Robert H. Broyles

Two erythropoietic microenvironments and two larval red cell lines in bullfrog tadpoles

Abstract Previous work has shown that Rana catesbeiana tadpoles have two erythropoietic sites: mesonephric kidney and liver. Both sites apparently are active throughout larval life. Among the pleomorphic larval red blood cells (RBCs) we observe two morphological types which differ in the larval hemoglobins (Hb) that they contain. One type is an oblong, oval RBC with an acentric nucleus and contains the larval Hb which is slowest migrating on electrophoresis at basic pH. This RBC type emanates from the kidneys. The other type, which is elliptical in shape, with a central nucleus, contains three other larval Hbs and emanates from the liver. Histological sections of kidney and liver reveal erythropoietic foci of differing cellular composition and configuration. We have hypothesized that choice of RBC and Hb types is determined in part by the different erythropoietic microenvironments. In support of this hypothesis is our finding that, in organ cultures, liver cocultured with kidney undergoes a shift in its Hb pattern. Part of the shift is toward the Hb type which is the product of kidney erythropoiesis.

Improved polyacrylamide gel electrophoresis with different amino acids as the trailing constituent.

Abstract Using a polyacrylamide disc gel electrophoretic system similar to that described by J. T. Clarke (1964, Ann. N. Y. Acad. Sci.121, 428–436), we have achieved an improved separation of hemoglobins from Rana catesbeiana tadpoles by substituting one of several amino acids in the place of glycine in the electrode chamber buffer. The relative migrations (Rf) and degree of separation of these similar hemoglobins are proportional to the pK′ of the α-amino group of the amino acid used in the buffer. Specifically, for these proteins, log (Rf × 100) was found to be directly proportional to the pK′2 of the amino acid divided by the volume conductivity (specific conductance) of the electrode chamber buffer. For example, improved separation of these hemoglobins in short electrophoretic times can be achieved, at low cost, by using dl -alanine instead of glycine in the buffer. Improved separation of other proteins which migrate at basic pH might be achieved by a similar approach.

In vivo regulation of hemoglobin phenotypes of developing Rana catesbeiana.

We have examined the effects of phenylhydrazine-induced anemia on the in vivo synthesis of specific hemoglobins at larval, metamorphic, and post-metamorphic stages of the bullfrog Rana catesbeiana, and have found that at all stages the animals qualitatively and quantitatively regenerate their pre-anemia hemoglobin profiles, with one exception: Animals approaching or undergoing the metamorphic hemoglobin switch synthesize only adult hemoglobin during recovery from anemia. We conclude that the ontogenetic progression of hemoglobins in R. catesbeiana is regulated at the level of differentiation of distinct erythroid cell lines, each committed to expressing a particular hemoglobin phenotype; this regulation is unperturbed by anemia.

Determination of hemoglobin expression patterns in erythroid cells of Rana catesbeiana tadpoles

1. Rana catesbeiana (bullfrog) tadpoles are heterogeneous in the relative amounts of four major tadpole hemoglobins (Hbs), as well as in the relative amounts of two tadpole red blood cell types in the peripheral blood. 2. Previous work has shown that this heterogeneity is present at all stages of larval development and growth. 3. Although some tadpoles lack one of the Hbs in their peripheral blood (i.e. the electrophoretically slowest form, Td-4), the missing Hb can be found in the erythropoietic organ from which it emanates (the kidneys), indicating that the heterogeneity results from quantitative differences in gene expression. 4. We wished to know whether this in vivo regulation is subject to external environmental perturbation and report that tadpoles of known Hb phenotypes regenerate precisely the pre-anemia Hb profile during early as well as late stages of recovery from phenylhydrazine-induced anemia. 5. These and other results indicate that the in vivo mechanism for regulating the pattern of Hb expression has become firmly determined in the erythropoietic system by the earliest larval stage of development.

Intracellular signals for developmental hemoglobin switching

We have detected trans-acting factors that regulate developmental hemoglobin switching by fusing erythroid cells of different developmental programs. Adult erythroid cells of one anuran species, Xenopus laevis, were fused with tadpole erythroid cells of another frog, Rana catesbeiana. In a second set of experiments, dimethyl sulfoxide-induced murine erythroleukemia cells, which express only adult mouse globins, were fused with Rana tadpole erythroid cells, which express only embryonic and fetal-like globins. Adult Rana globin gene expression was detected in both sets of transient heterokaryons at 6 hr after fusion. Dot blots and Northern blots of total RNA from the heterokaryons contained material that reacted with an adult Rana alpha-globin probe; newly synthesized adult Rana hemoglobin tetramers were detected with native polyacrylamide gel electrophoresis. These results show that developmental stage-specific transacting factors for globin genes can function across vertebrate classes (mammalia to amphibia) and suggest that the mechanisms that regulate developmental hemoglobin switching are highly conserved.

Nuclear reprogramming by cell fusion.

The use of cell fusion to study exchange of information at the molecular level between the nucleus and the cytoplasm of cells during regulation of gene expression was pioneered by Harris and Ringertz more than three decades ago. The ability to make heterokaryons with cells from different species or genetic strains is especially useful because genetic differences in gene products allow the origin of trans-acting regulatory factors to be determined. Heterokaryons between adult nucleated erythroid cells of one species and embryonic/larval nucleated erythroid cells of another species, for example, show cross-induction between the two types of nuclei, resulting in reprogramming of the adult nucleus to embryonic/larval globin gene expression and/or reprogramming of the embryonic/larval cell nucleus to adult globin expression. These experiments provided definitive evidence that developmental program switching is mediated by trans-acting factors. Other possible uses of this cell fusion protocol in stem cell biology and transplantation of genetically engineered cells for tissue regeneration are briefly discussed.