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The Snail protein family regulates neuroblast expression of inscuteable and string, genes involved in asymmetry and cell division in Drosophila

Program in Molecular Medicine and Department of Cell Biology, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA

View larger version (89K): [in a new window]   Fig. 1. Expression of snail and worniu in early CNS. (A,B,E-H) RNA in situ hybridization of worniu. (C) RNA in situ of snail. (D) Antibody staining for Snail. (B) Double staining with Prospero antibody. (A-E) Wild-type embryos; the genotypes of the embryos in F-H are as indicated. The orientation of the embryos in this and following figures is anterior towards the left. (A-D) Lateral views; (E-H) Dorsal-ventral views. All the embryos are approximately at stage 9. wor RNA is expressed extensively in neuroblasts but in only a small number of GMCs, indicated by arrows (A). At a similar stage, many GMCs have formed, as indicated by the Prospero protein staining (arrows, B). (C,D) sna RNA and protein expression is also restricted predominantly to neuroblasts. (E-H) wor RNA expression is defective in embryos mutant for different pro-neural genes.

View larger version (166K): [in a new window]   Fig. 2. inscuteable expression is dependent on the Snail family of proteins. (A,C,E,G) Stage 9 embryos. (B,D,F,H) Stage 10 embryos. All embryos are ventral views. RNA in situ hybridization reveals the mRNA expression of inscuteable is significantly lower in osp29 mutant (C,D) than in wild-type embryos (A,B). Note that some localized mRNA is still present in osp29 embryos. (E-H) Loss of inscuteable expression can be efficiently rescued in embryos expressing transgenic Snail family of proteins. (E,F) Embryos with P[snail] transgene; (G,H) embryos with P[wor, esg] transgenes. The P[wor, esg] was generated by recombination of the two individual transgenes (Ashraf et al., 1999), with each under the control of 2.8 kb snail promoter, including the neuroblast expression element (Ip et al., 1992; Ip et al., 1994). For this and following figures, some of the mutant embryos shown have morphological defects that are due to the requirement of Snail in gastrulation. The morphological phenotype is used whenever possible to identify embryos that harbor the mutation. The gastrulation phenotype has no direct consequence on the expression of CNS markers. This is based on the observations that rescue of morphological defect by snail driven by mesoderm promoter alone cannot rescue the CNS defect, and worniu and escargot transgenes can rescue CNS defect but not gastrulation defect (e.g. see G,H).

View larger version (88K): [in a new window]   Fig. 3. Defective prospero mRNA localization in the deletion mutant. (A,B,E,F) Ventral views of embryos at low magnification. (C,D) Sagittal views of embryos at medium magnification. (G-J) Sagittal views of embryos at high magnification. (A,B) RNA in situ hybridization for miranda mRNA expression shows normal miranda mRNA expression and subcellular localization in wild-type and osp29 mutant embryos (arrows indicate cells with RNA localization). (C,D) The Miranda protein is detectable, although less abundant and in fewer cells, in the deletion mutant (arrows indicate cells with subcellular localization). (E,F) The expression of prospero mRNA is detectable in the neuroblasts of mutant embryos albeit slightly lower in early stage; the intense midline (ML) staining in the mutant is probably due to expansion of midline cell fate in the absence of Snail in the blastoderm. Older mutant embryos (H) accumulate higher levels of prospero RNA, similar to that of wild type (G). The apparently lower level in the mutant in earlier stages may also be due to the loss of subcellular localization (compare G with H, arrows indicate localization). This localization defect can be rescued (arrows) in the presence of P[wor, esg] transgenes (I). In inscuteable mutant embryos (J), the Prospero protein expression is clearly seen in some GMCs (arrowhead) and neuroblast (arrow) nuclei. This phenotype is different from Prospero protein pattern in osp29 embryos (compare with Fig. 4B).

View larger version (65K): [in a new window]   Fig. 4. GMC formation is defective in the absence of Snail family of proteins. All embryos are approximately at stage 11. (A-C) Ventral view. (D-F) Sagittal views of embryos. (A,D) Wild type; (B,E) Df(2L)osp29 mutant; (C,F) osp29 with P[wor, esg] transgenes. The Prospero protein staining is largely absent in the deletion mutant (B). More cells show staining in the midline (ML), probably owing to the derepression of midline determinants in the absence of Snail (see also Fig. 3F). Transgenes of worniu and escargot rescued Prospero expression efficiently (C). Similar results are also observed in the presence of snail transgene (data not shown). Hunchback (Hb) at this stage is present in many GMCs (D). The brackets in D-F indicate where Hb-positive GMCs are seen in wild-type and rescued embryos but absent in osp29 mutant embryos.

View larger version (66K): [in a new window]   Fig. 5. Snail family of proteins regulate string expression in neuroblasts. RNA in situ hybridization was carried out using antisense string probe on wild-type (A), osp29 mutant (B) and P[wor, esg]-carrying mutant (C) embryos. After in situ hybridization, the embryos were embedded in Epon plastic and 3 µm sections were cut and representative sections are shown here. The arrows in panels A and C indicate RNA expression of string in neuroblasts. Staining is also seen in ectodermal cells. The neuroblast layer is located between the ectoderm and the mesoderm (A). The osp29 mutant embryos also have more folding, indicating gastrulation defects. Nonetheless, the ectodermal staining is clear but the neuroblast staining is largely absent (B). The transgenes can partially rescue expression of string in the neuroblasts (C).

View larger version (117K): [in a new window]   Fig. 6. string-lacZ reporter expression is regulated by the Snail protein family. RNA in situ hybridization using an antisense lacZ probe reveals string promoter-lacZ reporter expression. Two different string-lacZ reporters were used. (A-F) Embryos expressing string 5.3-lacZ. (G-L) embryos expressing string 6.4-lacZ. (A,B,G,H) Wild-type embryos. (C,I,J) osp29 embryos. (D) Another deletion do-1 embryo (Ashraf et al., 1999). (E,F,K,L) osp29 embryos carrying the indicated rescue transgenes. In the deletion mutants, the lacZ expression is almost abolished. A single copy of each of the indicated transgenes was sufficient to confer a clear rescue of the reporter expression.

View larger version (117K): [in a new window]   Fig. 7. Cell division defects and rescue of Prospero GMC expression. (A-F) Embryos stained for phosphorylated-histone H3 (brown). (C-F) Embryos also stained for prospero RNA (blue). (A-D) Ventral views of embryos shown at medium magnification; (E-F) sections of similar embryos shown at high magnification. The brackets in A,B indicate the ventral neurogenic region where most neuroblasts are located. There is clear staining of phosphorylated H3 in several cells in wild-type embryo (A), indicating mitosis. Much less staining is seen in the osp29 mutant embryo (B). Similar embryos double stained for prospero RNA showing the neuroblast layer (C,D). The arrowheads indicate some of the phosphorylated-H3 staining. Sections of similar embryos shown in E,F indicate that the neuroblast cell layer with prospero RNA staining also show mitosis occurring in wild-type embryos, but very rarely in osp29 embryos. The results suggest that the deletion mutants undergo less mitosis. (G,H) Staining of Prospero protein in GMCs. The expression of Prospero protein can be detected in many GMC nuclei along the two sides of the expanded midline in an embryo containing the string and inscuteable transgenes (H). The embryo containing the transgenes still has expanded midline, indicating the lack of Snail activity in blastoderm.

View larger version (116K): [in a new window]   Fig. 8. Snail function in neuroblasts requires the dCtBP co-repressor interaction motif. The schematic shows the protein structure of Snail. The embryos show RNA in situ hybridization for ftz (A-C,E,G) and inscuteable (D,F,H). All the embryos are osp29 mutants, except in A (wild type). (C,D) Embryos expressing a P[snail] transgene in which the N-terminal dCtBP interaction motif is mutated (M1). (E,F) Embryos containing the M2 mutant; (G,H) Embryos containing the M12 double mutant. There is much lower rescue of ftz and inscuteable in the absence of both dCtBP interaction motifs.

View larger version (15K): [in a new window]   Fig. 9. Snail family of proteins play an essential role in neuroblast development. Many pro-neural genes control the expression of snail and worniu in neuroblasts. Snail and Worniu, and to a lesser extent Escargot, normally function to regulate the asymmetry and cell division of neuroblasts by controlling the expression of inscuteable and string, respectively. The correct expression of these two genes is required for proper segregation of Prospero into GMC, where Prospero functions as a crucial factor for cell fate determination. The arrows indicate genetic hierarchy, not necessarily direct regulation. The Snail family of proteins likely function as repressors. Thus, the regulation of inscuteable and string may be through the repression of another repressor, leading to gene activation. It is also possible that the Snail family of proteins can directly activate the expression of these two genes.