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

First published online January 23, 2009
doi: 10.1242/10.1242/dev.031195

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 Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Spirov, A. Articles by Baumgartner, S. Search for Related Content PubMed Articles by Spirov, A. Articles by Baumgartner, S. Social Bookmarking                         What's this?

Formation of the bicoid morphogen gradient: an mRNA gradient dictates the protein gradient

Alexander Spirov¹, Khalid Fahmy^2,*, Martina Schneider^2,, Erich Frei³, Markus Noll^3, and Stefan Baumgartner^2,

¹ Center for Developmental Genetics, Stony Brook University, Stony Brook, NY 11794-5140, USA.
² Department of Experimental Medical Sciences, Lund University, BMC B13, S-22184 Lund, Sweden.
³ Institut für Molekularbiologie, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

View larger version (76K): [in this window] [in a new window]   Fig. 1. Visualization of the bcd mRNA gradient by established in situ hybridization methods. (A,B) Dark- (A) and bright- (B) field photomicrographs of a frozen midsagittal section through a Drosophila embryo at nuclear cycle 14, hybridized in situ with a 3H-labeled bcd cDNA probe [fig. 5e,f of Frigerio et al. (Frigerio et al., 1986), reproduced with permission]. The inset in B is a magnified view of the anterior region, as marked with a bracket, that shows the silver grains (black) in the apical periplasm of nuclei. (C) The bcd mRNA gradient. Silver grains in the apical periplasm of each dorsal nucleus in B were counted under a microscope and plotted against the position of the nucleus along the anteroposterior axis [nucleus 1 at the anterior tip corresponds to 0% egg length (EL)]. Irregularities and small gaps at nuclei 10-20 were caused by shearing forces during sectioning. (D-F) Transcripts detected after in situ hybridization with a DIG-labeled probes of a bcd cDNA (D,E) or of nos and bcd cDNAs (F) by alkaline phosphatase and exposure to its substrate for 30 minutes (D,F) or by horseradish peroxidase and exposure to its substrate for 2 minutes (E) in embryos at nuclear cycle 10 (D), nuclear cycle 14 (E), or during early cleavage stage (F). The inset in E is a magnified view of nuclei close to the anterior tip of the embryo, where localization of bcd mRNA in the apical periplasm is clearly visible. (G-I) ImageJ scans along the anteroposterior axis (between the dashed red lines) of embryos in D-F, demonstrating the bcd mRNA gradients. The bcd mRNA profiles shown in G-I are derived from the embryos in D-F, respectively. All embryos in this figure are oriented with their dorsal side up and anterior to the left.

View larger version (86K): [in this window] [in a new window]   Fig. 2. Formation of bcd mRNA gradient analyzed by confocal microscopy. Confocal images (taken with a Leica TCS SP microscope) at midsagittal planes of Drosophila embryos oriented with their dorsal side up and anterior to the left. (A-I) bcd transcripts are detected by FISH with a DIG-labeled bcd cDNA in an unfertilized (unf) egg (A), and in embryos during interphase of nuclear cycle 3 (B), 6 (C), 9 (D), 10 (E), mitosis of nuclear cycle 11 (F), interphase of nuclear cycle 13 (G), and 4 (H) and 10 (I) minutes after onset of nuclear cycle 14. During cleavage stage, nuclear cycles were determined by counting the nuclei stained with DAPI (B-D). Insets in G-I show magnifications of the anterior region of the embryos. (J) Bcd protein gradient in an embryo 10 minutes after onset of nuclear cycle 14, visualized by an anti-Bcd antiserum and fluorescent immunostaining. (K,L) Magnified views of the dorsal anterior region of the embryos shown in H and I, respectively, visualizing the posterior extent of the bcd mRNA gradients using a color scale from 10 to 120, as shown to the right. For color conversion and interpretation of signal intensities, the OsiriX DICOM program was used (Rosset et al., 2004).

View larger version (27K): [in this window] [in a new window]   Fig. 3. Quantitative analysis of bcd mRNA gradient formation. (A-I) Distribution of basal and apical bcd transcripts along the dorsal midline of the anteroposterior axis in unfertilized eggs (A) and during early embryonic development (B-I) of Drosophila. The graphs, which were derived from raw data obtained by confocal microscopy of embryos shown in Fig. 2A-I (in the manner illustrated in L), show the intensities (arbitrary units) of basal (blue) and apical (pink) bcd transcripts. (J,K) Nuclear Bcd protein intensities, similarly measured along the anteroposterior axis of the embryo at nuclear cycle 14 shown in Fig. 2J, plotted against a linear (J) or base-10 logarithmic (K) scale to illustrate the exponential decay of the gradient between 10% and 65% EL. In both graphs, a background level of 58.9, reached at 65% EL and producing the widest range of exponentiality, was subtracted. (L) Overview of an embryo (above) with enlarged view of its surface (beneath), illustrating the positions at which basal (blue lines) and apical (pink lines) bcd transcript levels were measured in overlapping circular discs along the dorsal cortex of the embryos shown in Fig. 2A-I.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Batch analysis of the bcd mRNA gradient of embryos at nuclear cycles 13 and 14 and the exponentiality of the gradient. (A-F) Basal (A,C,E) and apical (B,D,F) bcd transcript profiles of 15 Drosophila embryos at nuclear cycle 13 (A,B), 46 embryos at 0-4 minutes of nuclear cycle 14 (C,D), and of nine embryos at 8-12 minutes of nuclear cycle 14 (E,F), were measured as illustrated in Fig. 3L. Insets show the same profiles with intensities of transcripts plotted on a base-10 logarithmic scale.

View larger version (42K): [in this window] [in a new window]   Fig. 5. Transport of bcd mRNA from basal to apical nuclear periplasm, and its degradation during early nuclear cycle 14. Anterior regions of Drosophila embryos during interphase (A) and mitosis (B) of nuclear cycle 13, at the onset of nuclear cycle 14 (C), and 4 (D), 10 (E) or 16 (F) minutes after onset of nuclear cycle 14, are shown in midsagittal planes with dorsal to the left. Embryos were collected from the same FISH experiment and analyzed in the same confocal session. Fluorescence intensities, reflecting bcd mRNA concentrations, were converted to a color scale, as shown to the right. For color conversion and interpretation of signal intensities, the OsiriX DICOM program was used. Insets are differential interference contrast (DIC) images of enlarged views of the cortical region with nuclei that reveal the stage (A,B) and the position of the leading edge (arrow) of the progressing cellular membranes (C-F), thereby permitting precise determination of the time (in minutes) after onset of nuclear cycle 14 (Foe and Alberts, 1983; Gutjahr et al., 1993). Confocal images were taken with a Leica TCS SP microscope.

View larger version (51K): [in this window] [in a new window]   Fig. 6. Similarity of Bcd protein, bcd mRNA and Stau gradients. (A-I) Similarity of bcd mRNA and protein gradients. Patterns of bcd mRNA and Bcd protein are visualized by double-staining of bcd mRNA with Alexa 647 (A,D,G, green) and Bcd protein with Alexa 555 (B,E,H, red) in Drosophila embryos during interphase of nuclear cycle 5 (A-C) and 13 (D-F), and 10 minutes after onset of nuclear cycle 14 (G-I; enlarged views of the anterior of the embryo), with the merge shown to the right (C,F,I). (J-L) Colocalization of bcd mRNA and Stau protein. Drosophila embryos were stained with an anti-Stau antiserum during interphase of nuclear cycle 5 (J) and 13 (K), and 10 minutes after onset of nuclear cycle 14 (L). Inset in L is a DIC image (see Fig. 5). All embryos are oriented with anterior to the left and dorsal side up. Confocal images were taken with a Zeiss LSM Pascal (A-I) or a Leica TCS SP (J-L) microscope.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter