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A positive role for Short gastrulation in modulating BMP signaling during dorsoventral patterning in the Drosophila embryo

Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA

View larger version (65K): [in a new window]   Fig. 1. sog transcription is reduced in sogP129D embryos. sog expression in wild-type (A,B) or sogP129D (C,D) cellular blastoderm embryos. Lateral views (A,C) and ventral views (B,D). To directly compare levels of sog expression, wild-type and sogP129D embryos were mixed in the same tube and assayed for both sog and Race transcription (both in blue). The lack of Race expression in the presumptive amnioserosa of sogP129D embryos was used to differentiate the two genotypes.

View larger version (58K): [in a new window]   Fig. 2. The amnioserosa is not properly patterned in sogP129D embryos derived from MadES1/+ females. (A) A darkfield photomicrograph of a cuticle of a sogP129D embryo (lateral view, dorsal up, anterior left). The ventral-most ectodermal cells form the neurogenic ectoderm and are characterized by a segmentally repeated pattern of denticle bands. Dorsolateral cells differentiate dorsal hairs, only faintly visible in the cuticle preparation. The filzkörper, respiratory structures of the tail, are derived from cells in a dorsolateral position in the blastoderm. The amnioserosa, which does not contribute to the embryonic cuticle, can be visualized in a dorsal view of a sogP129D stage 13 embryo (B) after staining for ß-galactosidase activity from a P[Kr-lacZ] construct expressed in this tissue. (C) Lateral view of a cuticle from a sogP129D embryo derived from a MadES1/+ female. The internalized filzkörper and the lack of head elements are indicative of a weakly ventralized embryo. The internalization of the filzkörper results from defects in germband extension in embryos that lack a fully functional amnioserosa. The embryo was raised at 25°C. (D) ß-galactosidase activity from the P[Kr-lacZ] construct in a stage 13 sogP129D embryo derived from a MadES1/+ female. This embryo shows a large reduction in the amount of amnioserosa. Embryos in B and D were raised at 29°C.

View larger version (36K): [in a new window]   Fig. 3. Synergistic lethality between weak alleles of zen and sog. (A) At 25°C, sogP129D and zen1 are both homozygous viable. Occasional sogP129D; zen1 double mutant flies arise from a sogP129D/FM7; zen1/TM3 stock maintained at 18°C. When these flies are transferred to 25°C, they produce dead embryos with a partially ventralized phenotype (as seen in B).

View larger version (62K): [in a new window]   Fig. 4. zen expression is strongly reduced in sogP129D embryos derived from MadES1/+ females. Because zen transcription is very dynamic, it was assayed in stage 6a embryos. At this stage, which lasts approximately 5 minutes, the cephalic furrow is evident but the invagination of the mesodermal primordium is not yet visible. All embryos were grown at 29°C. All in situ hybridizations in A-E were carried out in parallel, as were the hybridizations in G-H, with the same concentration of probe and were developed for exactly the same amount of time. (A) Dorsal view of the distribution of zen mRNA in a wild-type embryo. At this stage zen is expressed only in the 10% most dorsal tissue. (B) Expression of zen in a sogP129D embryo. Note a small reduction in the intensity of transcription, but a larger spatial domain of expression. (C) zen expression in an embryo derived from a MadES1/+ female. Note that zen is expressed in the same spatial domain as in wild type, but with reduced intensity. (D-F) Reduction in zen expression in sogP129D heterozygous and homozygous embryos derived from sogP129D/FM7; MadES1/+ females crossed to sogP129D males. 66% (n=35) of the stage 6a embryos analyzed displayed a reduction or elimination of zen transcription; the remaining embryos, of putative genotype FM7, were identical to those in C. (G) zen expression in a sogYSO6 embryo. (H) zen expression in a sogYSO6 embryo derived from a Mad12/+ female. The intensity of zen transcription was found to be variable to an equivalent degree in embryos of both genotypes. In both cases, the embryos shown have an intermediate intensity of zen transcription.

View larger version (72K): [in a new window]   Fig. 5. Injection of sog mRNA locally inhibits the transcription of a dorsal-specific gene, but activates its transcription at a distance from the site of injection. Lateral views of snake (snk) mutant embryos hybridized with Race (blue) and sog (brown) riboprobes. Late cellularization (A,C) and post gastrulation (stage 7 by developmental time; B,D). (A,B) Race expression in uninjected embryos is restricted to the anterior domain, and no endogenous sog transcription is present. (C,D) Embryos injected in a dorsal anterior position (marked by arrow) with a small bolus of sog mRNA (brown). Race expression is inhibited by sog at the site of injection, and is activated at a distance from the source of sog, both more posteriorly (bracket) and at a ventral-anterior position. Note that the white ‘halo’ is more pronounced in the older embryo, corresponding to inhibition of endogenous Race expression outside the domain of detectable sog mRNA.

View larger version (135K): [in a new window]   Fig. 6. Race expression in dorsalized embryos injected with different amounts of sog or chordin mRNAs. Embryos laid by snk females were injected with decreasing amounts of sog mRNA (A-E) or chordin mRNA (F-H) and stained after injection for Race expression (blue). Each embryo illustrates the most frequent outcome after the injection of a particular mRNA concentration, the total numbers are given in Table 2 for sog mRNA and Table 3 for chd mRNA. Injections were performed in an anterior dorsal position (similar to that in Fig. 5) and the size of the bolus of injected mRNA was held constant in all injections. (A) Lateral view of an embryo injected with 40 µg/µl of sog mRNA. The inhibition of endogenous Race expression is strong and no ectopic Race expression is observed. (B) Lateral view of an embryo injected with 0.8 µg/µl of sog mRNA. Race expression is inhibited at the site of injection and ectopic Race expression is detected at a distance from the site of injection. (C) Dorsal view of the embryo shown in B. (D) Dorsal view of an embryo injected with 0.2 µg/µl of sog mRNA. Race expression is inhibited locally but no ectopic expression is observed. (E) Dorsal view of an embryo injected with 0.08 µg/µl of sog mRNA. No inhibition of Race is observed. (F) Lateral view of an embryo injected with 4.5 µg/µl of chd mRNA showing complete inhibition of Race expression. (G) Dorsal view of an embryo injected with 2.25 µg/µl of chd mRNA showing local inhibition but no ectopic expression of Race. (H) Dorsal view of an embryo injected with 1.5 µg/µl of chd mRNA. No inhibition of Race expression is observed.

View larger version (91K): [in a new window]   Fig. 7. Elevation of dpp+ dosage restores amnioserosa in sogYSO6 embryos. (A-C) Dorsal views of stage 6a embryos hybridized with digoxigenin-labeled Race riboprobes showing Race expression in the presumptive amnioserosa. (A) Distribution of Race mRNA in a wild-type embryo. At this stage Race mRNA is present only in the 10% dorsal-most cells. (B) Race mRNA is absent in the presumptive amnioserosa in a sogYSO6 embryo. Weak staining is present in the anterior dorsal region of the embryo. (C) Race expression is partially restored along the AP axis in a sogYSO6 embryo that carries an extra copy of Mad12. Race expression is expanded compared to wild type in the dorsolateral regions of the embryo. From a cross of sogYSO6/FM7 females with Dp(2;2)DTD48 males, 51% (n=78) of the sogYSO6 embryos showed partially restored Race staining. (D-F) Lateral views of stage 13 embryos stained for ß-galactosidase activity driven by a P[Kr-lacZ] construct expressed in the amnioserosa. (D) Wild-type embryo. (E) sogYSO6 mutant embryos do not differentiate amnioserosa as evidenced by the absence of ß-galactosidase activity. (F) Restoration of amnioserosa in a sogYSO6 embryo by the presence of an extra copy of dpp+. From the same cross as above, 51% (n=70) of the sogYSO6 embryos showed restoration of ß-galactosidase staining in the amnioserosa.

View larger version (127K): [in a new window]   Fig. 8. Elevation of screw activity does not rescue a sogYSO6 mutant. (A) Lateral view of a stage 13 sogYSO6; P[Kr-lacZ] embryo after dorsal injection of 2.7 µg/µl of scw mRNA embryo. No ß-galactosidase staining is observed, indicating that the overexpression of scw mRNA does not restore amnioserosa in sog embryos (0%; n=38). (B) Lateral view of a Df(2L)OD16 P[Kr-lacZ] stage 13 embryo lacking scw, stained for ß-galactosidase activity after injection of 27 ng/µl of scw mRNA. While uninjected scw mutants fail to develop amnioserosa, injection of 27 ng/µl of scw mRNA resulted in restoration of amnioserosa (83%; n=34).

View larger version (72K): [in a new window]   Fig. 9. chordin mRNA injection can block the activity of injected scw mRNA but not that of injected dpp mRNA. (A-D) Lateral views of stage 13 Df(2L)OD16 P[Kr-lacZ] embryos lacking scw. Each embryo was injected dorsally with a different combination of mRNAs before the blastoderm stage and stained for ß-galactosidase activity. (A) Injection of 30 ng/µl of scw mRNA resulted in restoration of amnioserosa in 81% (n=114) of scw embryos. (C) Co-injection of 4.5 µg/µl of chd mRNA completely abolished the ability of injected scw mRNA to restore amnioserosa (0%; n=107). (B) Injection of 370 ng/µl of dpp mRNA resulted in restoration of amnioserosa in 83% (n=83) of scw embryos, however, co-injection of 4.5 µg/µl of chd mRNA with the dpp mRNA (D) was not sufficient to block amnioserosa formation (85%; n=68).