We have developed a non-radioactive in situ hybridization technique for the localization of RNA in whole mount Drosophila embryos. After fixation, whole embryos are hybridized in situ with a DNA probe which has been labeled with digoxygenin. The hybridization products are detected by using a phosphatase-coupled antibody against digoxygenin. In parallel experiments, embryos can be treated with an antibody directed against the corresponding protein product to allow the detection of its distribution using standard immunochemical techniques. We have used this approach to compare the spatial and temporal distribution patterns of the RNA and protein products of the segmentation gene hunchback (hb) during the early stages of embryogenesis. This comparison revealed translational control of the maternally derived hb mRNA, which was difficult to detect by conventional techniques. The non-radioactive in situ hybridization method is as sensitive as conventional methods, but is faster and easier to perform. This may make it a useful tool for a variety of other systems.

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A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback.

The metameric organization of the Drosophila embryo is generated in the first 5 h after fertilization. An initially rather simple pattern provides the foundation for subsequent development and diversification of the segmented part of the body. Many of the genes that control the formation of this pattern have been identified and at least twenty have been cloned. By combining the techniques of genetics, molecular biology and experimental embryology, it is becoming possible to unravel the role played by each of these genes. The repeating segment pattern is defined by the persistent expression of engrailed and of other genes of the 'segment polarity' class. The establishment of this pattern is directed by a transient molecular prepattern that is generated in the blastoderm by the activity of the 'pair-rule' genes. Maternal determinants at the poles of the egg coordinate this prepattern and define the anteroposterior sequence of pattern elements. The primary effect of these determinants is not known, but genes required for their production have been identified and the product of one of these, bicoid is known to be localized at the anterior of the egg. One early consequence of their activity is to define domains along the A-P axis within which a series of 'cardinal' genes are transcribed. The activity of the cardinal genes is required both to coordinate the process of segmentation and to define the early domains of homeotic gene expression. Further interactions between the homeotic genes and other classes of segmentation genes refine the initial establishment of segment identities.

The molecular basis for metameric pattern in the Drosophila embryo.

https://www.cell.com/cell/pdf/0092-8674(88)90182-1.pdf

A gradient of bicoid protein in Drosophila embryos

Analysis of the genes that control the early events of Drosophila embryogenesis is providing details of the molecular processes underlying the positional specification of cells. There are two distinct phases: the first precedes the cellularization of the blastoderm embryo and is associated with a cascade of interactions between transcriptional regulators; the second occurs after cellularization and depends on communication between cells. These processes may be conserved in a wide range of invertebrates and vertebrates.

The molecular genetics of embryonic pattern formation in Drosophila

The A, B and C lamins are the major proteins of the nuclear envelope. The complete nucleotide sequence of the coding region of the A and C lamins shows that these proteins are identical except for their carboxy termini. The most prominent structural feature of both lamins is an alpha-helical region of repeating heptads of amino acids that shows striking homology with the entire family of cytoplasmic intermediate filament proteins. These features suggest that the nuclear envelope is made up of a network of coiled-coil polymers.

Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins.

Using differential interference contrast optics, combined with cinematography, we have studied the morphological changes that the living, syncytial embryo undergoes from stage 10 through 14 of Drosophila embryogenesis, that is just prior to and during formation of the cellular blastoderm. We have supplemented these studies with data collected from fixed, stained, whole embryos. The following information has been obtained. The average duration of nuclear cycles 10, 11, 12 and 13 is about 9, 10, 12 and 21 min, respectively (25 degrees C). In these four cycles, the duration of that portion of the mitotic period that lacks a discrete nuclear envelope is 3, 3, 3 and 5 min, respectively. The length of nuclear cycle 14 varies in a position-specific manner throughout the embryo, the shortest cycles being of 65 min duration. During nuclear cycles 10 through 13, it is commonly observed in living embryos that the syncytial blastoderm nuclei enter (and leave) mitosis in one of two waves that originate nearly simultaneously from the opposite anterior and posterior poles of the embryo, and terminate in its midregion. From our preparations of quick-frozen embryos, we estimate that these mitotic waves take on average about half a minute to travel over the embryonic surface from pole to equator. The yolk nuclei, which remain in the core of the embryo when the rest of the nuclei migrate to the periphery, divide in synchrony with the migrating nuclei at nuclear cycles 8 and 9, and just after the now peripherally located nuclei at nuclear cycle 10. After cycle 10, these yolk nuclei cease dividing and become polyploid. The syncytial embryo has at least three distinct levels of cytoskeletal organization: structured domains of cytoplasm are organized around each blastoderm nucleus; radially directed tracks orient colchicine-sensitive saltatory transport throughout the peripheral cytoplasm; and a long-range organization of the core of the embryo makes possible coherent movements of the large inner yolk mass in concert with each nuclear cycle. This highly organized cytoplasm may be involved in providing positional information for the important process of nuclear determination that is known to occur during these stages.

V.E. Foe

Papers

Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis