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First published online 13 December 2006
doi: 10.1242/dev.02735

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Muscle-dependent maturation of tendon cells is induced by post-transcriptional regulation of stripeA

¹ Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
² Institut für Neurobiologie, Universität Münster, Badestrasse 9, D-48149 Münster, Germany.

View larger version (85K): [in this window] [in a new window]   Fig. 1. stripeA and stripeB mRNAs show distinct expression profiles during embryonic development. Upper panel: schematic representation of the coding sequences of StripeA and StripeB isoforms, and the genomic region of the stripe gene (non-coding exons are shown in black and coding exons in white). Lower panel: whole embryo staining of stage 14 embryo, before muscle-tendon interaction (A-D), stage 15 (E-H), where part of the muscles are attached, or stage 17 embryos, where muscle-tendon interaction has been established (I-L). Staining was with anti-StripeA (red, A,E,I); anti-Stripe (blue, B,F,J; an antibody recognizing both Stripe isoforms); and anti-Myosin heavy chain (MHC, green, C,G,K). D,H and L are the corresponding merged images. Arrowheads in I point to the three muscle-bound tendon cells expressing high StripeA levels. The inset in L represents high magnification of the bracket showing about 14 blue-labeled tendon precursor cells, from which only three cells that are in close proximity with the three lateral transverse muscles express StripeA. NLS, nuclear localization signal; ZF, zinc finger.

View larger version (129K): [in this window] [in a new window]   Fig. 2. StripeA is hardly detected when muscle-tendon interaction is abrogated. Dorsal view of stage 16 wild-type (A-D) or myospheroid (mys) mutant (E-H) embryos or embryos overexpressing Hid in muscle cells (I-L), stained with anti-StripeA (red, A,E,I) anti-Stripe (Blue, B,F,J) anti-Myosin heavy chain (MHC; green, C,G,K). The corresponding merged panels are shown in D,H and L. Arrowheads (E,F,H) or arrows (I,J,L) show that in mys mutants, or in regions where muscle are lacking, all tendon precursors express StripeB, but StripeA is significantly reduced or completely absent.

View larger version (86K): [in this window] [in a new window]   Fig. 3. Overexpression of StripeA leads to severe disruption of the somatic muscle pattern, and arrest of germ band retraction. Wild-type embryos (A,B) or embryos overexpressing StripeA (C-F) or StripeB (G,H), using the pan ectodermal driver 69B-gal4, were double labeled with Myosin heavy chain (MHC; green), or with Stripe (red). Panels A,C,E,G show the somatic muscle pattern, whereas panels B,D,F,H show the corresponding merge of MHC and Stripe labeling. Note the disruption of muscle pattern (E,F) and the inhibition of germ band retraction (C,D) in embryos overexpressing StripeA.

View larger version (167K): [in this window] [in a new window]   Fig. 4. Differential effect of StripeA and StripeB on Short stop levels. Stage 14-15 wild-type embryos (A,D,D'), or embryos overexpressing StripeA (B,E,E') or StripeB (C,F,F') together with GFP driven by ptc-gal4 were labeled for GFP (green, A,B,C) and Shot (red, D-F'). Only StripeA induced the ectopic expression of Shot. Arrowheads and brackets mark the ptc-gal4 domain. D',E' and F' represent higher magnification of D,E,F. Note the change in cell shape induced by StripeA (arrows in E,F and white outlines marking a single cell in each panel).

View larger version (74K): [in this window] [in a new window]   Fig. 5. StripeA and StripeB exhibit differential transcriptional output. (A-F) Stage 15 wild-type embryos (WT; A,B) or embryos overexpressing StripeB (C,D) or StripeA (E,F) driven by the pan-ectodermal driver 69B-gal4 were double-labeled for Slit (red, A,C,E) and for Stripe (green) (merged view is shown in B,D,F). Arrows in A and B show Slit expression pattern close to the Stripe-expressing cells. A significant elevation of Slit is detected in both overexpressing embryos. (G-L) In situ analysis of wild-type embryos (G,J) or embryos overexpressing StripeB (H,K) or StripeA (I,L) using en-gal4 driver with probes specific to how(L) (G-I) or how(S) (J-L) mRNAs. While both StripeA and StripeB induce the expression of how(L) mRNA, only StripeA induces the expression of how(S) mRNA. (Brackets indicate the segmental border expression domain). (M) The effect of StripeA or StripeB overexpression driven by ptc-gal4 driver on the endogenous levels of stripeA or stripeB mRNA levels in 13-16-hour-old embryos, as measured by RT-PCR performed on RNA samples using primers specific for the endogenous stripeA or stripeB mRNA.

View larger version (93K): [in this window] [in a new window]   Fig. 6. How(S) is required for stripeA elevation. (A-H) Wild-type embryo (A,C,E,G) or howstru mutant embryo (B,D,F,H) at early stage 16 stained for anti-StripeA (red, A,B) and anti-Stripe (blue, C,D). C and D are the corresponding merged images of A and B. Embryos stained for anti-Shot (red, E,F) and anti-Stripe (blue, G,H). G and H are the corresponding merged images of E and F. Tendon cells of the ventral longitudinal muscles are shown. A significant reduction of StripeA and Shot is detected in the how mutant embryos. (I-K) Embryos carrying either the 69B-gal4 driver alone (I), together with How(L) (J) or together with How(S) (K) stained with anti-StripeA antibody. Tendons of the ventral longitudinal muscles are shown. (L) RT-PCR performed on RNA extracted from howstru mutant embryos (selected by their negative GFP staining) or wild-type embryos at the age of 14-16 hours AEL, using primers specific for either stripeA (left panel) or stripeB (right panel). The total RNA of each sample was normalized against tubulin levels. (M) RT-PCR with stripeA-(left panel) or stripeB-(right panel) specific primers, performed on RNA extracted from embryos overexpressing How(S) using the stripe-gal4 driver. The embryos were at 14-16 hours AEL.

View larger version (28K): [in this window] [in a new window]   Fig. 7. How(S) facilitates the splicing of stripeA minigene. (A) S-2 (Schneider) cells transfected with stripeA minigene containing the intronic sequences of stripeA flanked by partial sequences of its 3' an 5' exons, were co-transfected with different forms of How proteins. (B) The splicing of the two exons was monitored by RT-PCR using primers specific for the two flanking exons (509F and 5020R, see scheme). All RNA samples were normalized using tubulin-specific primers. In the presence of wild-type How(S) the splicing reaction was facilitated by threefold. By contrast, a mutant form of How(S) containing an NLS did not induce such elevation, nor did the nuclear-specific How(L) form. A mutant How(L), which is also cytoplasmic, did induce a mild elevation (1.5-fold). (C) Western analysis of the transfected S-2 crude extracts using anti-HA antibody to detect transfected How constructs. (D) A protein-RNA binding assay was performed on in vitro transcribed stripeA intronic RNA sequences labeled with biotin and precipitated with HA-tagged in vitro translated How(S) or mutated form of How(S), How(S)e44, which does not bind RNA. Western analysis shows the presence of HA-tagged How(S) in the beads only in the presence of stripeA intronic sequences, while its levels are equal in all the binding reactions.

View larger version (69K): [in this window] [in a new window]   Fig. 8. stripeA mRNA levels are specifically reduced in crn mutant embryos. (A) RT-PCR with stripeA- and stripeB-specific primers, performed on RNA extracted from crn homozygous mutant or from wild-type embryos at 14-16 hours AEL. The mRNA levels were normalized against tubulin mRNA levels in each sample. A specific reduction of stripeA is detected. (B-E) Stage 16 wild-type (B) or crn mutant (D) embryos stained for Shot (red) and Stripe (Blue, C,D); C and D are the corresponding merged images. A significant reduction of Shot is detected in crn mutant embryos. (F-I) Stage 16 wild-type (F) or crn mutant (H) embryos stained for How (red) and Stripe (blue, G,I); G and I are the corresponding merged images. Note that there is no change in How levels in crn mutants. Arrowheads mark the tendon cells of the ventral longitudinal muscles.

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