Anton W. Neff

Pattern formation in amphibian embryos prevented from undergoing the classical “rotation response” to egg activation☆

Fertile Xenopus laevis eggs were immobilized so that they were prevented from undergoing the rotation response to activation. Many of those unrotated eggs developed through organogenesis, indicating that egg rotation is not a prerequisite for normal early embryogenesis. Various aspects of the regulation of pattern formation were analyzed in unrotated eggs: It was discovered that a substantial rearrangement of yolk platelets occurred without affecting subsequent pattern formation. The germ plasm, however, remained localized in the vegetal hemisphere in inverted eggs. Cleavage furrows and the site of involution were both often observed in novel locations in inverted eggs which were prevented from rotating during activation.

Early development of Xenopus embryos is affected by simulated gravity.

Early amphibian (Xenopus laevis) development under clinostat-simulated weightlessness and centrifuge-simulated hypergravity was studied. The results revealed significant effects on (i) morphological patterning such as the cleavage furrow pattern in the vegetal hemisphere at the eight-cell stage and the shape of the dorsal lip in early gastrulae and (ii) the timing of embryonic events such as the third cleavage furrow completion and the dorsal lip appearance. Substantial variations in sensitivity to simulated force fields were observed, which should be considered in interpreting spaceflight data.

The influence of gravity on the process of development of animal systems

The development of animal systems is described in terms of a series of overlapping phases: pattern specification; differentiation; growth; and aging. The extent to which altered (micro) gravity (g) affects those phases is briefly reviewed for several animal systems. As a model, amphibian egg/early embryo is described. Recent data derived from clinostat protocols indicates that microgravity simulation alters early pattern specification (dorsal/ventral polarity) but does not adversely influence subsequent morphogenesis. Possible explanations for the absence of catastrophic microgravity effects on amphibian embryogenesis are discussed.

Amphibian (urodele) myotomes display transitory anterior/posterior and medial/lateral differentiation patterns

Myotome differentiation during Mexican axolotl (Ambystoma mexicanum) somitogenesis was analyzed by employing anti-actin and anti-myosin monoclonal antibodies as molecular probes. Myotome differentiation occurs after segmentation and proceeds in the cranial-to-caudal direction along the somite file. Within individual somites myotome differentiation displays distinct polarities. Examination of the somite file at the tailbud stage revealed that soon after segmentation, actin/myosin accumulate predominantly in the anterior and medial region of the myotome initially. Subsequently, cells within the myotome differentiate in an anterior-to-posterior and medial-to-lateral direction. Experimental analysis of presomitic paraxial mesoderm grafts before segmentation revealed that this transient myotome polarity is autonomous. Comparative analyses indicate that this myotome differentiation pattern is urodele specific. Cynops pyrrhogaster undergoes myotome differentiation like the axolotl, while two anurans, Xenopus laevis and Bombina orientalis, do not.

Expression of the axolotl homologue of mouse chaperonin t-complex protein-1 during early development

Molecular chaperones assist in the folding of proteins, but their role during development is not well understood. Here we report the temporal and spatial expression pattern of the axolotl homologue of mouse chaperonin TCP-1 during normal amphibian embryogenesis and in several models of abnormal embryogenesis. A partial axolotl TCP-1 cDNA (646 bp; 519 coding bp) isolated by 3 RACE PCR shows considerable homology to mouse TCP-1. Developmental Northerns and RT-PCR analyses of whole axolot1 embryos revealed a low level of maternal TCP-1 transcripts in fertilized eggs. The maternal transcripts were down-regulated to a non-detectable level in early gastrulae. Zygotic TCP-1 transcripts first appeared during gastrulation. They were mainly expressed in mid-neurula and later stage embryos. Whole-mount in situ hybridization studies showed abundant TCP-1 transcripts in the blastopore at the mid-gastrula stage and in the brain and spinal cord beginning at the neurula stage, and in the somites (myotomes) at the tailbud stage. RT-PCR analysis of TCP-1 expression in axolotl embryos treated with either high salt (causing exogastrulation) or ultraviolet (UV) irradiation (causing ventralization) substantiated the correlation between TCP-1 expression and neural and somitic development. In high salt-induced exogastrulated embryos TCP-1 mRNA was detectable in the ectoderm part (with neural tissues) but not in its exogastrulated endoderm part. Lower levels of TCP-1 expression were detected in UV-irradiated, ventralized embryos with smaller head and reduced neural and somitic tissues. Normal levels of TCP-1 expression were detected in embryos with double axes/heads. These studies provide strong evidence that at the transcript level axolotl chaperonin TCP-1 is regulated both temporally and spatially during embryogenesis, especially in neural and somitic development.

The amphibian egg as a model system for analyzing gravity effects

Amphibian eggs provide several advantageous features as a model system for analyzing the effects of gravity on single cells. Those features include large size, readily tracked intracellular inclusions, and ease of experimental manipulation. Employing novel gravity orientation as a tool, a substantial data base is being developed. That information is being used to construct a 3-D model of the frog (Xenopus laevis) egg. Internal cytoplasmic organization (rather than surface features) are being emphasized. Several cytoplasmic compartments (domains) have been elucidated, and their behavior in inverted eggs monitored. They have been incorporated into the model, and serve as a point of departure for further inquiry and speculation.

Amphibian egg cytoplasm response to altered g-forces and gravity orientation

Elucidation of dorsal/ventral polarity and primary embryonic axis development in amphibian embryos requires an understanding of cytoplasmic rearrangements in fertile eggs at the biophysical, physiological, and biochemical levels. Evidence is presented that amphibian egg cytoplasmic components are compartmentalized. The effects of altered orientation to the gravitational vector (i.e., egg inversion) and alterations in gravity force ranging from hypergravity (centrifugation) to simulated microgravity (i.e., horizontal clinostat rotation) on cytoplasmic compartment rearrangements are reviewed. The behavior of yolk compartments as well as a newly defined (with monoclonal antibody) non-yolk cytoplasmic compartment, in inverted eggs and in eggs rotated on horizontal clinostats at their buoyant density, is discussed.

Effects of gravity perturbation on developing animal systems.

Developing systems provide unique opportunities for analyzing the effects of microgravity on animals. Several unusual types of cells as well as various extraordinary cellular behavior patterns characterize the embryos of most animals. Those features have been exploited as test systems for space flight. The data from previous experiments are reviewed, and considerations for the design of future experiments are presented.