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First published online 2 February 2005
doi: 10.1242/dev.01650

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SoxB1 downregulation in vegetal lineages of sea urchin embryos is achieved by both transcriptional repression and selective protein turnover

Lynne M. Angerer^*,, Laurel A. Newman and Robert C. Angerer^*

Department of Biology, University of Rochester, Rochester, NY 14627, USA

View larger version (82K): [in a new window]   Fig. 1. SoxB1 protein encoded from mRNA microinjected at the one-cell stage does not persist in vegetal cells. (A) Translation of SoxB1 mRNA is efficiently blocked in embryos that develop from zygotes injected with SoxB1MO, as shown by the absence of staining with an antibody specific for SoxB1 (green, compare with B and C). Red staining detects the 6e10 extracellular epitope produced by primary mesenchyme cells. This embryo is at the temporal equivalent of prism stage but has failed to differentiate an archenteron (Kenny et al., 2003). (B) Co-injection of the SoxB1 morpholino and MO-immune SoxB1 synthetic mRNA rescues differentiation of gut and coelomic rudiments (white arrowheads). SoxB1 protein translated from the microinjected mRNA (green) accumulates and persists in the ectoderm, but does not persist in secondary mesenchyme (white arrowheads) or endoderm (except for low levels in the foregut). (C) An embryo at the hatched blastula stage treated as in B also clears SoxB1 protein (green signal, left) from vegetal blastomeres, despite the fact that the microinjected mRNA is present in them, as shown by whole-mount in situ hybridization to SoxB1 mRNA in the same embryo (blue signal, right). The embryo is stained for SoxB1 (FITC) and DNA (DAPI); the merged fluorescent signals over SoxB1-positive nuclei are blue-green, whereas those of SoxB1-negative nuclei are blue. Immunofluorescence images are shown on the left; DIC images, right. A and V indicate animal and vegetal poles, respectively. Scale bars: in B, 20 µm for A,B; in C, 20 µm.

View larger version (27K): [in a new window]   Fig. 2. Vegetal turnover of SoxB1GFP requires nuclear ß-catenin and can be driven by Pmar1, but is not affected by Krl. (A) Embryos at the 16-cell stage derived from fertilized eggs that had been injected with mRNA encoding SoxB1-GFP. Images were obtained with a Nikon inverted microscope equipped with epifluoresence optics and Kodak Elite 35 mm slide film. The embryo on the left is shown in a slightly tilted, vegetal pole view that shows the four micromeres, eight macromeres and one mesomere. The embryo is oriented so that mesomeres at the animal pole are up and micromeres at the vegetal pole are down. (B-G) Zygotes were injected with mRNA encoding SoxB1-GFP and the indicated proteins, allowed to develop to the temporal equivalent of mesenchyme blastula stage, deciliated, and fluorescence images were captured from live embryos by laser confocal microscopy. (B) Control embryo injected with SoxB1-GFP mRNA and glycerol, demonstrating that the SoxB1-GFP fusion protein mimics the vegetal turnover exhibited by the endogenous SoxB1. The arrow indicates PMCs that have ingressed from the vegetal plate. (C) Embryos in which nuclearization of ß-catenin is blocked by co-injection of C-cadherin mRNA do not clear SoxB1GFP from vegetal blastomeres. These embryos lack PMCs (Logan et al., 1999). (D) By contrast, upregulation of nuclear ß-catenin activity with mRNA encoding stabilized ß-catenin vegetalizes the embryo and also expands the vegetal domain of SoxB1-GFP degradation. (E,F) Mis/overexpression of Krl (E) also appears to expand the SoxB1-GFP degradation domain, consistent with its strong vegetalizing effect (Howard et al., 2001), but knockdown of Krl by means of Krl MO injection (F) does not detectably alter SoxB1 turnover. (G) Mis/overexpression of Pmar1 converts most of the cells to a PMC-like fate and upregulates SoxB1-GFP turnover throughout the embryo. Scale bar: 20 µm.

View larger version (39K): [in a new window]   Fig. 3. The SoxB1 C-terminal region contains multiple sequence signals for turnover in macromere lineages that are not required for degadation in micromere progeny. Zygotes were injected with GFP-tagged (green box) SoxB1, from which residues were deleted as indicated by the thinner black lines in the diagrams of the constructs shown on the left. The DNA-binding domain (light blue box) is bordered on each side by a nuclear localization signal motif (dark blue). Embryos (at least 30) were examined for depletion of the protein in micromere derivatives (primary mesenchyme cells) and macromere progeny (the vegetal plate). All embryos treated with the same mRNA showed a consistent phenotype, which is illustrated in the middle column. An asterisk marks the center of the vegetal plate of each embryo. (A) Control, intact SoxB1-GFP. (B) SoxB1-GFP from which almost the entire sequence 3' of the DNA-binding domain was deleted was not destabilized preferentially in vegetal plate cells. (C-E) Different portions of the C-terminal region are each sufficient to mediate vegetal clearance. Scale bar: 20 µm.

View larger version (81K): [in a new window]   Fig. 4. SoxB1 protein is released into the cytoplasm during mitosis. Two blastomeres of embryos at the 16-cell stage are shown in interphase (left side) or in mitosis (right). Measurements of pixel densities show that the cytoplasmic signal increases 1.7-fold in blastomeres in mitosis (arrowheads). Chromosomes in these cells lack detectable SoxB1 staining, whereas most of the SoxB1 signal in interphase cells is in nuclei. Scale bar: 5 µm.

View larger version (43K): [in a new window]   Fig. 5. Entry into nuclei is required for SoxB1 degradation in macromere progeny, but not in primary mesenchyme cells. The assay was performed as described in the legend to Fig. 3. (A) SoxB1-GFP from which the DNA-binding domain and flanking NLS sequences were removed was cleared from micromere derivatives, but not from macromere progeny. (B) SoxB1 deleted of the DNA-binding domain but retaining the NLS sequences is preferentially degraded in both micromere and macromere progeny. (C) SoxB1-GFP deleted of the DNA-binding domain and 5'NLS sequence, but retaining the 3'NLS sequence. The arrows indicate clusters of ingressed primary mesenchyme cells. Scale bar: 20 µm.

View larger version (51K): [in a new window]   Fig. 6. SoxB1 functions in a negative-feedback loop. Zygotes were injected with glycerol as a control (A) or with SoxB1 MO (B) and allowed to develop to the mesenchyme blastula stage or its temporal equivalent. They were then subjected to whole-mount in situ hybridization with a SoxB1 probe. In order to observe the spatial pattern of expression, the enzymatic signal development was 4-fold longer for controls than for experimental embryos. Signals were consistently and significantly elevated in SoxB1 MO embryos, but only in presumptive ectoderm. Scale bar: 20 µm.

View larger version (98K): [in a new window]   Fig. 7. Downregulation of SoxB1 mRNA in vegetal blastomeres requires ß-catenin, but not Krl. Zygotes were injected with glycerol (control), with mRNA encoding the indicated mRNAs, or with Krl-MO, allowed to develop to the hatched blastula stage, and then assayed by whole-mount in situ hybridization with a SoxB1 probe. (A) Blocking nuclearization of ß-catenin by injection of C-cadherin mRNA blocks downregulation of SoxB1 mRNA in vegetal blastomeres. (B) Neither knockdown of Krl translation with a KrlMO nor its mis/overexpression by mRNA injection (Krl MOE) detectably affects downregulation of SoxB1 message in vegetal blastomeres. However, in the animal hemisphere, loss of Krl function leads to a decrease in SoxB1 mRNA, whereas MOE up regulates it. This effect is likely to reflect the operation of the SoxB1 negative-autoregulatory loop, as discussed in the text and illustrated in Fig. 5. Scale bar: 20 µm.

View larger version (28K): [in a new window]   Fig. 8. Summary of evidence for the multiple mechanisms that regulate SoxB1 accumulation along the animal-vegetal axis. (A) Uniformly distributed SoxB1 is asymmetrically partitioned at fourth cleavage among different sized blastomeres in proportion to their cytoplasmic volume. [Image of an embryo doubly stained with SoxB1 antibody and DAPI between 16 and 32-cell stages reproduced, with permission, from Kenny et al. (Kenny et al., 1999)]. (B,C) SoxB1 is selectively degraded in micromeres and macromeres via different mechanisms, both of which depend on nuclear ß-catenin function. SoxB1 peptides lacking the NLS sequences or the 3'-terminal region are eliminated from micromeres but not macromres (B), whereas SoxB1 variants retaining the NLSs and at least one out of three regions in the C-terminal domain clear from both micromere and macromere derivatives (C). (D) SoxB1 mRNA levels remain high in vegetal blastomeres in the absence of nuclear ß-catenin. (E) SoxB1 mRNA concentrations are elevated in animal blastomeres in embryos lacking SoxB1 protein.

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