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

First published online 29 August 2007
doi: 10.1242/dev.006080

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 Related articles in Development 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 Google Scholar Google Scholar Articles by Toledano-Katchalski, H. Articles by Volk, T. Search for Related Content PubMed PubMed Citation Articles by Toledano-Katchalski, H. Articles by Volk, T.

Post-transcriptional repression of the Drosophila midkine and pleiotrophin homolog miple by HOW is essential for correct mesoderm spreading

Hila Toledano-Katchalski^*, Ronit Nir, Gloria Volohonsky and Talila Volk

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

View larger version (72K): [in this window] [in a new window]   Fig. 1. Defects in mesoderm spreading in how germline clone embryos. Whole-mount wild-type (WT; A-E) or how germline clone (F-J) embryos stained for Twist (TWI; A,F), Tinman (TIN; B,G), EVE (C,H), EVE (green) + MEF2 (red) (D,I) (D' and I' represent respective single EVE staining of the merged images in D,I), and Myosin heavy chain (MHC; E,J). Arrows indicate uneven mesoderm spreading (F), segments lacking TIN-positive heart cells (G), segments lacking EVE-positive cells (H), uneven distribution of cardioblasts (I) and segments lacking dorsal muscles (H). Arrowhead in I shows a region lacking both cardioblasts and EVE-positive pericardial cells, while more ventral MEF2-positive cells are present. The images in D-I' were taken in threefold higher magnification to detect the details of the distinct cell types.

View larger version (62K): [in this window] [in a new window]   Fig. 2. HOW(L) is the major HOW isoform at stages 6-10. Whole-mount wild-type embryo double labeled for Twist (A) and for HOW (B). The merged image (C) indicates that HOW is expressed in the nuclei of the Twist-positive mesodermal cells. (D) Western analysis of embryos at 0-1, 3-5 and 14-16 hours after egg laying with anti-HOW antibodies that recognize both the HOW(L) and HOW(S) isoforms. At early stages, only the HOW(L) isoform is detected. (E,F) In situ hybridization of wild-type embryos at stage 10 with how(L)-specific probe (E) or with how(S)-specific probe (F).

View larger version (91K): [in this window] [in a new window]   Fig. 3. The four mRNA targets of HOW are elevated in the mesoderm of how germline clone embryos and exhibit direct binding to HOW. Whole-mount wild-type (A-D) or how germline clone (E-H) embryos hybridized with each of the four mRNAs identified in the microarray screen: miple (A,E), lap (B,F), CG31638 (C,G) and falten (D,H). Arrowheads in E-H indicate the specific increase in the levels of the distinct mRNAs in the mesoderm of how germline clone embryos. (I) HOW-RNA binding assay with in vitro-transcribed RNA of the 3' UTRs of miple, lap, CG31638, falten and punt. HOWR185C is a form of HOW that is mutated in the KH RNA-binding domain and served as a control for non-specific binding. The first two lanes on the left represent total HOW protein levels. Each binding experiment shown is representative of three repetitions.

View larger version (85K): [in this window] [in a new window]   Fig. 4. Overexpression of Miple leads to mesoderm spreading defects. Embryos overexpressing Miple-HA under the twist-gal4 driver labeled for Twist (TWI; A), EVE (B), Tinman (TIN; C), or Myosin heavy chain (MHC; D). (E,F) Higher magnification of an embryo overexpressing Miple-HA double-labeled for EVE (E) and MEF2 (F is the merged image of both). Arrowheads indicate uneven mesoderm distribution (A), segments lacking EVE cells (B), heart cells (C), dorsal muscles (D), or a region in which both cardioblast and EVE-positive pericardial cells are missing or mislocalized (E,F). (G) Western analysis of the embryos overexpressing Miple-HA driven by the twist-gal4 driver indicates a specific HA-positive band. Miple-HA from these embryos bound and eluted specifically from a Sepharose-heparin column (third lane from left). (H) Western blot of SR+ cell extract with anti-GFP, -Actin and -HA. The cells were transfected with HOW(L)-HA together with either GFP fused to wild-type miple 3' UTR (gfp-miple 3' UTR) or to miple 3'UTR mutated at the HOW-binding site (gfp-miple 3' UTR*).

View larger version (71K): [in this window] [in a new window]   Fig. 5. Ectopic MAPK activation in embryos overexpressing Miple. Whole-mount wild type (WT; A-D), embryos overexpressing Miple under the twist-gal4 driver (E-H) or how germline clone embryos (I-L) double labeled for Twist (green, A,E,I) and dpERK (red, B,F,J), and their merged images (C,G,K), are shown. (D,H,L) Single sections of the framed areas of the embryos shown in C,G,K at higher magnification. Arrowheads indicate the most dorsal MAPK-positive cells (D) or the scattered MAPK-positive cells (H,L).

View larger version (59K): [in this window] [in a new window]   Fig. 6. Increased number of EVE-positive cells in embryos overexpressing Miple appears to depend on HTL. Wild-type embryo (WT; A), embryo overexpressing Miple under the mef2-gal4 driver (B), or a htl mutant embryo overexpressing Miple under the mef2-gal4 driver (C) were stained with anti-EVE. The arrow in A shows three EVE-positive cells in the dorsal mesoderm, whereas the arrow in B indicates nine EVE-positive cells.

View larger version (9K): [in this window] [in a new window]   Fig. 7. Summary of HOW functions in early mesoderm development. Following mesoderm specification, levels of HOW(L) are increased in the presumptive mesoderm in order to inhibit the levels of string (cdc25) mRNA and to therefore arrest cell divisions during mesoderm invagination. During mesoderm spreading, HOW is essential to repress the levels of a set of maternal mRNAs (falten, CG31638 and lap), as well as miple, the high levels of which might induce ectopic MAPK activation in the mesoderm during spreading.