Insulin-like growth factor I (IGF I) in the red spotted newt, Notophthalmus viridescens: Description of larval limb development; localization of IGF I in larval and adult newt limbs; and effects of IGF I on epimorphic regeneration of an adult newt appenda

This study initiates research into the effects of IGF I on epimorphic appendage regeneration in the adult newt, Notophthalmus viridescens. As regeneration in this animal is often considered a recapitulation of development, the current research compares the expression of IGF I in developing larval forelimbs and regenerating adult forelimbs. To facilitate this comparison, a description of larval forelimb development was undertaken. This thesis is the first comprehensive record staging limb development in N. viridescens. In addition to the morphological descriptions, whole-mount and histological analyses of cellular development were made. This study determined that IGF I is a naturally occurring growth factor in this animal's liver, as observed among many vertebrates. This investigation also demonstrates that IGF I could not be localized in proliferating or differentiating cells of either larval (developing) forelimbs or adult regenerating forelimbs. However, the localization of IGF I in the early stages of adult newt forelimb regeneration suggests a role for this factor during wound healing. These observations were further examined utilizing in vitro manipulation of regenerating newt tail explants. Insulin-like growth factor I had little proliferative effect on regenerating explants, nor did it promote cartilage differentiation in these regenerates. This contrasts with insulin, which is supportive of adult newt appendage regeneration. Additionally, the combination of IGF I and growth hormone (GH) in vitro was not found to be supportive of cartilage formation, a process commonly attributed to IGF I and GH interactions. In the regenerating adult newt, this combination appeared to be detrimental to forelimb regeneration. This may be due to the increase in osteoclast numbers observed at the distal bone stump within regenerating explant. Placing IGF I and GH within the context of a more complete Hormonal Milieu (a multi-hormone culture environment including hydrocortisone and thyroxine) retained osteoclast numbers at pre-explantation levels, although it did little to advance regeneration. However, when prolactin was substituted for GH in an IGF-containing Hormonal Milieu, this culture environment supported not only pre-explantation osteoclast numbers, but also growth and differentiation of the adult newt explants.