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Two new roles for the Drosophila AP patterning system in early morphogenesis

Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA

View larger version (84K): [in a new window]   Fig. 1. Three domains of yolk-stalk diameters and depth of cellularization. Wild-type embryos stained for Myosin. (A) Cross-section of late-cellularization embryo showing depth of cellularization. (B) Same embryo as in A observed in grazing section at the depth of the furrow canals. (C) Higher magnification view of B. Arrows in B,C indicate anterior domain, arrowheads indicate pre-CF domain. (D-F) 1 µm optical sections of wild-type embryo proceeding from interior of embryo (D) towards surface-most section (F). Each section is separated from the next by 2 µm. Arrows indicate pre-CF domain. (G,H) +/CyO, hb-lacZ embryo stained for Myosin (basal localization, same as in A) and ß-gal (cytoplasmic). These embryos are normal for cellularization, but the cytoplasmic ß-gal allows for easier visualization of cell depth. The great difference in the depth of cellularization between the anterior pole (15-20 µm) and the rest of the embryo (35 µm) means that this difference can be seen in a cross-sectional view of the embryo. The small difference in depth between the pre-CF domain and the posterior domain (a difference of 2-3 µm) means that this difference can not be easily viewed in cross-section (G,H), but can be visualized with optical sectioning technique (D-F).

View larger version (76K): [in a new window]   Fig. 2. The AP patterning system specifies the spatial pattern of cellularization. (A) An embryo from a bcd nos tsl/+ female. (B,C) Embryos from bcd nos tsl homozygous females. (A-C) First column: cross section through embryo showing stage of cellularization. Second column: focal plane through nuclei showing slightly anterior shift of Eve stripes (A), ubiquitous Eve (B), and minus the green channel (C) for better resolution of nuclei. Third column: focal plane through furrow canals showing uniform stalk diameters in (B,C). Red, Myosin; Green, Eve; Blue, DNA (Hoechst). The arrow marks the position of the first stripe of Eve in embryos in which the green channel has been removed so that yolk stalks may be seen more easily. Embryos from D,E are derived from females carrying six copies of bicoid. (D-E) First column: cross section through embryo showing stage of cellularization. Second and third columns: two different focal planes of furrow canals. Red, Myosin; Green, DNA; Blue, Eve. The embryos in the third column of D,E have had the blue channel removed so the yolk stalks can be clearly seen. The thin strips in the third row are higher magnifications of yolk stalks from the anterior domain (ad), pre-CF domain (pcf) and posterior domain (pd). (F,G) Cross sections through embryos showing stage of cellularization and depth of cellularization front. (F) Embryo from bcd nos tsl/+ female; (G) a bcd nos tsl homozygous female. Note deeper extent of cellularization front in the anterior in G than in the anterior in F.

View larger version (90K): [in a new window]   Fig. 3. Prd expression overlaps with the pre-CF domain and paired is required for the pre-CF domain to form. Cross-section (A) and surface view (B) through a very early cycle 14 embryo showing Myosin (green) and the early single stripe expression of Prd (red). Optical sections from basal side of yolk stalks (C), through yolk stalks (D) and apical side of yolk stalks (E) showing overlap of first stripe of Prd expression (green) with pre-CF domain (deeper band of cellularization front as marked by Myosin in red). A lateral part of embryo (D) has had the green channel removed so the smaller yolk stalks of the pre-CF domain can be seen. (F,G) Representative example of prd/+ embryo (F, classified as 3.5 on 0-5 scale), and its homozygous prd sibling (G, classification of 1.0) stained with Myosin (green) and Neurotactin (a general cell surface marker in red). Embryos were also stained against Prd in far-red channel for genotyping purposes (not shown). (F,G) Embryos are a composite of a cross-section focal plane with the furrow canal focal plane super-imposed. The thin strips in the third row are higher magnifications of yolk stalks from the anterior domain (ad), pre-CF domain (pcf) and posterior domain (pd). The remaining inhomogeneity in the region of the pre-CF domain in G may reflect the role of additional AP patterning genes in the specification of this domain. The graph shows the results of a blind scoring of the pre-CF domain in prd/+ and prd– homozygous embryos. The data illustrates that although a minority of prd heterozygotes possess a poor pre-CF domain, no prd– homozygotes form a normal pre-CF domain (a rating of 3.5 or higher).

View larger version (99K): [in a new window]   Fig. 4. An anterior domain of lower nuclear densities is formed before the start of cellularization. Embryos are stained against Myosin (red) and DNA (green). First column: cross-section showing absence of cellularization front. Second column: focal plane though nuclei. Numbers across the top represent nuclei counts in a square of constant size (70x70 pixel box) positioned at corresponding points along the AP axis of the embryo. (A) A pre-cellularization, cycle 14 wild-type embryo shows a lower density of nuclei in the anterior. (B) A late-stage cellularizing embryo shows a similar anterior nuclear density as in A, but also has an additional slight clustering of nuclei in the region of the pre-CF domain (arrow).

View larger version (77K): [in a new window]   Fig. 5. Left-hand six pictures: the anterior domain of lower nuclear densities arises by cycle 11. To facilitate visualization of the anterior domain, and to reduce the impact of the complicating geometry of the curvature at the anterior pole, embryos with a larger anterior domain (from females carrying six copies of bicoid) were scored for the distribution of nuclei (marked by oli-green). Similar results were obtained from the nuclear counts of wild-type embryos (GFP-histone embryo, and data not shown). The numbers represent nuclei counts in a square of constant size (70x70 pixel box) positioned at corresponding points along the AP axis of the embryo. The final image in the fixed embryos series shows Eve stripes (blue). The arrowhead marks the position of the border of the anterior domain, several nuclei to the anterior of the first stripe of Eve. The right-hand six pictures are a time-lapse series of a single embryo carrying a GFP-histone transgene. Owing to differences in magnification from using two different microscopes to capture the images, the same size square yields different densities between the fixed embryo series and the live embryo series, as well as in Fig. 4. Note that the relative difference between the anterior domain and the rest of the embryo is similar. The position of the CF can be seen in the final image of the GFP-histone embryo (gastrulation), and this position can be used as reference point for the AP axis in the other embryos as the same focal plane was kept at all time points.

View larger version (36K): [in a new window]   Fig. 6. Actin caps in the anterior domain possess larger actin caps than in the rest of the embryo. Actin caps in cycle 10, cycle 11 and cycle 12 wild-type embryos were visualized by Alexa-568 phalloidin (red). Nuclei (DNA marked by oli-green) are in green.

View larger version (127K): [in a new window]   Fig. 7. The AP patterning system regulates the anterior domain of lower nuclear densities through transcription. (A) Wild-type embryo with anterior lower nuclear density. (B) Embryo from bcd nos tsl female with uniform nuclear density. (C) Embryo from female carrying six copies of bicoid showing expanded domain of lower nuclear densities. Arrowheads in A,C indicate the approximate border of the anterior domain. (D) Control wild-type embryo injected with water exhibiting an anterior domain. (E,F) Wild-type embryos injected with -amanitin demonstrating uniform nuclear densities. The images are a focal plane through nuclei (green, as marked by DNA stain), and stained against Eve (blue in A-C). Note the uniform Eve in embryo from bcd nos tsl female (B) and the posterior shift in the Eve stripes in embryo from female carrying six copies of bicoid (C).

View larger version (101K): [in a new window]   Fig. 8. prd embryos have a delay in initiator cell behavior and CF formation. (A-D) Scanning electron microscopy of wild-type (A,C), prd (B,D), eve (E) and btd (F) embryos. Embryos are viewed on their lateral sides and are at early gastrula (A,B) and at mid-germband extension (C-F). (G-L) Embryos are stained with neurotactin (green), Eve (red) and Prd (blue). prd homozygotes were identified based on the absence of Prd staining. prd (H,J,L) or non-prd sibling (G,I,K) embryos of similar stages of gastrulation are shown. (G,H) Embryos are at the very onset of gastrulation. (I,J) Approximately 15 minutes into gastrulation; (K,L) early germband extension. Prd is expressed in the cell immediately anterior to the initiator cell, which corresponds to the middle cell of the first stripe of Eve. Eve and Prd therefore overlap in a single cell. Arrows mark either the initiator cell or the cell that would be an initiator cell in a wild-type embryo, as marked by localization relative to Eve expression. Note that the embryo in L, although at early germband extension, has barely begun a basal bulge of the CF and only on one side.