Anterior determinants in embryos of Chironomus samoensis: characterization by rescue bioassay.

Embryos of Chironomus samoensis are programmed, by anterior u.v. irradiation, to form the abnormal body pattern 'double abdomen'. Most double abdomen embryos show a mirror-image duplication of abdominal segments in the absence of cephalic or thoracic segments. Such embryos can be 'rescued', i.e. restored to normal development, by microinjection of cytoplasm or RNA from unirradiated donor embryos. Most of the rescued embryos look completely normal and many of them hatch spontaneously. The rescuing activity decreases from the anterior to the posterior pole in the donor cytoplasm and must be delivered near the anterior pole of the recipient for maximum efficiency. Rescuing activity is present in total RNA extracted from whole, unirradiated embryos. Upon fractionation, the activity is associated with poly(A)+ RNA, with LiCl precipitate depleted of RNA smaller than 250 nucleotides (nt) and with a sucrose gradient fraction depleted of RNA larger than 500 nt. Corresponding fractions of RNA from Xenopus oocytes have no rescuing activity. The activity of Chironomus RNA is sensitive to u.v. irradiation with low fluence affecting less than 2% of the pyrimidine bases. Rescuing activity is present in cytoplasm until the blastoderm stage but disappears earlier from poly(A)+ RNA. Rescuing activity is also present, and localized, in cytoplasm of embryos from two related dipterans, Smittia sp. and Drosophila melanogaster, although the extent of rescue observed in Chironomus decreases with the phylogenetic distance between donor and recipient. The results of these and previous experiments indicate that dipteran embryos contain localized RNP particles acting as anterior determinants. In Chironomus, the activity of these particles seems to depend on the integrity of polyadenylated RNA of about 250 to 500 nt length.

Photoreversible inhibition by ultraviolet light of germ line development in Smittia sp. (Chironomidae, Diptera).

Pole cell formation in embryos of the parthenogenetic midge, Smittia sp., can be delayed or inhibited by irradiation of the posterior egg pole with ultraviolet light (uv). This leaves the schedule of nuclear divisions and chromosome eliminations virtually unaffected. However, uv irradiation delays the precocious migration to the posterior pole of one nucleus, which normally becomes included in the first pole cell. This effect is photoreversible, i.e., mitigated by application of blue light after uv. Photoreversibility indicates that a nucleic acid component is involved as an effective target. During normal development of Smittia a number of chromosomes are eliminated during mitosis V, not only from somatic nuclei but also in the germ line. In the latter, this mitosis takes place during the first gonial division in the larva. After uv irradiation, the first pole cell nucleus has undergone supernumerary mitoses before pole cell formation and, as a result, is driven into mitosis V precociously as the pole cell divides. This is frequently associated with chromosome elimination from pole cells, which in turn is correlated with subsequent disappearance of already formed pole cells. Adults derived from embryos without pole cells do not form ovaries. Pole cell formation, pole cell preservation, and ovary development are separately inhibited by uv, and inhibition of each step is photoreversible. The results are discussed in the context of germ cell determination, protection against chromosome elimination, and the role of chromosomes limited to the germ line.

Inhibition by ultraviolet light of pole cell formation in Smittia sp (Chironomidae, Diptera): action spectrum and photoreversibility.

The formation of pole cells (primordial germ cells) in Smittia sp can be inhibited by ultraviolet (uv) irradiation without causing significant mortality. Until 70 min after egg deposition, pole cells are suppressed by low uv doses applied to the posterior pole region. Microbeam irradiation of a target area including the oosome inhibits pole cell formation; this is not observed after irradiation of other target areas. The action spectrum for uv inhibition of pole cells shows a distinct peak at 260 nm; its shape suggests that a nucleic acid-protein complex acts as an effective target. Independent evidence for the involvement of a nucleic acid moiety is derived from the fact that uv inhibition of pole cell formation is photoreversible. The results are discussed in the context of pole cell determination by localized cytoplasmic components.

Synthesis of a posterior indicator protein in normal embryos and double abdomens of Smittia sp. (Chironomidae, Diptera).

In embryos of the chironomid midge Smittia, synthesis of a posterior indicator protein designated PI1 (Mr approximately 50,000; pI approximately 5.5) forecasts development of an abdomen as opposed to head and thorax. The protein is synthesized several hours before germ anlage formation. In normal embryos at early blastoderm stages, synthesis of PI1 is restricted to posterior embryonic fragments but not to pole cells. In "double-abdomen" embryos, a mirror-image duplication of the abdomen is formed by cells that would otherwise develop into head and thorax. Embryos were programmed for double-abdomen development by UV irradiation of the anterior pole, and half of them were reprogrammed for normal development by subsequent exposure to visible light (photoreversal). Correspondingly, PI1 was synthesized in anterior fragments of UV-irradiated embryos but not after photoreversal. In a control experiment, UV irradiation of the posterior pole caused neither double-abdomen formation nor PI1 synthesis in anterior fragments. The identity of PI1 formed in anterior fragments of prospective double abdomens with the protein found in posterior fragments was revealed by two-dimensional gel electrophoresis and limited proteolysis. Suppression of PI1 synthesis in anterior fragments of normal embryos is ascribed to the activity of cytoplasmic ribonucleoprotein particles thought to act as anterior determinants.

Complete reversal of antero-posterior polarity in a centrifuged insect embryo.

Spatial pattern formation during embryogenesis is ascribed to differential gene expression, which in turn is thought to result in part from interactions of nuclei with cytoplasmic determinants. In the chironomid midge Smittia, and probably in other dipterans as well, blastoderm cells seem to make an early decision as to whether they contribute to cephalic and thoracic or to abdominal (and possibly thoracic) structures. Inactivation or translocation of cytoplasmic components involved in this antero-posterior decision could conceivably lead to duplications of head and thorax, or abdomen, or to complete but inverted embryos forming the head posteriorly and the abdomen anteriorly in the egg. Whereas the former two malformations have been described, completely inverted embryos are reported for the first time, to our knowledge, in this letter. Reversal of partial germ bands has previously been observed following combined ligation and cytoplasmic translocation in eggs of the leaf hopper, Euscelis plebejus.

Pattern formation in early insect embryogenesis - data calling for modification of a recent model.

A mathematical model of biological pattern formation based upon lateral inhibition has recently been applied by Meinhardt to insect embryogenesis. This model has stimulated a re-evaluation of previous results, and new experiments designed to test the validity of the model. Split u.v. dose experiments with eggs of the chironomid midge Smittia show that the effective targets for the production of the aberrant 'double abdomen' are not subject to the rapid turnover which is required by the model in its currently published version. Certain types of segment pattern, and differences in the length of segments as predicted by the model could not be observed. Other data conflict with the rather unusual type of photoreversal and the particular view of determination associated with the model. The model can be reconciled with part of the conflicting data if the effective targets for double abdomen induction are regarded as morphogen-producing structures, rather than the morphogen itself which specifies the segment pattern (Meinhardt, personal communication). This version of the model, however, is still at variance with some of the data discussed here. A complementary explanation is proposed taking into account relevant aspects of homoeotic transformations.

RNase sensitivity of an anterior morphogenetic determinant in an insect egg (Smittia sp., Chironomidae, Diptera).

In chironomid midges, the development of head and thorax in the embryo requires the function of cytoplasmic determinants localized near the anterior pole of the egg. Experimental inactivation of these determinants causes a dramatic switch in the developmental program of the embryo. Instead of the normal segment pattern, the aberrant pattern "double abdomen" is formed. Head, thorax, and anterior abdominal segments are then replaced by an additional set of posterior abdominal segments joined in mirror image symmetry to the original abdomen. Such double abdomens have been produced, with a maximum yield of 29%, by application of RNase (ribonuclease I, ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) to the anterior pole region of the egg. This was achieved by microinjection or puncturing the eggs during submersion in RNase. Control experiments with inactive RNase S fragments reliably proved that double abdomen formation resulted from RNase activity. Neither application of other enzymes to the anterior pole region nor application of RNase to other egg regions produced double abdomens in significant yields. The effects of RNase concentration and stage of development were determined. The data from these and earlier experiments are compatible with the idea that stored cytoplasmic RNP particles act as anterior determinants. Similarities to genetically caused switches in developmental pathways (homeotic mutations) are discussed.