Progress toward the culture and transformation of chicken blastodermal cells.

Chicken blastodermal cells can be cultured for short periods of time and retain the ability to contribute to somatic and germline tissues when injected into gamma-irradiated stage X embryos. Such a method has yet to yield a germline transgenic bird, in part due to the low rate of transgene integration into the avian genome. In addition, the short culture period precludes the identification and expansion of those cells that carry an integrated transgene. In this study, two methods were developed that produced blastodermal cells isolated from stage X Barred Plymouth Rock embryos bearing an integrated transgene. Addition of chick embryo extract to the culture medium enabled expansion of single colonies for multiple passages. Southern blot analysis indicated that the transgenes had integrated as a single copy in most of the clones. Cells from passaged, transgenic embryo cells were injected into irradiated stage X White Leghorn embryos, producing hatched chicks that bore the donor cells in their somatic tissues. Transgene sequences were detected in sperm DNA; however, breeding of chimeras did not result in germline transmission of the transgene, indicating that the contribution of the transgenic cells to the germline was either nonexistent or very low.

Restriction of proliferation of primordial germ cells by the irradiation of Japanese quail embryos with soft X-rays.

Primordial germ cells (PGCs) are the progenitor cells for the gametes. Avian PGCs are located in the central region of the area pellucida at the blastoderm stage. Shortly after further incubation, they migrate to the extra-embryonic germinal crescent, and then as soon as the blood vessels form, they enter the circulation and finally settle in the gonadal primordium. We have developed a simple method using soft X-ray irradiation (18 kV power, 20 cm distance) to reduce the number of PGCs in Japanese quail embryos, which should be useful in preparing recipient embryos for PGC-transfer studies. When embryos were exposed to the soft X-rays for 40 s before incubation, the concentration of circulating PGCs was less than one-fifth that in controls after 2 days of incubation. Embryos at day 6 of incubation contained approximately half the number of PGCs compared to controls when they were exposed before or at day 2 of incubation. Irradiation for 40 s is recommended taking into consideration the restriction of proliferation of PGCs, and viability and hatchability.

Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos.

Stage-X blastoderms, within intact eggs from White Leghorn hens, were exposed to 500-700 rads of gamma radiation from a 60Co source prior to injection, into the subgerminal cavity, of approximately 100 or 200-400 dispersed cells from stage-X blastoderms isolated from eggs laid by Barred Plymouth Rock hens. Embryos developing past day 14 of incubation and hatched chicks were assessed for donor and recipient cell contribution to the melanocyte population through examination of black and yellow down pigmentation, respectively (Barred Plymouth Rocks have a recessive allele at the I locus while the White Leghorns have a dominant allele at the I locus). Of the 809 embryos injected with approximately 100 cells, 192 developed past day 14 and black pigmentation, indicating somatic chimerism, was observed on 118 of the 192 (58%) embryos and chicks. Of the 296 embryos injected with 200-400 donor cells, 86 developed past day 14 of incubation. Somatic chimerism was observed on 55 of the 86 (64%) embryos and chicks. To test for germline chimerism, birds surviving to maturity were mated to Barred Plymouth Rocks. Five somatically chimeric females were produced when approximately 100 cells were injected, and one was a germline chimera. Six somatic female chimeras were produced following the injection of 200-400 cells, three of which proved to be germline chimeras by the presence of Barred Rock chicks among their offspring. Two of the nine males produced by injecting approximately 100 cells were germline chimeras.(ABSTRACT TRUNCATED AT 250 WORDS)

Temporal regulation of gene expression in the blastoderm Drosophila embryo.

The Drosophila embryo undergoes a developmental transition during cycle 14 when it initiates asynchronous mitotic cycles and markedly increases its rate of zygotic transcription. The nucleo-cytoplasmic ratio has been proposed to be the single factor that temporally regulates this developmental transition. We altered the ratio in the embryo and analyzed the consequences on the cell cycle program and on the transcripts of specific genes. These genes were chosen because their transcripts normally undergo changes in pattern during cycle 14. We found evidence that the nucleo-cytoplasmic ratio is read and interpreted locally to regulate the cell cycle program. Based on the response of the transcripts to changes in the ratio, we found evidence that at least two classes of temporal regulatory mechanisms control these transcripts. We therefore propose two corresponding classes of transcripts: (1) nucleo-cytoplasmic ratio dependent; and (2) nucleo-cytoplasmic ratio independent or time correlated. The temporal regulation of the ratio-independent transcripts may be dependent on developmental time. We conclude that multiple modes of temporal regulation underlie the events of the developmental transition in Drosophila embryogenesis.

The cell lineage of the muscles of the Drosophila head.

Using a cell marker mutation the cell lineage of the muscles of the Drosophila head are traced out. Three sets of muscles separated by lineage restrictions are observed, even when cells are marked as early as the blastoderm stage. Each set underlies the derivatives of one of the three pairs of imaginal discs which differentiate to form the epidermis of the adult head. Clones of the homoeotic mutation engrailed (en10) were apparently normal in the muscles of the head. The muscle clone frequency, at the blastoderm stage, in each hemisegment of the fly is similar, indicating an equal partitioning of cells during segmentation.

Cell lineage of the thoracic muscles of Drosophila.

The thorax of the adult Drosophila contains about 80 muscles, which develop from the mesoderm. A new genetic marker was used to map the cell lineage of the myoblasts that form these muscles. Clones of marked cells were produced by irradiation of embryos and larvae, and these were detected in the adult by histochemical staining. The principal findings are that the muscles of each segment have separate origins, and that each becomes compartmented precisely into a dorsal-lineage and a ventral-lineage set of muscles, each set probably being formed by the adepithelial cells found in one imaginal disc. In contrast with the epidermis, the muscles of each thoracic segment are not subdivided into anterior and posterior compartments, and clones of muscle cells that are homozygous for recessive-lethal alleles of engrailed develop normally.

[Germ cells of the bird embryo: early localization, migration and colonization of gonadal anlagen]. / Les cellules germinales de l'embryon d'oiseau: localisation précoce, migration et colonisation des ébauches gonadiques.

Several experimental and morphological papers are analyzed concerning germ cell origin, migration and proliferation within sexually undifferentiated gonads. The germ material which is first localized in both anterior and posterior halves of the unincubated blastoderm assembles in the anterior area only at the beginning of incubation. In gonads the germinal population does not depend on the initial quantity of germ cells in the early blastoderm: its value seems to be determined by the somatic cells of the gonadal stroma. During the migratory phase, it is suggested that regulated interactions occur between the glycoconjugates of the moving germ cells and the physico-chemical characteristics of the extracellular matrices acting as substrates for the movement.

[Germ cell population of chick embryos incubated 5 1/2 days after ultraviolet irradiation of young blastoderms]. / Peuplement en cellules gerinales d'embryons de Poulet de 5 jours et demi d'incubation après irradiation aux rayons ultraviolet des jeunes blastodermes

Chick blastoderms, incubated for 5 hrs., were wholly irradiated with ultraviolet light (2 537 A). With ultraviolet doses of 14,400 and 16,200 erg/mm2, the embryos, ofter morphologically abnormal, which developed from these blastoderms, were shown to contain, at 5 1/2 days of incubation, very few or no germ cells in the region of their primordia.

The development of Drosophila embryos after partial u.v. irradiation.

U.v. irradiation of the anterior pole of nuclear multiplication stage Drosophila eggs produces embryos with defective anterior structures. At a low frequency embryos resembling some phenotypes of the bicaudal syndrome of Drosophila were observed. These embryos had no head or thorax and the eight abdominal segments were spread to the anterior of the embryo. Sometimes spiracles, characteristic of the most posterior embryonic segment were observed at the anterior of the embryo. The development of these embryos was followed, and abnormalities occurred as early as blastoderm formation. The extent of the blastoderm defects correlated well with the final abnormality in the embryo.

[The localization of primordial germ cells in Japanese quail embryos using a technic of ultraviolet irradiation]. / Etude de la localisation des cellules germinales primordiales chez l'embryon de caille japonaise au moyen d'une technique d'irradiation aux rayons ultraviolets

In the Japanese quail embryo at the stage of 6 pairs of somites, ultraviolet irradiation of the extraembryonic crescent-shaped area anterior to the level of the first somites does not lead to total sterilization of the developing gonads. There are always subsisting primordial germ cells which apparently can restore the population of gonocytes. On the contrary, complete sterilization can be obtained by irradiating the area extending posteriorly to the level of the Hensen node.

[Regulation of the number of gonocytes in the genital ridges of duck embryos after partial destruction of the initial stock of germ cells]. / Régulation du nombre des gonocytes dans les ébauches gonadiques de l'embryon de Canard après destruction partielle du stock initial des cellules germinales

X rays irradiation of anterior or posterior halves of Duck blastoderms at the non incubated stage is followed by a decrease of the number of germ cells in the germinal crescent at the stage of 10 somites. However, after 6 days of incubation, the genital ridges of such embryos contain the same number of gonocytes as those of normal embryos. A regulation of the number of gonocytes occures at the level of the genital ridges.