2009 Group Project 2

Stages of Drosophila Development

The stages listed below are adapted from Bownes stages (1975) by Ortega and Hartenstein (1985), as there were some dissimilarities and inconsistencies observed in Bownes stages. The following times given for the stages are an average recorded room temperature (25°C).

Stage 1

0hr - 0:25hr

In the first stage after fertilisation, the first 2 nuclear divisions occur. The Embryo is dark in the centre and lighter at periphery. This is due to the centre containing yolk granules evenly distributed throughout. In the periphery, the periplasm and pronucleus, containing no yolk granules, are pushed to the side.

Stage 2

0:25hr - 1:5hr

In this stage, syncytial divisions 3-8 occur, along with a retraction of egg cytoplasm, becoming separated from the vitelline envelope. The retraction of the cytoplasm results in the appearance of two empty spaces at anterior and posterior poles. They are the anterior and posterior spaces. Retraction initiates at the presence of four zygotic nuclei. During the syncytial divisions, the zygotic nuclei begin to move posteriorly, after which they move to the periphery. At the completion of the 8th syncytial division there will be approximately 200 nuclei at the periphery, and 50 nuclei in the centre, which form vitellophages (yolk nuclei).

Stage 3

1:5hr - 1:20hr

Syncytial division 9 occurs in this stage. Polar buds are formed by the budding off of 2 protuberances, at end of 8th syncytial division, into the posterior space, where they then proceed to divide once. The appearance of cytoplasmic periphery indicates end of stage. The conclusion of the stage is signified by the appearance of a clear cytoplasmic ring at the periphery of the embryo.

Stage 4

1:20hr-2:10hr

Syncytial divisions 10-13 occur in stage four. The polar buds increase significantly in number and begin to form polar cells in the posterior space. Blastoderm nuclei are discernible in the clear rim periphery after performing final divisions. The peripheral location of blastoderm nuclei cause the surface to bulge during divisions, resulting in the appearance of ‘somatic buds.’

Stage 5

2:10hr-2:50hr

Cellularization begins in stage 5. Spherical blastoderm nuclei begin to elongate to triple their original size. Pole cells begin moving dorsally. The anterior space formed in stage 2 disappears.

Stage 6

2:50hr-3:00hr

Insert non-formatted text hereStage 6 signifies the onset of gastrulation, and the formation of the ventral and cephalic furrows. The pole cells shift dorsally. Mesodermal and endodermal cells originate at ventral furrow, and then start to invaginate. Blastoderm cells at posterior pole shift their position to form dorsal plate, where pole cells adhere. The cephalic furrow is visible at approximately 2/3rd of the embryo length. The ventral furrow continues extending until it covers the majority of the embryos length.

Stage 7

3:00hr-3:10hr

Stage 7 begins when cell plate at the posterior pole reaches a horizontal orientation. Completion of gastrulation occurs. The endoderm primordia of anterior and posterior midgut and the primordium of the hindgut begin to invaginate. As a result of this invagination three dorsal folds become clearly visible diverging in a dorsoventral direction. The cephalic fold is a deep invagination, persisting until stage 9, whereas the anterior transversal fold and posterior transversal fold are superficial and short lasting. Pole cells are no longer visible unless viewed by histological sectioning and examination. Cells caudal to invaginating midgut surround the superficial opening of the midgut, forming a neck of cells, which become the hindgut.

Stage 8

3:10hr-3:40hr

Beginning of stage signified by formation of amnioproctodeal invagination, and the rapid phase of germ band expansion to 60% egg length. At the conclusion of germ band expansion the proctodeal opening is located at about 60% embryo length. The germ bands consist of a mesodermal layer on the interior, and an ectodermal layer on the exterior, which are almost indistinguishable. At this point, the cells remaining on the outer surface of the embryo form the ectoderm and amnioserosa.

Stage 9

3:40hr-4:20hr

First neuroblasts delaminate from ectoderm giving a clearly demarcated layering to the germ band; mesoderm-neuroblasts-outer ectoderm. Anterior pole separates from vitelline envelope through ventral retraction. This gives rise to the stomodeal cell plate, which begins to invaginate at the end of stage 9. Germ band expansion continues.

Stage 10

4:20hr-5:20hr

Stomodeum invaginates, forming the foregut. Stomodeal plate cells divide mitotically, and form a regular single layered epithelium which tilts posteriorly, establishing contact with the cell mass of the anterior midgut primordium. Germ band expansion reaches maximum length at 75% of embryo length. Pole cells leave the posterior midgut, and come to rest on both sides of posterior midgut outside yolk sac. Parasegmental grooves appear in epidermis of the trunk.

Stage 11

5:20-7:20hr

Segmental furrows become apparent in this stage. Ten tracheal pits arise, with the anterior-most pits opening into the 1st and 2nd thoracic segment boundary, giving rise to anterior spiracles. Posterior most pits open into 8th abdominal segment, forming posterior spiracles. Remaining pits grow and fuse to form tracheal tree. Posterior midgut invagination bends ventrally to reach posterior pole. Malpighian tubules begin forming as two buds at the junction of the posterior midgut and the hindgut. Apoptosis begins in this stage. Stage ends at first signs of germ band retraction.

Stage 12

7:20hr-9:20hr

Germ bands begin retracting caudally, with an associated increase in the width of the ferm bands to one and a half times their original size. The anterior midgut and posterior midgut fuse together. The yolk sac is moved to dorsal embryo, and retracts from cephalic regions. Amnioserosa covers the yolk sac. Three invaginations form on the roof of the foregut, which precede the development of stomatogastric nervous system. The tracheal tubes from stage 11 begin to fuse together. The ventral cord separates from epidermis. Stage 12 is completed once the germ bands complete retraction.

Stage 13

9:20hr-10:20hr

The head of the embryo begins involution. The anal plate occupies the posterior pole. A triangular gap begins to form ventrally at the anterior pole as a result of the clypeolabrum becoming thinner and beginning to retract. The labium moves to the midline on the ventral side. The yolk sac protrudes dorsally, becoming convex in shape.

Stage 14

10:20hr-11:20hr

Head continues involution. The dorsum and midgut of the embryo begins closing, as does the midgut. The labium moves inwardly, followed by the opening of the salivary glands into the mouth. The dorsal spiracles become evident.

Stage 15

12:20hr-13:00hr

In this stage the epidermis and gut close completely, leaving the yolk sac completely enclosed within the gut. Epidermal segmentation is completed. Dorsal ridge reaches 85% of the embryo length. Involution of head continues

Stage 16

13:00hr-16:00hr

Stage 16 begins once intersegmental grooves are distinguishable at middorsal levels. Cuticle begins to be secreted in the epidermis, tracheal tree, foregut and hindgut. Somatic musculature and sensory organs become visible, and heart forms in a mid-dorsal position. Four gastric caecae evaginate from the midgut. The stage ends with the dorsal ridge overgrowing the top of the clypeolabrum, including it in the atrium.

Stage 17

Continues until hatching of embryo

Air starts to infiltrate the tracheal tree. The ventral cord continues to retract. Movement of the embryo is evident within the vitelline envelope. There is no clear difference between stages 16 and 17.

History of Drosophila Embryological Model Use

• Ectomologist Charles W. Woodworth was the first to breed the Drosophila at Harvard university and suggested to W.E. Castle they could be used in studies of genetics.

• In 1908 T.H Morgan, an American geneticist and embryologist, was looking for an inexpensive that could be breed quickly and in limited space and Castle suggested the drosophila. Through Morgan’s studies of heredity he discovered the white-eyed mutation in the drosophila.

• By 1910, at Columbia University T.H Morgan and his students work on the top floor of the Schermerhorn Hall and t became known as the fly room. Students of the Fly Room were A.H. Sturtevant, C.B Bridges and H.J. Muller.

• In 1913 Sturtevant published a paper with the first genetic map and clearly laid out the logic for genetic mapping.

• In 1927 Muller ionized radiation and found it caused genetic damage.

• 1930’s feasability of generating deficiences and duplications were exploited in 1970 to generate duplicates and initiating whole-genome scanning.

• In 1933 Morgan won the Nobel Prize for his discovery that genes are carried on chromosomes and are the mechanical basis of hereditary.

• Also in 1933 Heitz and Bauer fly Bibio hortulanus studies discovered salivary gland chromosomes.

• 1934 saw the 1st published drawings of Drosophila melanogaster polytene chromosomes by T.S painter.

• In 1935 and 1938 Bridges publish polytene maps that are still used today.

• In 1946 Muller received the Nobel prize for his work, showing mutation induced by x-rays.

• In 1972 D.S. Hogness of Stanford Universtiy put in a grant application for modern genome reseach and in 1974 he made the 1st random clone of any organism.

• In 1975, clone libraries representing the entire genome had been generated and screened for clones carrying specific sequences.

• In 1980 C. Nusseiln-Volhard and E. Wieschaus attempt to identify all genes involved fundamental processes leading to the discovery of major signalling pathways. In 1995 they shared a Nobel Prize for their work.

• In 1981 a breakthrough of first rescue of a mutant phenotype in an animal by gene transfer. This led to the use of enhancer traps to screen for genes based on pattern of expression.

References

1. Campos-Ortega, Hartenstein (1985) The Embryonic Development of Drosophila melanogaster. Springer-Verlag berlin Heidelberg. pp9-84
2. Katrin Weigmann, Robert Klapper, Thomas Strasser, Christof Rickert, Gerd Technau, Herbert Jäckle, Wilfried Janning and Christian Klämbt: FlyMove – a new way to look at development of Drosophila.Trends Genet. In press. http://flymove.uni-muenster.de
3. Sturtevant, A. H. 1913. The linear arrangement of six sex-linked factors in Drosophila, as shown by their mode of association. Journal of Experimental Zoology, 14: 43-59.

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