Newt Loken jumping near Crisler Arena sign

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Cloning of actin genes from a genomic library of the newt, Notophthalmus viridescens

The regenerating urodele limb is a useful model system in which to study, in vivo, the controls of cell proliferation and differentiation. Techniques are available which enable one to experimentally manipulate mitogenic influences upon the blastema, as well the morphogenesis of the regenerating 11mb. Although classical regeneration studies have generated a wealth of knowledge concerning tissue interactions, little 1s known about the process at the level of gene expression. The aim of this project was to clone potentially developmentally regulated genes from a newt genomic library for use in future studies of gene expression during limb regeneration. We decided to clone the cytoskeletal actin gene for the following reasons: 1. its expression reflects the proliferative and differentiatlve states of cells in other systems 2. the high copy number of cytoplasmic actin pseudogenes in other vertebrates and the high degree of evolutionary sequence conservation among actin genes increased the chance of cloning one of the newt cytoplasmic actin genes. 3. Preliminary experiments indicated that a newt actin could probably be identified using an available chick ~-actln gene for a molecular probe. Two independent recombinant phage clones, containing actin homologous inserts, were isolated from a newt genomic library by hybridization with the chick actin probe. Restriction mapping identified actin homologous sequences within the newt DNA inserts which were subcloned into the plasmid pTZ19R. The recombinant plasmids were transformed into the Escherichia coli strain, DHsa. Detailed restriction maps were produced of the 5.7Kb and 3.1Kb newt DNA inserts in the plasmids, designated pTNAl and pTNA2. The short «1.3 Kb) length of the actin homologous sequence in pTNA2 indicated that it was possibly a reverse transcript pseudogene. Problems associated with molecular cloning of DNA sequences from N. viridescens are discussed with respect to the large genome size and abundant highly repetitive DNA sequences.

Transfection of newt blastema mesenchyme using the techniques of lipofection and direct gene transfer

The regenerating amphibian limb provides a useful system for studying genes involved in the establishment of positional information. While a number of candidate genes that may playa role in pattern formation have been identified, their function in vivo is unknown in this system. To better ascertain the role of these genes, it would be useful to be able to alter their normal patterns of expression in vivo and to assess the effects of this misexpression on limb pattern. In order to achieve this, a method of introducing a plasmid containing the eDNA of a gene of interest into a newt blastema (a growth zone of mesenchymal progenitor cells) is needed. Unfortunately, most commonly used transfection techniques cannot be used with newt blastema cells. In this study, I have used the techniques of lipofection and direct gene transfer to introduce plasmid DNA containing reporter genes into the cells of a regenerating newt limb. The technique of lipofection was most effective when the blastema cells were transfected in vitro. The optimal ratio for transfection was shown to be 1:3 DNA:Lipofectin (W/w) , and an increase in the amount of DNA present in the mixture (1:3 ratio maintained) resulted in a corresponding increase in gene expression. The technique of direct gene transfer was used to transfect newt blastema cells with and without prior complex formation with Lipofectin. Injection of plasmid DNA alone provided the most 3 promising results. It was possible to introduce plasmid DNA containing the reporter gene ~-galactosidase and achieve significant gene expression in cells associated with the injection site. In the future, it would be interesting to use this technique to inject plasmid DNA containing a gene which may have a role in pattern formation into specific areas of the newt blastema and to analyze the resulting limb pattern that emerges.

An examination of transcriptional and translational regulation of the 70kDa heat shock proteins following amputation of the tail and forelimb in the newt Notophthalmus viridescens

Several stresses to tissues including hyperthermia, ischemia, mechanical trauma and heavy metals have been demonstrated to affect the regulation of a subset of the family of heat shock proteins of70kOa (hsp70). In several organisms following some of these traumas, the levels of hsp70 mRNA and proteins are dramatically upregulated. However, the effects of the stress on limb and tail amputation in the newt Notophthalmus viridescens, involving mechanical tissue damage, have not adequately been examined. In the present study, three techniques were utilized to quantitate the levels of hsp70 mRNA and protein in the tissues of the forelimbs and tails of newts during the early post-traumatic events following surgical resection of these:: appendages. These included quantitative Western blotting of proteins separated by both one and twodimensional SDS-polyacrylamide gel electrophoresis and quantitative Northern blot analysis of total RNA. In tissues of both the limb and tail one hour after amputation, there were no significant differences in the levels of hsp70 protein measured by one-dimensional SOSPAGE followed by Western blotting, when compared to the levels measured in the unamputated limb. A 30 minute heat shock at 35°C failed to elicit an increase in the levels of hsp70 protein in these tissues. Further analysis using the more sensitive 20 PAGE separation of stump tissue proteins revealed that at least some of the five hsp70 isoforms of the newt may be differentially regulated in limbs and tails in response to trauma. It appears also that amputation of the tail and limb tissues leads to slight 3 elevation in the levels of HSP70 mRNA when compared to those of their respective unstressed tissues.

The role of microRNAs and retinoid signaling during spinal cord regeneration in the adult newt.

The molecular events after spinal cord injury that lead to the establishment of a permissive environment and epimorphic regeneration remain unclear. Two molecular pathway regulators that may converge to create a spinal cord regeneration-permissive environment in the urodele are retinoic acid (RA) and microRNAs (miRNAs). Recent evidence suggests that RARβ-mediated signaling is necessary for tail and caudal spinal cord regeneration in the adult newt. MicroRNAs are attractive candidates as mediators of retinoid signaling during regeneration, as their pleiotropic effects are vital in situations where global changes in gene expression are required. Thus, the overall aim of this thesis was to determine if miRNAs are involved in tail and caudal spinal cord regeneration in the adult newt, and if they act as regulators and/or effectors of retinoid signaling during this process. I have demonstrated here, for the first time, that multiple miRNAs are dysregulated in response to spinal cord injury in the adult newt, as well as in response to inhibition of retinoid signaling. Two of these miRNAs, miR-133a and miR-1, appear to target RARβ2 transcripts both in vivo and in vitro. Inhibition of RA signaling via RARβ with a selective antagonist, LE135, alters the pattern of expression of these miRNAs, which leads to an inhibition of tail regeneration. These data are indicative of a negative feed back loop, albeit potentially an indirect one. I also aimed to examine which miRNAs are affected by inhibiting RA synthesis during regeneration, and provided a long list of miRNAs that are dysregulated. These data provide the foundation for future studies on the putative roles of these miRNAs, as well as their function in retinoid signaling. Overall, these studies provide the first evidence for a role for miRNAs as mediators of retinoid signaling during caudal spinal cord regeneration in any system.

Population genetic diversity of the endemic Sardinian newt Euproctus platycephalus: implications for conservation

The Sardinian mountain newt Euproctus platycephalus, endemic to the island of Sardinia, (Italy), is considered a rare and threatened species and is classed as critically endangered by IUCN. It inhabits streams, small lakes and pools on the main mountain systems of the island. Threats from climatic and anthropogenic factors have raised concerns for the long-term survival of newt populations on the island. MtDNA sequencing was used to investigate the genetic population structure and phylogeography of this endemic species. Patterns of genetic variation were assessed by sequencing the complete Dloop region and part of the 12SrRNA, from 74 individuals representing four different populations. Analyses of molecular variance suggest that populations are significantly differentiated, and the distribution of haplotypes across the island shows strong geographical structuring. However, phylogenetic analyses also suggest that the Sardinian population consists of two distinct mtDNA groups, which may reflect ancient isolation and expansion events. Population structure, evolutionary history of the species and implications for the conservation of newt populations are discussed.

Habitat correlates of distribution and local population decline of the endemic Sardinian newt Euproctus platycephalus

Habitat-based statistical models relating patterns of presence and absence of species to habitat variables could be useful to resolve conservation-related problems and highlight the causes of population declines. In this paper, we apply such a modelling approach to an endemic amphibian, the Sardinian mountain newt Euproctus platycephalus, considered by IUCN a critically endangered species. Sardinian newts inhabit freshwater habitat in streams, small lakes and pools on the island of Sardinia (Italy). Reported declines of newt populations are not yet supported by quantitative data, however, they are perceived or suspected across the species' historical range. This study represents a first attempt trying to statistically relate habitat characteristics to Sardinian newt occurrence and persistence. Linear regression analysis revealed that newts are more likely to be found in sites with colder water temperature, less riparian vegetation and, marginally, absence of fish. The implications of the results for the conservation of the species are discussed, and suggestions for the short-term management of newt inhabited sites suggested. (C) 2003 Elsevier Ltd. All rights reserved.

Expression of Sonic hedgehog gene in regenerating newt limb blastemas recapitulates that in developing limb buds

This study aimed at characterizing the Sonic hedgehog (shh) gene in newt limbs, which encodes a signaling molecule of the zone of polarizing activity (ZPA) responsible for determining the anterior–posterior axis of the embryonic chicken and mouse limbs. The reverse transcription–PCR showed that adult newt regenerating limbs express shh genes. In situ hybridization experiments demonstrated that shh genes were expressed in mesenchymal cells of the posterior region of both embryonic buds and regenerating blastemas of newt limbs, strongly suggesting the presence of ZPA in these tissues. Experiments of the axial reversal graft of blastemas further supported this suggestion. The grafted blastemas regenerated supernumerary limbs, and this has been explained by three models: the polar coordinate model, the boundary model, and the polarizing zone model. In favor of the third model, the shh gene was expressed not only in the original region (new anterior region) of the graft, but also ectopically in the other region (new posterior region) of the same graft. This study implies that the regenerating limb blastema produces ZPA as the signaling center of the AP patterning as in the developing limb bud and, therefore, supports the notion that the limb regeneration recapitulates the limb development.

The newt ribozyme is part of a riboprotein complex

Strand-specific transcripts of a satellite DNA of the newts, Notophthalmus and Triturus, are present in cells in monomeric and multimeric sizes. These transcripts undergo self-catalyzed, site-specific cleavage in vitro: the reaction requires Mg2+ and is mediated by a “hammerhead” domain. Transcription of the newt ribozyme appears to be performed by RNA polymerase II under the control of a proximal sequence element and a distal sequence element. In vitro, the newt ribozyme can cleave in trans an RNA substrate, suggesting that in vivo it might be involved in RNA processing events, perhaps as a riboprotein complex. Here we show that the newt ribozyme is in fact present as a riboprotein particle of about 12 S in the oocytes of Triturus. In addition, reconstitution experiments and gel-shift analyses show that a complex is assembled in vitro on the monomeric ribozyme molecules. UV cross-linking studies identify a few polypeptide species, ranging from 31 to 65 kDa, associated to the newt ribozyme with different affinities. Finally, we find that an appropriate oligoribonucleotide substrate is specifically cleaved by the riboproteic activity in S-100 ovary extracts.

Cloning and characterization of cDNAs for matrix metalloproteinases of regenerating newt limbs.

Matrix metalloproteinases (MMPs) of regenerating urodele limbs have been suggested to play crucial roles in the process of the dedifferentiation of cells in the damaged tissues and the ensuing blastema formation because the activation of MMPs is an early and conspicuous event occurring in the amputated limb. MMP cDNAs were cloned as products of the reverse transcription-PCR from cDNA libraries of newt limbs, and their structures were characterized. Three cDNAs encoding newt MMPs (2D-1, 2D-19, and 2D-24) have been cloned from second day postamputation regenerating limbs, and a cDNA (EB-1) was cloned from early bud-stage regenerating limbs. These cDNAs included the full-length coding regions. The deduced amino acid sequences of 2D-1, 2D-19, 2D-24, and EB-1 had a homology with mammalian MMP9, MMP3/10, MMP3/10, and MMP13, respectively. The basic motif of these newt MMP genes was similar to mammalian counterparts and contained regions encoding a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. However, some unique molecular evolutionary features were also found in the newt MMPs. cDNAs of 2D-19 and 2D-24 contained a specific insertion and deletion, respectively. The insertion of 2D-19 is threonine-rich, similar to the threonine cluster found in the collagenase-like sea urchin hatching enzyme. Northern blot analysis showed that the expression levels of the newt MMPs were dramatically increased after amputation, suggesting that they play an important role(s) in tissue remodeling of the regenerating limb.

1963 Men's Gymnastics Team,

Back Row: manager Lewis Hyman, Alex Frecsa, Barry Spicer, Jim Hynds, Paul Levy, Phil Bolton, Gil Larose, head coach Newt Loken

Front Row: Mike Henderson, John Hamilton, Arno Lascari, Fred Sanders, Gary Erwin

1965 Men's Gymnastics Team,

Front Row: Cliff Chilvers, coach Newt Loken, Dr. Earl Ziegler, Gary Erwin, John Hamilton, Chip Fuller, Phil Fuller,

Back Row: Rich Blanton, Mike Henderson, Ken Williams, Dick Stone, Chris Vanden Broek, John Cashman, Gary VanderVoort, Art Bassler, Fred Sanders

Not in photo: Ned Duke