Permeabilization, staining and culture of living Drosophila embryos.

The organic solvent octane has been used routinely to permeabilize the hydrophobic vitelline membrane surrounding the Drosophila embryo, thereby allowing the movement of small molecules into the egg. We present evidence that hexane is a more effective permeabilizing agent than octane and compare the effects of these solvents on uniformity of permeabilization and embryonic viability. The ability of each solvent to make the embryo accessible to a range of biological stains was compared. The effect of octane versus hexane permeabilization on subsequent embryonic viability was measured at seven different stages during early embryogenesis. We found that although hexane is a superior solvent for permeabilizing the vitelline membrane, it decreases the viability of embryos exposed between 0 and 3 hr of age. Older embryos treated with either hexane or octane are usually viable. We also showed that molecules with a molecular mass of 984 Daltons or more did not diffuse into the embryo following treatment with either hexane or octane. Results presented here challenge a phase-partition model that has been proposed previously to explain the molecular basis of permeabilization of the Drosophila egg. An alternative model is described as well as an optimized protocol for permeabilizing and staining Drosophila embryos at any stage during early embryogenesis while maintaining viability for subsequent culture.

Genetic control of cell fate in the termini of the Drosophila embryo.

Cell fates in the anterior and posterior termini of the Drosophila embryo are programmed by multiple zygotic genes that are regulated in response to a maternally encoded signal transduction pathway. These genes specify terminal as distinct from central cell fates, program pattern along the anteroposterior and dorsoventral axes of the termini, and also control endoderm specification and terminal morphogenetic movements. Here, we use a genetic interaction test to dissect the zygotic components of the terminal genetic hierarchy. We show that two genes, lines and empty spiracles, act downstream of tailless to repress central and promote terminal cell fates along the anteroposterior axis of the termini. Genes that control dorsoventral pattern in the termini and genes that program terminal morphogenesis act in distinct branches of the genetic hierarchy that are independent of tailless.

Zygotic genes that mediate torso receptor tyrosine kinase functions in the Drosophila melanogaster embryo.

The developmental signal that specifies the fates of cells at the anterior and posterior termini of the Drosophila embryo is transmitted by the torso receptor tyrosine kinase. This paper presents the results of a genetic interaction test for zygotic loci that act downstream of torso in the terminal genetic hierarchy. Tests of 26 zygotic mutants with defects in terminal development indicate that at least 14 reside in this hierarchy. The phenotypes associated with these genes fall into three classes, each of which represents a distinct aspect of terminal development and evolution. Four of the genes have been molecularly cloned and their products include an intercellular communication factor and three kinds of transcription factors.

Reciprocal effects of hyper- and hypoactivity mutations in the Drosophila pattern gene torso.

In Drosophila, five "terminal" polarity genes must be active in females in order for them to produce embryos with normal anterior and posterior ends. Hypoactivity mutations in one such gene, torso, result in the loss of the most posterior domain of fushi tarazu expression and the terminal cuticular structures. In contrast, a torso hyperactivity mutation causes the loss of central fushi tarazu expression and central cuticular structures. Cytoplasmic leakage, transplantation, and temperature-shift experiments suggest that the latter effect is caused by abnormal persistence of the torso product in the central region of the embryo during early development. Thus, the amount and timing of torso activity is key to distinguishing the central and terminal regions of the embryo. Mutations in the tailless terminal gene act as dominant maternal suppressors of the hyperactive torso allele, indicating that the torso product acts through, or in concert with, the tailless product.

Graded requirement for the zygotic terminal gene, tailless, in the brain and tail region of the Drosophila embryo.

We have used hypomorphic and null tailless (tll) alleles to carry out a detailed analysis of the effects of the lack of tll gene activity on anterior and posterior regions of the embryo. The arrangement of tll alleles into a continuous series clarifies the relationship between the anterior and posterior functions of the tll gene and indicates that there is a graded sensitivity of anterior and posterior structures to a decrease in tll gene activity. With the deletion of both anterior and posterior pattern domains in tll null embryos, there is a poleward expansion of the remaining pattern. Using anti-horseradish peroxidase staining, we show that the formation of the embryonic brain requires tll. A phenotypic and genetic study of other pattern mutants places the tll gene within the hierarchy of maternal and zygotic genes required for the formation of the normal body pattern. Analysis of mutants doubly deficient in tll and maternal terminal genes is consistent with the idea that these genes act together in a common pathway to establish the domains at opposite ends of the embryo. We propose that tll establishes anterior and posterior subdomains (acron and tail regions, respectively) within the larger pattern regions affected by the maternal terminal genes.

The zygotic mutant tailless affects the anterior and posterior ectodermal regions of the Drosophila embryo.

The recessive zygotic lethal mutation tailless maps to region 100A5,6-B1,2 at the tip of the right arm of chromosome 3, and results in shortened pharyngeal ridges in the head skeleton of the mature embryo and the elimination of the eighth abdominal segment and telson. Although they have a normal body length, tailless embryos have a smaller number of abdominal segments, some of which are larger than normal. The mutant phenotype is seen as early as 8 hr postfertilization, when tailless embryos are observed to have fewer tracheal pits than wildtype. At 9 hr, tailless embryos appear to be missing segments A8, A9, and A10 and have an abnormal clypeolabrum, optic lobes, and procephalic lobe. Segments A4, A5, A6, and A7 appear larger in tailless embryos than wildtype at this stage. The tailless mutation, although affecting anterior and posterior ectodermal structures in the mature embryo, does not affect the formation of pole cells, the posterior midgut, or the proctodeum, which arise from the most posterior region of the embryo. The mutation does result, however, in the failure of Malpighian tubule formation. Consistent with its effect on ectodermal segments, tailless leads to a reduction in the number of segmented, paired ganglia in the ventral nerve cord as well as to an abrupt alteration in the posterior region of the tracheal system. The role the tailless gene may play in the formation of the most anterior and posterior regions of the embryo's ectodermal body plan is discussed.

A critical review of the McCredie-McBride hypothesis of neural crest influence on limb morphogenesis.

A number of hypotheses have been presented to explain the mechanism of action of thalidomide. The most widely published and apparently widely accepted hypothesis is that of embryonic neuropathy advanced by McCredie and McBride. This paper reviews the points of the hypothesis and analyzes it in light of known limb embryology supported with recent experimental evidence which directly tests the hypothesis. The hypothesis may be considered as being composed of two separate parts. The first part proposes that thalidomide-induced limb defects exhibit a segmental pattern. The second proposes that the segmental pattern of limb defects is a result of segmental peripheral neuropathy. We are in complete agreement with the first portion of the hypothesis, but find the second portion quite unlikely.

Peripheral nerves do not play a trophic role in limb skeletal morphogenesis.

Research was undertaken to test the hypothesis that thalidomide-induced limb defects resulted from damage to the neural crest or peripheral nerves and that normal limb development depends upon either the quality (level specific) or quantity of peripheral nerves. Barriers which were placed into early chick embryos to block brachial plexus-level neural crest cells from reaching the limb resulted in normal limb skeletons. These data agree with previous work in suggesting that skeletal morphology is independent of innervation.