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Thyroid hormone deficiency may cause severe neurological disorders resulting from developmental deficits of the central nervous system. The mutant hyt/hyt mouse, characterized by fetal-onset, life-long hypothyroidism resulting from a point mutation of the thyroid-stimulating hormone receptor of the thyroid gland, displays a variety of abnormalities in motor behavior that are likely associated with dysfunctions of specific brain regions and a defective corticospinal tract (CST). To test the hypothesis that fetal and neonatal hypothyroidism cause abnormal CST development, the growth of the CST was investigated in hypothyroid hyt/hyt mice and their euthyroid progenitors, the BALB/cByJ mice. Anterograde labeling with biotinylated dextran amine demonstrated a decrease in the number of CST axons in the hyt/hyt mouse at the first lumbar level at postnatal day (P) 10. After retrograde tracing with fast blue (FB), fewer FB-labeled neurons were found in the motor cortex, the red nucleus, and the lateral vestibular nucleus of the hyt/hyt mouse. At the fourth lumbar level, the hyt/hyt mouse also showed smaller CST cross-sectional areas and significantly lower numbers of unmyelinated axons, myelinated axons, and growth cones within the CST during postnatal development. At P10, the hyt/hyt mouse demonstrated significantly lower immunoreactivity of embryonic neural cell adhesion molecule in the CST at the seventh cervical level, whereas the expression of growth-associated protein 43 remained unchanged. Our study demonstrated an abnormal development of the CST in the hyt/hyt mouse, manifested by reduced axon quantity and retarded growth pattern at the lumbar spinal cord.

To understand better the role of growth-promoting and -inhibiting molecules in the development of the corticospinal tract (CST), temporospatial expression of embryonic neural cell adhesion molecule (E-NCAM), growth-associated protein-43 (GAP-43), and chondroitin sulfate proteoglycan (CSPG) was studied in developing rats. Transverse sections of the seventh cervical (C7), seventh thoracic (T7), and fourth lumbar (L4) segments were examined at postnatal days (P) 2, 6, 10, 14, and 28. The highest E-NCAM immunoreactivity appeared at the C7 level on P2 and shifted caudally to the T7 on P6 and L4 on P10, which correlated closely with the time course of CST development. The peak expression of GAP-43 emerged at C7 on P2 and shifted to the T7 and L4 levels at a relatively lagging pace compared with that of E-NCAM. Conversely, a transient reduction in CSPG immunoreactivity was found within the CST at the C7 level on P2, T7 level on P6, and L4 level on P10, corresponding well with the arrival of CST-leading axons at these levels. Interestingly, higher levels of CSPG were found to surround the growing CST, suggesting a repulsive environment that channels the growth of CST. Moreover, a transition from immature to mature astrocytes in a rostrocaudal direction during CST development was evidenced by anti-vimentin and anti-glial fibrillary acidic protein (GFAP) immunostaining, suggesting a guidance role of immature astroglia in axonal outgrowth. Our study thus demonstrated dynamic changes of multiple growth-related molecules and astroglial environment that contribute to postnatal development of the CST.

Rett syndrome is caused by mutation in MECP2, a gene located on Xq28 and subject to X-inactivation. MECP2 encodes methyl CpG-binding protein 2, a widely expressed transcriptional repressor of methylated DNA. Mutations in MECP2 are primarily de novo events in the male germ line and thus lead to an excess of affected females. Here we report the identification of a unique 47,XXX girl with relatively mild atypical Rett syndrome leading initially to a diagnosis of infantile autism with regression. Mutation analysis of the MECP2 gene identified a de novo MECP2 mutation, L100V. Examination of a panel of X-linked microsatellite markers indicated that her supernumerary X chromosome is maternally derived. X-inactivation patterns were determined by analysis of methylation of the androgen receptor locus, and indicated preferential inactivation of her paternal allele. The parental origin of her MECP2 mutation could not be determined because she was uninformative for intronic polymorphisms flanking her mutation. This is the first reported case of sex chromosome trisomy and MECP2 mutation in a female, and it illustrates the importance of allele dosage on the severity of Rett syndrome phenotype.

The hyt/hyt mouse has an autosomal recessive, fetal onset, characterized by severe hypothyroidism that persists throughout life and is a reliable model of human sporadic congenital hypothyroidism. The hypothyroidism in the hyt/hyt mouse reflects the hyporesponsiveness of the thyroid gland to thyrotropin (TSH). This is attributable to a point mutation of C to T at nucleotide position 1666, resulting in the replacement of a Pro with Leu at position 556 in transmembrane domain IV of the G protein-linked TSH receptor. This mutation leads to a reduction in all cAMP-regulated events, including thyroid hormone synthesis. The diminution in T3/T4 in serum and other organs, including the brain, also leads to alterations in the level and timing of expression of critical brain molecules, i.e. selected tubulin isoforms (M beta 5, M beta 2, and M alpha 1), microtubule associated proteins (MAPs), and myelin basic protein, as well as to changes in important neuronal cytoskeletal events, i.e. microtubule assembly and SCa and SCb axonal transport. In the hyt/hyt mouse, fetal hypothyroidism leads to reductions in M beta 5, M beta 2, and M alpha 1 mRNAs, important tubulin isoforms, and M beta 5 and M beta 2 proteins, which comprise the microtubules. These molecules are localized to layer V pyramidal neurons in the sensorimotor cortex, a site of differentiating neurons, as well as a site for localization of specific thyroid hormone receptors. These molecular abnormalities in specific cells and at specific times of development or maturation may contribute to the observed neuroanatomical abnormalities, i.e. altered neuronal process growth and maintenance, synaptogenesis, and myelination, in hypothyroid brain. Abnormal neuroanatomical development in selected brain regions may be the factor underlying the abnormalities in reflexive, locomotor, and adaptive behavior seen in the hyt/hyt mouse and other hypothyroid animals.

Thyroid hormone deficiency in the developing brain leads to disorders of neuronal process growth. This is evidenced by reduced axonal and dendritic size and complexity (Garza et al.: Developmental Brain Research 43:287-297, 1988; Ruiz-Marcos: Iodine and the Brain. New York; Plenum Press, pp 91-102, 1989). These findings may be related to alterations in the neuronal cytoskeleton in hypothyroidism, such as reduced or abnormal microtubular number and density (Faivre et al.: Developmental Brain Research 8:21-30, 1983), and altered assembly, stabilization, and composition of microtubule protein in the hypothyroid brain. Neurofilaments also contribute to axonal caliber and process stability. Similar to microtubules, certain properties of neurofilaments are altered in developing hypothyroid axons (Marc and Rabie: In-ternational Journal of Developmental Neuroscience 3:353-358, 1985; Faivre et al.: Developmental Brain Research 8:21-30, 1983) that may affect axonal caliber and process stability. Normal process growth is predicated on formation of appropriate numbers of microtubules and on the normal synthesis and axonal transport of cytoskeletal components [tubulin, microtubule associated proteins (MAPs), and neurofilament proteins]. Hypothyroidism might alter the neuronal cytoskeleton and neuronal growth either by affecting the developmental programs for expression of specific isoforms of cytoskeletal proteins or by changing the delivery of cytoskeletal proteins via slow axonal transport, particularly slow component a (SCa). Previous studies had demonstrated changes in the amount of specific microtubule protein isoforms and mRNAs (Stein et al.: Iodine and the Brain. New York: Plenum Press, pp 59-78, 1989a). To further elucidate the molecular basis for process growth abnormalities in the hypothyroid brain, we investigated slow axonal transport in the mouse to determine the effects of thyroid hormone deficiency on the rate and composition of SCa. Comparisons of SCa in the optic nerve of hyt/hyt hypothyroid mouse and euthyroid hyt/+ littermates and euthyroid progenitor strain, BALB/cBY +/+ mice, indicated that the velocity of SCa was significantly reduced in hytlhyt optic nerve relative to hyt/ + and +/+. The axonal transport rate for tubulin, which is carried in SCa, was 0.118 mm/day in the hyt/hyt optic nerves. This rate was significantly different for the tubulin rates for the hyt/ + optic nerves (0.127 mm/day) and for the +/ + optic nerves (0.138 mm/day). Neurofilament proteins, as measured by the 140,000 daltons component, NFM, also appeared to be reduced in velocity in the hytlhyt versus the hyt/ + and +/+ optic nerves. The ratio of tubulin/neurofilament and tubulin/actin transported was also reduced in the hyt/hyt optic nerves, suggesting a selective dimunition in the amount of axonally transported tubulin. Such changes in transport rate of cytoskeletal proteins would be expected to affect axonal growth and plasticity. When considered with previously reported changes in specific expression of tubulin isotypes and MAPs (Nunez et al.: Iodine and the Brain. New York: Plenum Press, pp 103-112, 1989), changes in the properties and availability of neuronal cytoskeletal elements, particularly tubulin and microtubule proteins, represent a potential mechanism for disturbances of process growth in hypothyroidism.

The growth of corticospinal tract (CST) axons was studied quantitatively at the 7th cervical (C7) and the 4th lumbar (L4) spinal segments in the balb/cByJ mice at the ages of postnatal day (P) 0, 2, 4, 6, 8, 10, 14, and 28. The cross-sectional area of the CST increased progressively with time. Unmyelinated axons, the most prominent CST element during early development, reached maximum at C7 and L4 on P14. Two phases of increase in the number of unmyelinated axons were observed at C7, while only one surge of axonal outgrowth was found at the L4 level. Pro-myelinated axons, defined as axons surrounded by only one layer of oligodendrocytic process, were first seen at P2 and P4 in the C7 and the L4 level, respectively, followed by a dramatic increase in the number of myelinated axons from P14 onwards at both spinal levels. Myelination of the CST axons occurred topographically in a dorsal-to-ventral pattern. The number of growth cones increased rapidly at the C7 level to reach its maximum at P4, while those at L4 increased steadily to the peak at P10. Growth cones with synapse-like junctions were occasionally observed in the growing CST. Degenerating axons and growth cones partly accounted for the massive axon loss at both spinal segments during CST development. Overall, the mouse CST elements changed dynamically in numbers during postnatal development, suggesting a vigorous growing and pruning activity in the tract. The mouse CST also showed a similar growth pattern to that of the rat CST.

The corticospinal tract (CST) plays an important role in the control of voluntary movements. Although the development of the CST has been studied extensively in other species, limited information is available on its development in mice. In the present study, the growth of corticospinal axons was characterized in developing mice using Phaseolus vulgaris leucoagglutinin (PHA-L). Our results indicate that the leading CST axons reach the 8th cervical segment at postnatal day (PD) 2, the 7th thoracic segment at PD4, the 13th thoracic segment at PD7, and the 5th lumbar segment at PD9. The arrival of corticospinal axons at the distal lumbar cord at PD9 was further confirmed by retrograde tracing using fast blue (FB). A waiting period of 2-3 days exists after the leading CST axons pass a particular segment before sending collaterals into the gray matter of that segment. The CST continues to increase in size in lower thoracic and lumbar areas up to PD14 when its adult appearance is achieved. In this study, the date of animal's sacrifice was used as the specific postnatal date to demonstrate the growth of the CST. This definition gives a more reliable indication of the exact location of the CST at a specific developmental time point since the CST continues to grow after tracer injections and since the dye is transported much faster than axonal growth. We suggest that these findings can be used as a template for studies on both normal and transgenic mice where some developmental significance is given to the CST.

Culturing neural progenitor cells from the adult rodent brain has become routine1,2 and is also possible from human fetal tissue3,4, but expansion of these cells from postnatal and adult human tissue, although preferred for ethical reasons, has encountered problems5,6,7,8. Here we describe the isolation and successful propagation of neural progenitor cells from human postmortem tissues and surgical specimens. Although the relative therapeutic merits of adult and fetal progenitor cells still need to be assessed, our results may extend the application of these progenitor cells in the treatment of neurodegenerative diseases.

The hyt/hyt mouse has a severe and pervasive primary inherited hypothyroidism with significantly depressed serum T4, elevated serum and pituitary TSH, and reduced thyroid gland iodide uptake. Previous ultrastructural and histologic analysis of the hyt/hyt thyroid gland along with these biochemical abnormalities support an inherited defect in TSH responsiveness of the hyt/hyt thyroid gland. In order to evaluate the potential site of the defect in the hyt/hyt mouse, we have studied the hyt/hyt gland and hyt/hyt TSH from a biochemical and molecular standpoint. Based on demonstrated bioactivity of hyt/hyt serum in the McKenzie bioassay, this reduced responsiveness to TSH in the hyt/hyt mouse is not due to reduced bioactivity of hyt/hyt TSH or a major structural abnormality in the hyt/hyt TSH molecule. In comparison to hyt/ + euthyroid littermates and +/+ BALB/cBY progenitor strain mice, the hyt/hyt mouse demonstrates a twofold reduction in thyroid gland basal cAMP and a markedly diminished response of adenylyl cyclase to exogenous TSH. However, hyt/hyt cAMP production is equivalent to the euthyroid mice after stimulation of thyroid glands by forskolin, cholera toxin, PGE1, and isoproterenol. These results support a defect in the TSH-G protein-adenylyl cyclase system in the hyt/hyt thyroid gland. Specifically, these findings suggest that the hyt/hyt mouse has a defect in TSH responsivity due to an inherited defect in the thyroid gland TSH receptor molecule. Since the hyt/hyt gland makes T3 and T4 but at diminished levels, the proposed defect in the TSH receptor would still impart partial function. Both hyt/hyt and euthyroid hyt/ + littermates make TSH receptor mRNAs of 5500 and 2400 base pairs. This suggests that the receptor defect does not represent a major structural abnormality of the gene. The receptor defect could represent a reduction in receptor number, receptor-TSH affinity, or TSH receptor-G protein coupling. The specificity of this effect on adenylyl cyclase-cAMP is shown by the reduction of TSH-cAMP regulated thyroid peroxidase (TPO) and thyroglobulin mRNAs in the hyt/hyt thyroid gland. Given the importance of TPO and thyroglobulin in normal thyroid hormone synthesis, the reductions in TPO and thyroglobulin mRNAs in the hyt/hyt thyroid gland may underlie the significant decrease in thyroid hormone production by the hyt/hyt mouse.

The hyt/hyt mouse (BALB/cBY-hyt, C.hytRF) provides a useful model for exploring the effect of inherited severe primary hypothyroidism. Studies were undertaken to try to define the basis of the primary