Rufus Richard Humphrey

Genetic and experimental studies on a mutant gene (c) determining absence of heart action in embryos of the Mexican axolotl (Ambystoma mexicanum).

Abstract A mutant gene, c (for “cardiac lethal”), carried by an adult Ambystoma mexicanum imported from Mexico, causes failure of onset of heart action at the usual time in hearts of apparently normal morphology. The trait appears in approximately 25% of the embryos of spawnings in which it occurs, and is an autosomal recessive. Heads and gills of affected embryos remain small and a marked ascites develops. The affected individuals hatch and show normal swimming and righting movements. They never eat, and die within a few days after hatching. Mutants united as embryos in parabiosis with normal may live indefinitely, their hearts disappearing in large part or remaining as a part of a vein. Transplants of various organ preprimordia of mutant into normal embryos develop normally. Transplants of the heart-forming mesoderm produce normally beating hearts, but in the reciprocal transplant (normal into mutant) the heart which develops never beats. Electron microscopy (Lemanski et al. , 1970) reveals that the hearts of mutants lack organized myofibrils. The failure of normal differentiation of these may result from lack of an inductive influence by the related entoderm or from an inhibitory effect of some type from that germ layer or other structures.

Nuclear transplantation in the axolotl

Abstract There are currently available in the axolotl several genes that affect development at times ranging from cleavage to larval stages. These genes would be extremely useful as markers in nuclear transplantation studies of nuclear differentiation. It is also possible that by the use of such genes the analysis of nuclear differentiation might eventually be refined to detect changes in the genes themselves, if they occur. In the past the exploitation of the axolotl material for work of this type has not been feasible because axolotl eggs, like those of other urodeles, have generally given very poor results in nuclear transplantation experiments. The present paper presents an analysis of the factors which determine whether or not nuclear transfers succeed in this species. This analysis shows that the conditions required for the transfers to be successful are: (1) activation of the egg by heat shock (35° C for 5 minutes)7; (2) enucleation by ultraviolet irradiation; and (3) nuclear transfer at 2 to 4 hours following the application of the heat shock. A method based on this analysis is described in detail on pages 151–154. Tests of the method were made by transplanting blastula nuclei into activated and enucleated eggs. Ninety-six per cent of the eggs cleaved and 77% developed into complete blastulae of normal appearance. The majority of these blastulae (66%) developed to postneurula stages. The validity of the method was tested by transplanting nuclei carrying the dominant gene D (for black pigmentation pattern) into enucleated eggs from a recessive white (dd) female. A total of 37 larvae were obtained. All showed the black phenotype, proving that the nuclear transfers were successful.

TRANSPLANTATION OF NUCLEI OF VARIOUS CELL TYPES FROM NEURULAE OF THE MEXICAN AXOLOTL (AMBYSTOMA MEXICANUM).

Abstract Nuclei from various regions of axolotl neurulae were transplanted into eggs which had previously been electrically activated and irradiated with UV to eliminate the egg chromosomes. The majority of the nuclei, regardless of their region of origin, elicited genuine cleavage but usually failed to promote development beyond blastula or early gastrula stages. A cytological study of the recipient embryos, fixed at various times following nuclear transfer, has revealed pronounced mitotic abnormalities in approximately 90% of the cases examined. Modifications of the transplantation medium, calculated to reduce the risk of technical damage to the nuclei, do not improve their capacity to promote the development of the recipient eggs. Triploid neurula nuclei do not differ from diploid nuclei in ability to promote the development of test eggs. Nuclei from the most recently differentiated (caudal) end of the notochord and endoderm, compared with nuclei from the more cephalic regions of the same structures, display a greater capacity to promote postgastrula development of the recipient eggs. The Discussion is focused mainly on the mitotic abnormalities exhibited by the majority of the transplanted somatic nuclei. The evidence at hand suggests that these abnormalities are expressions of a differentiated state of the nuclei, affecting their capacity to proceed normally through mitosis following transfer into egg cytoplasm. However, their exact mode of origin, and the problems they pose in the interpretation of nuclear transplantation experiments, remain to be worked out.

Studies on the maternal effect of the semilethal gene, v, in the Mexican axolotl. I. Influence of temperature on the expression of the effect. II. Cytological changes in the affected embryos.

Abstract The gene, v , in the Mexican axolotl has recently been shown to affect the egg cytoplasm during oogenesis in such a way as to lead to abnormalities in development following fertilization. Affected embryos frequently are arrested in blastula stages and display a remarkable appearance resulting from a stratification of the contents of the blastomeres. Or they may continue to later stages before the maternal effect becomes apparent ( Humphrey, 1962 ). The present paper is concerned with the influence of temperature on the expression of this maternal effect and with the cytological changes occurring in the affected embryos. The influence of temperature on the maternal effect was investigated by rearing the embryos at 25° C and at 14–20° C. (Both temperatures are well within the range permitting normal development of control eggs.) At 25° a large majority (88%) of the embryos were arrested in mid-blastula stages and exhibited a striking appearance due to a stratification of the contents of the blastomeres. At lower temperatures (14–20°) the expression of the maternal effect was delayed. Less than 1% of the embryos were arrested in mid-blastula stages. Approximately 70% developed to gastrula and neurula stages and 30% to more advanced embryonic stages. Stratification of cell contents was observed in the gastrulae and some neurulae but was less pronounced than that seen in arrested blastulae. The maternal effect results in a striking rearrangement of cell components in embryos arrested in blastula and gastrula stages. The first cytologically detectable change consists in a concentration of the RNA-containing cytoplasm into the upper (animal) parts of the cells. Nuclei are similarly displaced from their normal central location and are found in the upper parts of the cells, wholly or partly within the RNA-containing cytoplasm. These changes in the distribution of nuclei and of cytoplasmic RNA are followed by a sedimentation of yolk platelets and then of pigment granules. Eventually the cells take on a highly stratified appearance. For a time after the beginning of the cytoplasmic stratification the nuclei continue mitotic activity which, however, is usually of an abnormal type. The most common abnormality (at 25°) is an extensive polyploidization, indicating that chromosomal replication continues for a time after nuclear and cell division have been suppressed.

Experimental studies on two mutant genes, r and x, in the Mexican axolotl (Ambystoma mexicanum)

Abstract Mutant genes r and x, discovered in two unrelated axolotl stocks, are simple recessives determining autonomous cell lethal traits. These traits become recognizable by their characteristic gill and limb patterns which appear in each at about the same period of development. The life spans of the two mutants are approximately the same. Larvae homozygous for both mutants are easily recognized by their smaller size, reduced gill development, and unusually small eyes. None of the three mutant phenotypes (r/r, x/x, rr/xx) is benefited by parabiosis with a normal larva. Transplants of the forelimb area from all three usually were soon invaded by tissues of host origin, resulting in limbs ranging from those almost normal to those reduced to functionless stumps. Those from r/r donors produced the highest percentage of useful limbs. Transplants of the gill-forming area produced gills of the mutant type which, in all cases, regressed. Distention and rupture of gill vessels led to death of some animals. In others the gills became reduced to mere stubs or even disappeared. The failure of replacement of pharyngeal structures of mutant graft origin resulted in the death of all grafted animals from vascular accidents or by prevention of normal feeding or respiration.