NEURAL CREST IN DEVELOPMENT AND REPAIR

This thesis is focused on several aspects of neural crest biology, largely related to two main issues: 1) understanding its mechanisms of migration and 2) analyzing the potential of neural crest stem cells or their derivatives for nervous system repair. If repair is the aim, knowledge of both timing and events leading to axonal degeneration is required in order to block it and/or increase regeneration outcome. In these studies I have made use of mouse, rat and chicken in vivo and in vitro models to address specific questions regarding the scope of this thesis. In paper I, changes in distribution of myelin proteins are described after ligation of the sciatic nerve, an injury model which does not allow nerve regeneration to occur. Clustering of MBPs and P0 (the two most abundant peripheral myelin proteins) was shown to precede their degradation, involving subsequent roles of Schwann cells (SCs) and macrophages in the removal of debris distally to the injury zone. Both clustering and protein degradation steps were found to be accelerated in young rat animals when compared to adult ones. In paper II, similar kind of studies were applied in other two injury models allowing regeneration to occur: a transient compression (crush) of the nerve or an intraneural injection of colchicine, which transiently blocks axonal transport. Changes in distribution of myelin (MBPs) and axonal (PGP9.5) protein immunoreactivities were monitored for up to one month and degeneration/regeneration processes followed and compared in the two models. By applying the trophic factor apotransferrin we were able to block the degeneration produced by colchicine, likely through stabilization of axonal and/or SC cytoskeleton. In paper III, original culture protocols have been applied to efficiently generate highly pure cultures of mature SCs from boundary cap neural crest stem cells (bNCSCs). The in vitro SC differentiation from stem cells was found to resemble the in vivo process when looking at combinations of markers changing expression during SC lineage. Stem cell-derived SCs were able to myelinate axons both in vitro and in vivo. These results prompted further work shown in paper V, in which the regenerative potential of neural crest stem cell-derived SCs has been tested in the contused spinal cord injury model. Even though in most cases grafted cells died soon after transplantation, when grafted alive they were found to improve locomotor behavior and to enhance endogenous regenerative mechanisms, producing both an increase in axonal re-growth and in recruitment of endogenous glial cells. In this study, evidences are shown suggesting that after injury ependymal cells of the spinal cord central canal can generate endogenous glial cells of myelinating phenotype, likely including some of the Schwann like cells which populate the injury zone allowing axonal re-growth. In paper IV, the scaffolding protein Nedd9 is shown to be expressed by multipotent migrating and post-migratory neural crest cells (NCCs). Nedd9 expression is induced in NCCs at the level of the dermamyotome dorsal lip. Loss-of-function and gain-offunction studies were consistent with a principal role for Nedd9 in NCC migration. Retinoic acid, known to be synthesized by the dermamyotome, was found to induce Nedd9 expression in NCCs. Nedd9 expression pattern in mouse is described in paper VI, including mesencephalic ventral midbrain as well as other discrete progenitor populations from diverse tissues and organs. LIST OF PUBLICATIONS I. Setton-Avruj C.P., Aquino J.B. , Goedelman C.J., Soto E.F. , Villar M.J. (2002) P0 and myelin basic protein-like immunoreactivities following ligation of the sciatic nerve in the rat. Neurochem Res. 27(11):1293-303. II. Aquino J.B. , Musolino P.L. , Coronel M.F. , Villar M.J., Setton-Avruj C.P. (2006) Nerve degeneration is prevented by a single intraneural apotransferrin injection into colchicine-injured sciatic nerves in the rat. Brain Res.