillating pattern of expression with a period of 10±1 hours.

View larger version (157K): [in a new window]   Fig. 3. Effects of foxa MASO on development. (A-L) Strongylocentrotus purpuratus embryos; (M-P) Lytechinus variegatus embryos. (A-C,J-L) Early-mid gastrula, (D-F) late gastrula, (G-I,M-P) pluteus stage larvae, respectively corresponding to 35 hours, 48 hours and 70 hours of development for S. purpuratus. (C,F,I,J,M,O) Control embryos treated as specified in each panel. (A,B,D,E,G,H,K,L,N,P) foxa MASO-treated embryos. The concentration of MASO injected is indicated in each panel. Black arrows (B) indicate that the SMCs reach their correct position even if a severe reduction of gut extension has occurred. (J) Fluorescent image of embryos injected with the 5'foxa-GFP fusion mRNA: all of the cells express GFP as shown by the (false) green color. (K,L) When foxa MASO is co-injected with 5'foxa-GFP mRNA embryos do not show any GFP fluorescence (K) but do display the foxa MASO phenotype (L). Fluorescent and bright field images of the same embryo. Red arrowheads (E,F,H,I) point to the foregut or to the location where the foregut should be. The increase in pigment cells occurring on MASO treatment can be seen by comparing control embryos (M,O) to MASO-treated embryos (N,P), where the pigment cells appear as dark red cells positioned throughout the aboral ectoderm on the anal side (M,N) and over the oral hood (O,P). The control embryo in O has a mouth, whereas the embryo in P injected with foxa MASO does not.

View larger version (76K): [in a new window]   Fig. 4. Mosaic analysis of the fates of specific blastomeres bearing foxa MASO. L. variegatus eggs were injected with foxa MASO and co-injected with rhodamine dextran as a dye tracer. Control donor embryos were injected with rhodamine dextran alone. At the 60-cell stage, two injected cells from each tier were transplanted to host embryos as indicated on the left diagrams. (Top row) Cells of the `an2' tier normally form ectoderm and foxa MASO-injected cells behaved like controls. (Second row) veg1 cells normally contribute to hindgut, midgut and vegetal ectoderm, and the foxa MASO-injected veg1 cells also contributed to gut and ectoderm. (Third row) veg2 cells normally contribute to foregut, midgut and SMCs. The foxa MASO cells contributed almost exclusively to SMCs. The red arrowhead points to the gut in both control and experimental embryos. (Bottom row) foxa MASO and control micromeres became normal PMCs.

View larger version (99K): [in a new window]   Fig. 5. Spatial expression of endomesoderm genes in foxa perturbed embryos. (A-O) S. purpuratus embryos at different time points injected with foxa (A-C,G-I) and control MASO (D-F,J-L) or mRNA (M-O) were hybridized with WMISH probes as indicated in the lower right corner of each panel; developmental time is indicated at lower left. Embryos are in lateral view with the vegetal pole towards the bottom of each panel, unless specified otherwise. AV, view from the apical plate; VV, view from the vegetal plate; OV, view from the oral ectoderm. An identical pattern of gcm expression is observed at mesenchyme blastula stage, in foxa MASO (A) and control (D) embryos. At 32 hours postfertilization, more cells stained positively for gcm are observed in the foxa MASO-injected embryo (B) than in control (E, see text for quantitation). The red arrowhead indicates a cell of the invaginating endoderm that is expressing gcm. (C,F) gatae expression at 48 hours in foxa MASO and control (late gastrula) embryos. At this stage, gatae is normally expressed in midgut, hindgut and coelomic pouches (Lee and Davidson, 2004); expression is nearly abolished in Foxa-depleted embryos (C). (G-L) Expression of foxa in foxa MASO and control embryos. At all three stages shown, the intensity of expression is increased by foxa MASO (G-I), compared with controls (J-L). The normal expression boundaries are maintained, however; although the remaining endoderm at 48 hours has failed to invaginate. (N,O) mRNA overexpression of Foxa. Complete repression of gcm relative to control embryos (M) is seen only at high concentrations (N) of injected mRNA.

View larger version (71K): [in a new window]   Fig. 6. Veg1 contributions in recombinant embryos bearing foxa MASO. (A,D) Diagram of recombinants consisting of an animal half plus veg1 (top part), recombined with veg2 plus micromeres (lower part). Control L. variegatus embryos were dyed green with fluorescein dextran, and experimental embryos were injected with foxa MASO and co-injected with RITC (red) before recombination. (B,C,E,F) In the recombinants, the control (green) endoderm from veg 1 (B,C) or from veg 2 (E,F) makes much of the gut; this is dependent upon whether veg2 (B,C) or veg1 (E,F) contains foxa MASO. Recombinations were made at the 60-cell stage. (B,C) Pluteus stage chimeras as in A. (E,F) Pluteus stage chimeras as in D. See text for interpretation.

View larger version (66K): [in a new window]   Fig. 7. Oral expression of foxa is required for mouth formation. Red indicates foxa MASO-injected cells; green indicates control cells. (A,D) Diagram of the experiment. Reciprocal combinations of animal and vegetal halves were recombined. (B,C) Control animal half, showing normal mouth (arrowhead, C). The foxA MASO vegetal half (red) produces a truncated gut, with most of the red cells becoming SMCs, and extra pigment cells (compare black cells in C and F). (E,F) foxa MASO-injected animal half (red) produces ectoderm with no mouth. The control vegetal half (green) produces a normal gut, including foregut that leads to a dead end. (B,E) DIC images superimposed on fluorescent images. (C,F) Fluorescent image only at a slightly higher magnification, focused on the mouth region.

View larger version (38K): [in a new window]   Fig. 8. foxa gene interactions in the endomesoderm regulatory network. Relevant network subcircuits are depicted as `views from the nuclei' (Bolouri and Davidson, 2002) up to beginning of gastrulation in veg2 SMC (left boxes) and veg2 endodermal cells (right boxes). Architecture is as presented previously (Davidson, 2006; Erwin and Davidson, 2002; Levine and Davidson, 2005; Oliveri and Davidson, 2004b) (for a current version see http://sugp.caltech.edu/endomes/), with the addition of data from this paper. (A) Blastula; (B) mesenchyme blastula to early gastrula. Solid connecting lines are predicted to be direct interactions between the product of a given regulatory gene and the target cis-regulatory module of other gene(s). The dashed line is an implied and possibly indirect interaction. Arrows indicate activating interactions and bars indicate repressive inputs. Bold colors are genes expressed or interactions operating in that cell type at that time; faded colors indicate genes that are inactive or interactions not then taking place. Yellow stars symbolize the Delta ligand and turquoise ovals the Notch receptor embedded in the diagrammatic membrane. White stars and white ovals indicate that Delta/Notch signaling is not active at a particular stage in a particular cell type (because the delta gene is not expressed there). At blastula stage (A), the genes gatae (blue) and otx (green) enter into a feedback-stabilizing loop (Yuh et al., 2004), and are responsible for initiating expression of the foxa gene (red) in all endomesodermal cells. In SMCs only, the gcm gene (orange) is activated in response to the Delta signal arriving from the adjacent micromeres (Sherwood and McClay, 1997; Sherwood and McClay, 1999). Gcm is then responsible for activating the pigment cell program represented by the pks gene (olive green). By mesenchyme blastula stage (B), gatae is expressed in veg2 endoderm as well as in mesoderm (Lee and Davidson, 2004). foxa expression becomes very strong and is restricted to endodermal cells. High levels of foxa are responsible for autorepression of foxa, and for repression of gcm. In the gcm expressing SMCs, foxa expression fades out; an unknown function downstream of gcm is inferred. The mesodermal cells now begin to express the Delta ligand that will signal to the adjacent veg2 endodermal cells, where Notch signal is used to drive the expression of gatae (P. Y. Lee and E.H.D., unpublished). At this time, gcm expression in response to Delta/Notch signaling is prevented in veg2 endoderm by the repressive action of Foxa. (C) Timeline of expression of the components in the model based on QPCR and WMISH data.