Neurotrophin-3 Induced by TriIodothyronine in Cerebellar Granule Cells Promotes Purkinje Cell Differentiation

brain development, but the mechanism(s) by which triiodothyronine (T3) mediates neuronal differentiation is poorly understood. Here we demonstrate that T3 regulates the neurotrophic factor, neurotrophin-3 (NT-3), in developing rat cerebellar granule cells both in cell culture and in vivo. In situ hybridization experiments showed that developing Purkinje cells do not express NT-3 mRNA but do express trkC, the putative neuronal receptor for NT-3. Addition of recombinant NT-3 to cerebellar cultures from embryonic rat brain induces hypertrophy and neurite sprouting of Purkinje cells, and upregulates the mRNA encoding the calcium-binding protein, calbindin-28 kD. The present study demonstrates a novel interaction between cerebellar granule neurons and developing Purkinje cells in which NT-3 induced by T3 in the granule cells promotes Purkinje cell differentiation. T HE development of the rat cerebellum mainly takes place postnatally and it is influenced by a variety of epigenetic signals such as growth factors and hormones. The external granule layer (EGL) t gives rise to the cerebellar granule neurons which divide and subsequently migrate inwards to reach the internal granule cell layer (IGL). Granule neurons establish synaptic contacts with the developing Purkinje cells in the molecular layer (ML) (Altman, 1972). Thyroid hormones are important for normal brain development (Legrand, 1979; Lindholm, 1984; Dussault and Ruel, 1987) and a lack of tri-iodothyronine (T3) is known to adversely influence the highly ordered structure of the developing rat cerebellum (Legrand, 1979). Thus, the differentiation of Purkinje cells which is characterized by an extensive branching of dendrites is retarded in hypothyroidism (Legrand, 1979). Moreover, migration, cell survival, and differentiation of cerebellar granule neurons are also influenced by T3 (Nicholson and Altman, 1972; Legrand, 1979; Heisenberg et al., 1992). Administration of T3 during early postnatal life is known to alleviate cellular changes in brain observed in hypothyroidism (Legrand, 1979; Dussault and Ruel, 1987). In the developing cerebellum, the first three Please address all correspondence to Dan Lindholm, Department of Neurochemistry, Max Planck Institute for Psychiatry, Am Klopferspitz 18A, D-8033 Martinsried Munich, Federal Republic of Germany. 1. Abbreviations used in this paper: BDNF, brain-derived neurotrophic factor; EGL, external granule layer; bFGF, basic FGF; IGL, internal granule layer; IL-I, interleukin-l; ML, molecdar layer; NT-3, neurotrophin-3; "1"3, tri-iodothyronine; "Irk C, tyrosine kinase receptor C. postnatal weeks seem to represent a period of particular sensitivity to thyroid hormones (Legrand, 1979). However, the mechanisms involved and the genes specifically regulated by T3 during cerebellar development are largely unknown (Samuels et al., 1988; Brent et al., 1991). Neurotrophin-3 (NT-3) is a recently discovered member of the NGF family (Ernfors et al., 1990; Holm et al., 1990; Jones and Reichardt, 1990; Kaisho et al., 1990; Maisonpierre et al., 1990a; Rosenthal et al., 1990) which in addition to NT-3 includes NGF, brain-derived neurotrophic factor (BDNF) (Leibroek et al., 1989), and neurotrophin-4/ neurotrophin-5 (Hallb66k et al., 1991; Berkemeier et al., 1991; Ip et al., 1992). NT-3 has been shown to support the survival and differentiation of various populations of peripheral, neural crest, and placode-derived, sensory neurons in vitro (Ernfors et al., 1990; Holm et al., 1990; Maisonpierre et al., 1990a). NT-3 is expressed in many different regions of the rat brain during embryonic development including the developing cerebellum (Maisonpierre et al., 1990b; Ernfors et al., 1992). The NT-3 expression is developmentally regulated, and in adult animals NT-3 mRNA expression is mainly confined to hiptx~ampus, cortex, and cerebellum (Holm et al., 1990; Maisonpierre et al., 1990a). The putative tyrosine kinase receptor (TrkC) for NT-3 has been shown to be expressed in the adult mouse brain (Larnballe et al., 1991) and recently also in the embryonic rat cerebellum (Ernfors et al., 1992). NT-3 at high concentrations also binds to trkB (Soppet et al., 1991; Squinto et al., 1991) which is the functional receptor for BDNF (Glass et al., 1991; Klein et al., 1991). However, using in situ hybridization, Ernfors et al. (1992) could not detect trkB in the em© The Rockefeller University Press, 0021-9525/93/07/443/8 $2.00 The Journal of Cell Biology, Volume 122, Number 2, July 1993 443-450 443 on O cber 8, 2017 jcb.rress. org D ow nladed fom