Richard A. Liversage

Molecular cloning of the Notophthalmus viridescens radical fringe cDNA and characterization of its expression during forelimb development and adult forelimb regeneration.

Larval and adult newts provide important experimental models to study limb development and regeneration. These animals have exceptional ability to regenerate their appendages, as well as other vital structures. Our research examines the role of the fringe gene (fng) in the developing and regenerating adult newt forelimb. Fringe codes for a secretory protein. It was first discovered in Drosophila, and later homologues were isolated in Xenopus laevis, chick and mouse. This gene has been highly conserved throughout evolution, indicating its crucial role in vertebrate and invertebrate development. We have isolated, cloned, and sequenced the full length of the Notophthalmus viridescens radical fringe cDNA (nrFng) by screening a newt forelimb blastema cDNA library with a 500‐bp fragment of the Xenopus lunatic fringe cDNA. The newt fringe cDNA codes for a 396 amino acid protein with a predicted N‐terminal signal sequence. Newt fringe shows high homology with radical fringe homologues of many species. Whole mount mRNA in situ hybridization on several stages of newt limb development reveals that nrFng is first expressed in the limb field, with intense expression as the limb bud develops. However, gene expression diminishes with more advanced digit development. A significant role in adult forelimb regeneration is also evident, as we isolated the cDNA from a regeneration–specific library and found it highly expressed during the regenerative phases of active cell division and then down regulated at sites undergoing differentiation and morphogenesis. Dev Dyn 1999;214:259–268.

Hox C6 expression during development and regeneration of forelimbs in larval Notophthalmus viridescens.

Abstract A central theme concerning the epimorphic regenerative potential of urodele amphibian appendages is that limb regeneration in the adult parallels larval limb development. Results of previous research have led to the suggestion that homeobox containing genes are ”re-expressed” during the epimorphic regeneration of forelimbs of adult Notophthalmus viridescens in patterns which retrace larval limb development. However, to date no literature exists concerning expression patterns of any homeobox containing genes during larval development of this species. The lack of such information has been a hindrance in exploring the similarities as well as differences which exist between limb regeneration in adults and limb development in larvae. Here we report the first such results of the localization of Hox C6 (formerly, NvHBox-1) in developing and regenerating forelimbs of N. viridescens larvae as demonstrated by whole-mount in situ hybridization. Inasmuch as the pattern of Hox C6 expression is similar in developing forelimb buds of larvae and epimorphically regenerating forelimb blastemata of both adults and larvae, our results support the paradigm that epimorphic regeneration in adult newts parallels larval forelimb development. However, in contrast with observations which document the presence of Hox C6 in both intact, as well as regenerating hindlimbs and tails of adult newts, our results reveal no such Hox C6 expression during larval development of hindlimbs or the tail. As such, our findings indicate that critical differences in larval hindlimb and tail development versus adult expression patterns of this gene in these two appendages may be due primarily to differences in gene regulation as opposed to gene function. Thus, the apparent ability of urodeles to regulate genes in such a highly co-ordinated fashion so as to replace lost, differentiated, appendicular structures in adult animals may assist, at least in part, in better elucidating the phenomenon of epimorphic regeneration.

Identification of cDNAs associated with late dedifferentiation in adult newt forelimb regeneration

Epimorphic limb regeneration in the adult newt involves the dedifferentiation of differentiated cells to yield a pluripotent blastemal cell. These mesenchymal‐like cells proliferate and subsequently respond to patterning and differentiation cues to form a new limb. Understanding the dedifferentiation process requires the selective identification of dedifferentiating cells within the heterogeneous population of cells in the regenerate. In this study, representational differences analysis was used to produce an enriched population of dedifferentiation‐associated cDNA fragments. Fifty‐nine unique cDNA fragments were identified, sequenced, and analyzed using bioinformatics tools and databases. Some of these clones demonstrate significant similarity to known genes in other species. Other clones can be linked by homology to pathways previously implicated in the dedifferentiation process. These data will form the basis for further analyses to elucidate the role of candidate genes in the dedifferentiation process during newt forelimb regeneration. Developmental Dynamics 233:347–355, 2005.

Insulin, glucagon and somatostatin localization in the pancreas of metamorphosed Xenopus laevis

The current study was designed to determine if insulin, glucagon and somatostatin-containing cells are present in the pancreas of adult Xenopus laevis. Localization methods utilized included cytochemical aldehyde fuchsin (AF) staining as well as the immunochemical peroxidase antiperoxidase (PAP) procedure for light microscopy. The results show numerous large clusters of AF-positive cells within a network of highly vascularized acinar tissue. PAP immunochemical localization with insulin antibody on adjacent sections demonstrates positive immunoreactivity to AF-positive cell groups and also the presence of immunoreactive insulin (IRI). Cells exhibiting this immunoreactivity are located in the central region of the islet-like structures. Serial sections not only show PAP immunoreactivity for IRI, but also for immunoreactive glucagon (IRG) and immunoreactive somatostatin (IRS) in the same islet-like structure. IRG and IRS-containing cells are situated around the periphery of the islet-like structures, surrounding the central core of IRI-containing cells. Antibody specificity was confirmed by homologous and heterologous antigen immuno-absorbance assays, as well as incubation of adjacent sections in preimmune sera. Based on this data we conclude that: the distribution of cells of the endocrine pancreas of metamorphosed Xenopus laevis is similar to that of many mammals and certain urodeles. Given the apparent specificity of the antigen-antibody reactions, it appears that Xenopus insulin, glucagon and somatostatin are structurally conserved.

In vitro studies of the influence of prolactin on tail regeneration in the adult newtNotophthalmus viridescens

SummaryIn vitro experiments were carried out to determine the effects of prolactin, and prolactin in combination with other hormones on the regeneration of adult newt tail blastemata. A total of 271 blastemata were explanted 13 days postamputation and were organ cultured for 96 h at 20 (±1)°C. Treatment with prolactin alone resulted in an increase in the blastema cell density of the tail regenerates. Cell accumulation and cell alignment were observed ventral to the reconstituted spinal cord. Prolactin and thyroxine, in combination, improved development of tail regenerates as compared with treatment with prolactin or thyroxine singly, supporting the results of earlier in vivo studies. Optimal development was obtained only when prolactin, insulin, thyroxine and hydrocortisone were added to the culture medium. Regeneration of tail explants maintained in medium augmented with the four hormones closely resembles that of in vivo tail blastemata 17 days post-amputation.

Regenerative response of amputated forelimbs of Xenopus laevis froglets to partial denervation

Xenopus laevis froglet forelimbs normally respond to amputational injury by forming a heteromorphic cartilaginous rod‐shaped outgrowth. However, partial denervation of a forelimb by ablation of the N. radialis or the N. ulnaris, followed in 2 days by amputation through the mid radius‐ulna, results in a size deficiency of the regenerative outgrowth 14 and 21 days postamputation. The decreasing quantity of forelimb innervation, as a result of partial denervation by 55 or 45%, apparently has a graded effect on the cell population and on the extent of cartilage development in the outgrowth. As a consequence of amputational injury, a nerve independent response of the periosteum was also found. This response produced considerable thickening in the periosteum and was due to cell proliferation in both the control and denervated cases.

Response of Amputated Xenopus Laevis Forelimbs to Augmentation of the Nerve Supply

This study concerns the effects of nerve augmentation on growth and development of forelimb regenerates in metamorphosed Xenopus laevis froglets. Augmentation was accomplished by implanting spinal ganglia from sibling donors near the base of the host regenerate. The results show that nerve augmentation increases the potential growth of regenerates and also leads to perturbations of the normal cartilaginous outgrowth. However, nerve augmentation does not appear to make a substantial contribution of the definitive pattern formation in the regenerate.

Xenopus adenine nucleotide translocase mRNA exhibits specific and dynamic patterns of expression during development

We report the isolation and characterization of the Xenopus homolog to human T1 ANT (adenine nucleotide translocase). The 1290-nucleotide sequence contains initiation and termination signals, and encodes a conceptual protein of 298 amino acids. The sequence shares high amino acid identity with the mammalian adenine translocases. The transcript is present in unfertilized eggs, and it is expressed at higher levels during formation of the antero-posterior dorsal axis in embryos. Although low levels are expressed constitutively except in endodermal cells, adenine nucleotide translocase (ANT) expression is dynamically regulated during neurulation. At this stage, expression in ectoderm rapidly diminishes as the neural folds form, and then ANT expression increases slightly in mesoderm. At the culmination of neurulation, the neural tube briefly expresses ANT, and thereafter its expression predominates in the somitic mesoderm and also the chordoneural hinge. In addition, ANT expression is particularly high in the prosencephalon, the mesencephalon, the branchial arches, eye, and the otic vesicle. Treatment of embryos with retinoic acid has the effect of diminishing constitutive expression of ANT, but microinjection studies demonstrate that immediate and local repression cannot be induced in dorsal structures.

In Vitro Effects of a Mitogenic Factor on Xenopus Laevis Forelimb Regeneration

The essential role of nerves in the support of amphibian forelimb regeneration is well documented (Singer, 1952; 1954). In vitro experiments (Globus and Vethamany-Globus, 1977; Tomlinson et al., 1981) suggest that the neurotrophic agent is a diffusible proteinaceous nerve growth factor of low molecular weight, which is inactivated by heat and trypsin (Singer, 1976). The current investigation was designed to test the effects of a chick brain growth factor (CBGF) on Xenopus laevis forelimb regeneration; specifically its role as a mitogenic factor for mesenchyme-like cells in the regenerate. CBGF is a low molecular weight (500–2, 000), relatively heat stable peptide (Carlone et al., 1988).

Insulin Localization in the Pancreas and in Forelimb Regenerates of the Adult Newt, Notophthalmus Viridescens

Metabolic hormones play an essential role in the regeneration of urodele appendages. Indeed, the involvement of insulin is essential as organ-cultured adult newt forelimb blastemata attain maximum growth and differentiation only when insulin is present in combination with thyroxine, hydrocortisone, and either growth hormone (1) or prolactin (2) . In experimentally induced diabetic adult newts, forelimb blastemata fail to accumulate the critical mass of cells reguired for the continuation of regeneration. Also, cartilage formation as well as growth and differentiation of the tissues are markedly reduced (3, 4). Given the importance of insulin to the process of regeneration, it was of interest to attempt localization of this hormone at its source (presumably the pancreas) and at its site of action in the regenerating forelimb.