3D Gene Expression

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Overview:
   Summary
   Project Goals

Methods:
   Staining & Mounting
   Probe Constructs
   Staging
   Imaging

Software:
   PointCloudXplore
   PointCloudToolBox
   PointCloudAlign
   NODE

File Formats:
   PointCloud (.pce)
   VirtualEmbryo (.vpc)
   Correspondence files

Staging

Embryos are staged by inspecting the embryos by phase contrast microscopy using a 20x objective lens on the confocal microscope. Our staging system is an extension of that of Campos-Ortega, J. A., and Hartenstein, V. (1997) "The Embryonic Development of Drosophila melanogaster", Second Edition, Berlin, Springer-Verlag.

The first number of the stage, stage 5, 6, 7 etc, denotes the same stage as Campos-Ortega and Hartenstein. After the stage number there may be a letter: a, b, c, etc, e.g. stage 4b. Letters from the beginning of the alphabet divide the stage into a temporal sequence of sub stages. The letter z is used for embryos that cannot be fit into the temporal series, either because of their orientation or because they fall between the clearly definable stages, e.g., 4z would be a stage 4 embryo undergoing a mitosis.

Descriptions of the features used to determine all sub stages are below. When the stage number and letter is followed by a value between 0-100% this indicates where within the sub stage the embryo lies. This value is optional. Examples of stages are thus: stage 5:50%; stage 7:30%; stage 6z; stage 12b. If a stage has designated sub stages (letters), then all embryos in that stage must be classed into a sub stage.

Specific stage descriptions

Stage 4a: Nuclear division 10: approximately 400 nuclei in the blastoderm.

Stage 4b: Nuclear division 11: approximately 800 nuclei in the blastoderm.

Stage 4c: Nuclear division 12: approximately 1500 nuclei in the blastoderm.

Stage 4d: Nuclear division 13: approximately 3000 nuclei in the blastoderm.

Stage 5: Nuclear division 14; approximately 6000 nuclei in the blastoderm.

This stage commences with the completion of the previous mitotic wave, comprises the initiation of cellularization, and is over once clear signs of cephalic furrow or ventral furrow formation are observed at the start of gastrulation, i.e., any embryo with a hint of cephalic furrow or ventral furrow formation is classed as stage 6.

During stage 5, the ~6000 nuclei elongate and the cell membranes begin to form, eventually surrounding the prospective cells. The cell membranes invaginate from the surface of the embryo, or the apical sides of the nuclei towards the yolk. The membranes reach the yolk and complete cellularization first ventrally towards the anterior of the embryo. The region where the cell membranes reach the yolk then grows along the ventral surface towards the posterior of the embryo. Cellularization has not yet completed in the dorsal region when the stage 6 begins, although sometime shortly after stage 5-50%, nuclear positions become increasingly irregular along the apical/basal direction.

The final % value is a measure of the extent of membrane invagination from the embryo surface to the yolk on the ventral side relative to the z-axis that is determined from analysis of the PointCloud. An empirical formula is used to calculate the actual extent of membrane invagination on the actual ventral side, as opposed to observed membrane invagination in phase contrast view.

    00%: no hint of the membranes is yet seen, the nuclei are often still rounded after the last nuclear division.

    01-07%: the membranes are seen as unclear "bubbles" at the apical ends of the increasingly columnar nuclei.

    10-40%: the clear line of the membrane edge has still not reached the basal end of the nucleus.

    45-99%: the nuclei elongate with the membrane edges basally towards the yolk.

    100%: Any stage 5 embryo where cellularization has reached the yolk at the anterior/ventral region.