Barry Fabian

The pluripotency of the extraembryonic mesodermal cells of the early chick blastoderm: Effects of the AP and AOV environments☆

This study investigates the developmental potential of the extraembryonic mesodermal cells of the early chick blastoderm. [3H]Thymidine-labeled mesodermal fragments from the extraembryonic area pellucida (AP) and area opaca vasculosa (AOV) were transplanted into the AP or AOV of nonlabeled host blastoderms in culture, and their fate followed autoradiographically. All the homotopically transplanted mesodermal cells differentiated in accordance with their normal fates. However, not all the heterotopically transplanted mesodermal cells did so, for some of the stage 8 AP extraembryonic mesodermal cells (normally nonerythropoietic) gave rise to blood cells when transplanted into the AOV. We also observed that the stage 4-5 AOV mesoderm continues to migrate peripherally when heterotopically transplanted into the AP, at a time when the AP mesodermal cells are nonmigratory. In support of our premise that the stage 8-9 AP extraembryonic mesoderm has the potential to form blood, we observed a clear-cut production of hemoglobin when the latter mesoderm was co-cultured on coverslips with stage 4 AOV endoderm.

The regulation of growth in the mesonephric kidney of adult Xenopus laevis by an endogenous inhibitor of proliferation.

Abstract The effect of dorsal lymph sac implanted tissue fragments, of a 100,000g kidney supernatant, and of various kidney-derived ultrafiltrate fractions on the percentage of DNA synthesizing cells in the mesonephric kidney of Xenopus laevis following partial unilateral nephrectomy was investigated autoradiographically. Using Amicon filters with cut-off values of MW 50,000 and 10,000, three ultrafiltrate fractions were obtained: a fraction containing molecules of MW 50,000 and less, a fraction containing molecules of MW 10,000 and less, and one containing molecules in the range of MW 10,000 to 50,000. The ultrafiltrates containing molecules of less than 10,000 MW were found to depress DNA synthetic activity on the sixth postoperative day by 30 to 40%, while the fraction containing molecules between MW 10,000 and 50,000 showed no significant effect. It has been concluded that an endogenous inhibitor of proliferation, with the attributes of a chalone, is present in the fraction of less than 10,000 MW. The loss of inhibitor action following Pronase treatment of the ultrafiltrate suggests that the inhibitor substance may be a protein or polypeptide, or that such constituent may be the carrier for the active agent. Since a depression in DNA synthetic activity of 60% was obtained in normal adult mesonephric kidneys following the injection of the ultrafiltrate, it is concluded that both compensatory growth and reparative growth in the kidney of Xenopus laevis are regulated by a G1 kidney chalone of less than 10,000 MW.

Metamorphosis in the frog Arthroleptella lightfooti (Anura, Ranidae) with emphasis on neuro-endocrine mechanisms

The Cape chirping frog, Arthroleptella lightfooti, has a type of direct development in which nonfeeding larval stages undergo an early metamorphosis resulting in the rapid attainment of the adult form. Precocious metamorphosis may be the result of the precocious activation of the same neuro-endocrine mechanisms known to regulate metamorphosis in species having free-living larvae. Alternatively there is evidence that during the course of evolution, metamorphosis in species with direct development has become largely independent of thyroid control. In this study we investigate this problem by comparing the timing of development of the median eminence in A. lightfooti relative to the timing of metamorphosis. The resulting data allow a comparison of the role of the median eminence during metamorphosis to be made between A. lightfooti and Xenopus laevis (a frog with free-living larvae). The results suggest that the brain — pituitary — thyroid axis is involved in the control of metamorphosis in A. lightfooti.

CELLULAR UBUNTU: ‘UMNTU NGUMNTU NGABANYE ABANTU’, AND OTHER PROBLEMS FOR CELL BIOLOGISTS IN THE NEW MILLENNIUM

Much has been written in South Africa in recent times about a way of communicating between people that recognizes a human quality known as ‘Ubuntu’. Ubuntu is an African concept that projects an image of mutual supportiveness under a communal umbrella. The core belief of ubuntu, as expressed in the Xhosa language, is that ‘a person only becomes a person through other persons’—‘umntu ngumntu ngabanye abantu’ (Khoza, 1993; Adonisi, 1993). In this sense, ubuntu may be seen as an orientation to life that is opposed to individualism. However, it is also not ‘comfortable with collectivism, where collectivism stresses the importance of the social unit to the point of depersonalizing the individual’ (Khoza, 1993; Adonisi, 1993). Both the individual and the social unit are thus mutually reflective and important. While ubuntu echoes that ‘no man is an island . . .’, it seems to go further in emphasizing a way of being that takes account of how the unique existence of an individual is shaped, supported and defined by the individual’s neighbours. However in the present context, what is the connection between ubuntu and cell biology? Does the ubuntu principle resonate at the cellular level of organization, especially as a way of thinking about the formative role that intercellular communications play in shaping and guiding the cells of the developing embryo? In this regard, it has become clear that a number of exquisite signalling strategies operate between

DNA synthesis during larval development and compensatory growth in the mesonephros of Xenopus laevis.

Abstract Autoradiography following tritiated thymidine administration to Xenopus laevis tadpoles of stages 45–48 of larval development has revealed that, as the cells of the mesonephric kidney differentiate during organogenesis, there is a marked decrease in the percentage of cells synthesizing DNA (from 100% at stage 45 to less than 9% at stage 48). In the adult this figure is of the order of 0.1%. This reduced DNA synthetic activity was found to take place in the cells of both the proximal and distal tubules of the nephrons. Special mucous cells which serve as markers of distal tubules were not observed to synthesize DNA after the onset of their differentiation at stage 48 of larval development. Through the partial extirpation of tissue in one kidney of adult Xenopus laevis males, DNA synthesis was reactivated in differentiated cells. The increased DNA synthetic activity following partial unilateral nephrectomy was found to be maximal after 6 days of recovery when 19% of cells synthesize DNA. This increased DNA synthetic activity was found to occur only in the cells of the distal tubules, both mucous and nonmucous cells, while the cells of the proximal tubules did not respond to this reactivation. The apparent inability of proximal tubule cells to synthesize DNA following partial unilateral nephrectomy is discussed.

Spectrin labeling during oogenesis in zebrafish (Danio rerio)

Progression through mitosis and meiosis during early zebrafish ovarian development is accompanied by highly regulated series of transformations in the architecture of oocytes. These cytoskeletal-dependent membrane events may be assumed to be brought about by deployment of proteins. While the cytoskeleton and its associated proteins play a pivotal role in each of these developmental transitions, it remains unclear how specific cytoskeletal proteins participate in regulating diverse processes of oocyte development in zebrafish. Results from this study show that a pool of spectrin accumulates during oogenesis and parallels an increase in volume of oocytes at pre-vitellogenic stages of development. Spectrin labeling is restricted to the surface of oogonia, the cortex of post-pachytene oocytes and later accumulates on the cytoplasm of pre-vitellogenic and vitellogenic oocytes. Results here suggest a correlation between spectrin labeling, increased cytoplasm volume of oocytes, an increase in the number of nucleoli and accumulation of cytoplasmic organelles. Overall, these results suggest that synthesis and storage of spectrin during pre-vitellogenic stages of oogenesis primes the egg with a pre-established pool of membrane-cytoskeletal precursors for use during embryogenesis, and that the presence of spectrin at the oocyte sub-cortex is essential for maintaining oocyte structure.

09-P060 Evaluation of acridine orange as a marker of germ cell developmental stages in the developing zebrafish ovary

molecular mechanism(s) of the regulation of Otx2 expression remains to be elucidated. We previously identified the cis-element that drives Otx2 expression in the forebrain and midbrain from E8.5, termed FM enhancer. In this study, we showed that the 157-bp core sequence in the FM enhancer was sufficient for the activation of reporter gene expression in the forebrain and midbrain. The core sequence contains the Tcf/Lef and Otx binding sites, which are deeply conserved among vertebrates. Physical interactions of Lef1 and Otx2 to these sites were observed by EMSA. Furthermore, mutations introduced in these sites caused reduction of the FM enhancer activity in the transient reporter analysis. We also demonstrated that another conserved 29-bp region in the 157-bp FM enhancer is necessary for the enhancer activity. By DNA affinity purification followed by mass spectrometry analyses, it was shown that classIII POU factors Brn1/2/4 interact with the 29-bp region. The homeodomain recognition site (TAATTA) in this region was shown to be necessary for the binding of the Brn1/2/4 invitro. Supershift analysis using antiBrn2 antibody suggested the binding of endogenous Brn2 to this region. From these results, it is inferred that brain regionalization is controlled by the orchestration of these transcription factors on the FM enhancer through regulating Otx2 expression.

Developmental biology and alternative life styles

Sexual differentiation is a paradigm case illustrating the potential of an animal to express alternative life styles. In this paper the morphogenesis of the male and female patterns are reviewed, especially in relation to the morphogenesis of the genital ducts. The stepwise events of morphogenesis are followed in terms of the cells as building blocks and attention is given to the plasticity of morphogenesis in this system. The suggestion is made that if the morphogenesis of this system is so plastic, then one could expect the inevitability of particular morphological and physiological arrangements becoming fixed following selection, as possibly seen, for example, in the diversity of species-specific variations found amongst the genitalia of insects. Following the discussion on the cellular strategies for the construction of the male and female ducts, the next part of this paper examines the underlying genetic structure upon which sexual dimorphism is based. The third theme poses the question as to whether there is an ontogenetic memory of past adaptations within the embryo which could be used to accelerate the rate of adaptation of a species to a new environmental challenge. In addressing this problem the example of the globin genes and globins, as well as of melanization, is considered, especially in relation to the fact that genes come in pieces.