QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Morisato, D. Search for Related Content PubMed PubMed Citation Articles by Morisato, D.

Spätzle regulates the shape of the Dorsal gradient in the Drosophila embryo

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA

View larger version (108K): [in a new window]   Fig. 1. Reduction in Egfr signaling expands the domain of pipe expression in the follicular epithelium. (A-C) Ovaries dissected from wild-type (A), Egfr1/Egfr1 (B), and grkWG/grkHG (C) females were visualized for the expression of pipe RNA. Panels show cross-sections of an egg chamber in mid-oogenesis at the position of the oocyte nucleus. Arrows indicate the dorsal borders of staining. In B,C, the domain of pipe expression appears uniformly expanded, with no distinct peaks apparent. The dorsal side is upwards in all figures. (D-F) Gastrulating embryos were stained for Twist protein. Panels show cross-sections of embryos at 50% egg length. Wild-type embryos (D) gastrulate with a single ventral furrow. Embryos produced by Egfr1 females (E) broaden the domain of cells expressing Twist, and a fraction gastrulate with two ventral furrows, as shown here. Embryos produced by grkWG/grkHG females (F) exhibit two distinct domains of cells that express Twist, and each domain invaginates as a distinct ventral furrow. (G-I) Cuticles of embryos produced by wild-type (G), Egfr1/Egfr1 (H) and grkWG/grkHG (I) females were dissected out of their vitelline membrane cases and are shown in dark-field illumination. The images in H,I are magnified 1.5x relative to G.

View larger version (185K): [in a new window]   Fig. 2. Increasing spätzle dose expands the Twist domain. Young cellularized embryos were stained for Twist protein. Panels show cross-sections of embryos at 50% egg length. (A,B) In wild-type embryos (A), Twist is expressed in the ventral 20% of the embryo. In embryos laid by spz- females that were injected with high levels of spz RNA (B), Twist expression is expanded to the ventral 40% of the embryo. (C,D) In embryos produced by Egfr1 females (C), Twist is expressed in the ventral 50% of the embryo. In embryos laid by Egfr1; spz- females that were injected with high levels of spz RNA (D), Twist expression is expanded to the ventral 70% of the embryo.

View larger version (96K): [in a new window]   Fig. 3. Changing spätzle dose does not affect the size of the sog domain. Young cellularized embryos were hybridized to detect expression of sog RNA. Panels show cross-sections of embryos at 50% egg length. In wild-type embryos (A), sog RNA is expressed in two stripes that together occupy 31.5% of the embryo circumference. In embryos produced by spz+/spz- females (B), sog is expressed in 30% of the embryo. In embryos produced by Egfr1 females (C), sog is expressed in 31% of the embryo. In most embryos laid by spz- females that were injected with high levels of spz RNA (D), sog is expressed in two stripes occupying 33% of the embryo. In a few embryos injected with high levels of spz RNA (E), sog is expressed in a single domain, although the RNA level appears lower at the fusion border.

View larger version (147K): [in a new window]   Fig. 4. High levels of Spätzle can generate two peaks of nuclear Dorsal and Twist expression. (A) Embryos laid by spz- females were injected with spz RNA and stained for Twist protein. Note the invagination of two ventral furrows. (B,C) Embryos laid by spz- females were injected with spz-(bcd 3'UTR) RNA and stained for Twist protein. (B) Two stripes of Twist are visible in this embryo, viewed from the ventral side. In cross-section (C), this embryo with two peaks of Twist expression is virtually indistinguishable from the embryo shown in F. (D,E) Embryos laid by spz- females were injected with spz-(bcd 3'UTR) RNA and stained for Dorsal protein. In a young embryo (D), the two peaks of nuclear Dorsal (arrows) are separated by several cells in which Dorsal protein is present in both the nucleus and the cytoplasm. (The region of non-staining (upper right) was caused by the incomplete removal of vitelline membrane before incubation with antibodies.) In an older embryo (E), the two peaks of nuclear Dorsal are clearly visible, as cytoplasmic Dorsal protein has been degraded. (F) Young cellularized embryos produced by grkWG/grkHG females were stained for Twist protein. Two peaks of Twist expression are apparent in this cross-section. (G) Cuticle of embryo laid by spz- female and injected with spz-(bcd 3'UTR) RNA is strongly ventralized. (H) Embryos laid by spz- females were injected with spzD1-(bcd 3'UTR) RNA and stained for Twist protein. Twist is expressed uniformly around the embryo circumference.

View larger version (52K): [in a new window]   Fig. 5. The Spätzle processing reaction in wild-type and Egfr1 embryos. (A) Extracts were prepared from 0-4 hour embryos laid by wild-type, spz-, ea-, and Toll- females. Proteins were separated on a 12.5% polyacrylamide gel and transferred to PVDF. The mobility of molecular weight markers is shown on left. The immunoblot was probed with antibodies directed against the N-terminal half of Spätzle protein. Full-length forms of Spätzle are marked by the bracket. Prominent proteolytically processed forms of N-terminal Spätzle, present in wild-type embryos and absent in embryos laid by ea- females, are indicated by arrows (migrating as 23 and 27 kDa proteins). (B) An immunoblot with the same samples as in A was probed with antibodies directed against the C-terminal half of Spätzle protein. The arrow marks the proteolytically processed form of C-terminal Spätzle (migrating as a 21 kDa protein). (C) On the left, extracts were prepared from sorted stage 4-5 embryos laid by wild-type or Egfr1 females. On the right, conventional extracts were prepared from 0-4 hour embryos laid by wild-type, spz-, and Toll- females. The immunoblot was probed with antibodies directed against the C-terminal half of Spätzle protein. (D) Cuticle of embryo laid by wild-type female and injected with N-spz RNA is strongly dorsalized. The bright outline is the vitelline membrane. (E) Cuticle of dorsalized embryo laid by spz- female.

View larger version (102K): [in a new window]   Fig. 6. Reduction of spätzle dose in Egfr1 background can generate two Twist peaks. (A,B) Cellularized embryos were stained for Twist protein. In embryos produced by Egfr1 females (A), Twist is expressed in the ventral 50% of the embryo. In embryos produced by Egfr1; spz+/spz- females (B), Twist is expressed in two stripes. (C,D) Young cellularized embryos were hybridized to detect expression of sog RNA. In embryos produced by Egfr1 females (C), sog is expressed in two stripes flanking the ventral Twist domain. In embryos produced by Egfr1; spz+/spz- females (D), sog expression is expanded ventrally, but is absent in two domains where Twist is expressed. (E) Cuticle produced by embryos laid by Egfr1; spz+/spz- females.

View larger version (26K): [in a new window]   Fig. 7. Model for generating two peaks of nuclear Dorsal. This cartoon depicts events within the perivitelline space at two different time points for embryos laid by wild-type and grk- females. The Spätzle cleavage reaction is hypothesized to produce an inhibitor (red), in addition to activated Spätzle (green). If processed Spätzle and the inhibitor possess different diffusion rates, the relative distribution of the two molecules would change from t0 to t1, giving rise to the final shape of the gradient characterized by one or two peaks of high nuclear Dorsal (blue).