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Sea urchin goosecoid function links fate specification along the animal-vegetal and oral-aboral embryonic axes

¹ Department of Biology, University of Rochester, Rochester, NY 14627, USA
² Department of Biochemistry and Molecular Biology, The University of Texas M.D. Anderson Cancer Center, and Graduate Program in Genes and Development, Houston, TX 77030, USA

View larger version (45K): [in a new window]   Fig. 1. (A) Goosecoid homeodomain sequence alignment. Red and blue residues indicate nonconservative and conservative amino acid substitutions, respectively. The asterisk indicates K50 a residue found in all bicoid-class homeodomain-containing proteins that is essential for binding to cognate cis elements. Vertebrate sequences are contained within the black rectangle. (B) Representative goosecoid proteins are aligned with respect to their homeodomain (HD) sequences. The relative positions of the conserved goosecoid-engrailed homology (GEH) domain that mediates repression are indicated by red boxes. An additional sequence that is similar between SpGsc and Drosophila Gsc is shown in gray. (C) Blot of sperm DNA from two individuals that has been digested with either EcoRI (a) or RsaI (b) and hybridized at reduced stringency with a 32P-labeled probe representing the homeodomain sequence (see Materials and Methods for details).

View larger version (42K): [in a new window]   Fig. 2. RNase protection assays show that SpGsc transcripts accumulate at mesenchyme blastula (MB), gastrula (G) and pluteus (P) stages. A negative control is provided by hybridization to yeast tRNA (ytRNA). Unhybridized probe (P), egg (E), 16-cell stage (16-c), VEB (very early blastula, 150 cells). The arrow indicates the band produced by the hybridized probe; trace amounts of undigested probe persist in some of the samples.

View larger version (106K): [in a new window]   Fig. 3. In situ hybridization with 33P-labeled antisense SpGsc probe shows that SpGsc mRNA accumulates in oral ectoderm throughout the mesenchyme blastula-pluteus period and transiently during blastula stages in presumptive mesenchyme cells in the vegetal plate. (A,C,E,G,I,K) Bright field images; (B,D,F,H,J,L) corresponding dark field images. (A,B) Egg; (C-H) early mesenchyme blastula; (I,J) gastrula; (K,L) pluteus. The arrowheads in C-H indicate recently ingressed primary mesenchyme cells. The black line in H indicates the approximate plane of section that would produce the hybridization pattern shown in C,D. The locations of oral and aboral ectoderm in I-L are marked with the labels oe and aoe, respectively. The distribution of SpGsc mRNAs is indicated in black in M (blastula) and N (pluteus). cm, coelomic mesenchyme; endo, endoderm; pmc, primary mesenchyme cells; smc, secondary mesenchyme cells. Embryos shown in C-L are oriented with the vegetal pole down. Scale bar: 20 µm.

View larger version (43K): [in a new window]   Fig. 4. Morpholino knockdown of SpGsc blocks oral endoderm and vegetal differentiation. (A) Confocal images of 3-day-old embryos injected either with 30% glycerol (top panels) or SpGsc morpholino in 30% glycerol (bottom panels). Embryos were stained either with antibodies against EctoV (red), which stains late oral ectoderm and foregut, and Spec1 (green), which stains aboral ectoderm at this stage (left side), or with antibody 6e10 that recognizes ingressed primary mesenchyme cells. (B) RT-PCR analysis of Endo16 mRNA levels in 24-hour embryos injected either with glycerol, or morpholinos against SpGsc (SpGsc-M) or SpKrl (SpKrl-M); RT, reverse transcriptase. Samples were analyzed at cycles 22 and 25 to verify that signals were compared during the linear phase of PCR amplification. Endo16 signals were normalized with respect to mitochondrial 12S rRNA values and these were set to a value of 1 for the positive control, which was RNA from glycerol-injected embryos. As SpKrl is required for Endo16 expression, SpKrl-M (morpholino) provides a negative control (Howard et al., 2001). (C) 2-day embryos that had been injected with glycerol (top) or the SpGsc morpholino (bottom) were stained with antibodies specific for the Sp1 epitope that is expressed on pigment cells (green) or for 6e10 (PMCs; red). Weakly Sp1-positive cells in SpGsc morpholino-injected embryos are indicated by arrowheads. Bars: 20 µm in A,C.

View larger version (86K): [in a new window]   Fig. 5. Misexpression of SpOtx and SpGsc drive ectoderm toward aboral and oral fate, respectively. Confocal images of 3-day-old embryos double-stained with anti-EctoV and anti-Spec1 that (top panels) identify oral (red) and aboral (green) ectodermal territories in normal embryos at this stage. Sibling embryos were injected with mRNAs encoding either SpGsc (middle panels) or a fusion protein consisting of the SpGsc DNA-binding domain linked to the VP16 transcriptional activation domain (bottom panels). Separate Spec1 (left panels) and EctoV (center panels) signals are merged in the right panels. Misexpression of SpGsc promotes expression of the oral ectoderm marker in all ectodermal cells while misexpression of the VP16 fusion protein has the reciprocal effect, driving these cells to express predominantly the aboral ectoderm marker. All these confocal images were obtained at the same photomultiplier sensitivity. Scale bar: 10 µm.

View larger version (30K): [in a new window]   Fig. 6. SpGsc competes for binding at SpOtx cis elements. (A) SpGsc binds with specificity to an SpOtx site from the Spec2a promoter (contained on the CII fragment). Bacterially produced GST-GSC fusion protein purified by glutathione affinity chromatography formed a complex that was competable with the sequences containing an intact SpOtx cis element (lane 2 versus lanes 3 and 7), but not with those in which this element was altered by point mutation (lane 5) or by sequence replacement as defined by Yuh et al. (Yuh et al., 2001) (lane 6). Addition of GST antibody (lane 4) supershifted a significant fraction of this complex. GSC protein derived from GSC-GST also formed specific complexes (lane 8) as shown by competition reactions with the probe sequence (lane 9), and those containing mutated Otx elements (lanes 10,11). (B) SpGsc and SpOtx compete for binding to CII. GSC/CII complexes are shown in lanes 2 and 3. GST-Otx/CII complexes (lane 4) are supershifted with Otx antibody (lane 5). Reactions containing mixtures of GSC and GST-Otx were carried out under limiting probe concentrations. Lanes 6-8, constant amounts of GST-Otx were mixed with increasing quantities of GSC; Lanes 10-13, constant amounts of GSC were mixed with increasing amounts of GST-Otx. (C) SpGsc down regulates the activity of a promoter driven by SpOtx. Embryos (100) were injected with a promoter/CAT transgene construct and either no (–) or 2x106 or 6x106 molecules of SpGsc mRNA. St refers to a positive control reaction containing chloramphenicol acetyl transferase.

View larger version (28K): [in a new window]   Fig. 7. Activation of SpGsc expression depends on cell-cell interactions and nuclear ß-catenin. (A) SpGsc transcription requires cell-cell interactions. Cultures of single cells obtained from embryos dissociated in Ca2+- and Mg2+-free sea water at the two-cell stage were analyzed by RNase protection assays at very early blastula (12 hours), mesenchyme blastula (25 hours) and gastrula (36 hours) stages for the levels of SpGsc and SpHE (hatching enzyme) transcripts. RNA samples were checked for concentration and quality by electrophoresis through formaldehyde-containing gels (bottom panel). Negative controls were carried out with yeast tRNA (ytRNA). (B) Cell-cell interactions are required for SpGsc transcription until early blastula stages. RNase protection assays were carried out with a mixture of SpGsc and SpHE probes on samples isolated from cultures of embryos dissociated into single cells at either two-, 32-, 60-, 200- or 500-cell stages and assayed at the 600-cell gastrula stage. C refers to embryos that remained intact throughout the experiment. (C) SpGsc expression depends on the canonical Wnt signaling pathway because transcript levels are strongly reduced in 24-hour embryos injected with cadherin mRNA. RT-PCRs with primers specific for the Endo16 vegetal plate marker or for SpGsc were sampled at either cycle 22 or cycle 25 and signals were quantitated by phosphorimagery and normalized for embryo number using the mitochondrial (Mito) 12S rRNA signal as described in the legend to Fig. 4. A similar analysis was carried out for the accumulation of SpBMP2/4 message that is not inhibited by loss of ß-catenin function. –RT refers to samples that were not reverse transcribed and Mito refers to detection of 12S mitochondrial rRNA to normalize for embryo loads among samples.