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Hypothyroidsm alters the development of radial glial cells in the term fetal and postnatal neocortex of the rat
Developmental Brain Research 153 (2004) 109 – 114 www.elsevier.com/locate/devbrainres
Research report
Hypothyroidsm alters the development of radial glial cells in the term fetal and postnatal neocortex of the rat Juan Ramon Martinez-Galana,*, Francisco Escobar del Reyb, Gabriela Morreale de Escobarb, Maria Santacanac, Antonio Ruiz-Marcosc a
Facultad de Medicina and Centro Regional de Investigacio´n Biome´dica, Universidad de Castilla-La Mancha, Avenida de Almansa s/n, 02071 Albacete, Spain b Unidad de Neuroanatomia, Instituto Cajal, Doctor Arce 37, Madrid, Spain c Unidad de Endocrinologia Experimental, Instituto de Investigaciones Biome´dicas Alberto Sols. CSIC and UAM, Arturo Duperier 4, Madrid, Spain Accepted 5 August 2004 Available online 11 September 2004
Abstract Alterations of thyroid function during human development are known to produce extensive damage to the central nervous system including severe mental retardation. Using immunohistochemistry to identify the intermediate filament nestin, we have studied the possible influence of fetal and neonatal hypothyroidism on neocortical neuronal migration by arresting the normal development of the radial glial scaffold. By embryonic day 21 (E21), hypothyroid animals had a significant decrease in the number of nestin immunoreactive processes in the presumptive visual cortex. By postnatal day 5 (P5), hypothyroid animals showed a significant increase in the number of glial processes in relation with controls, although only in the upper layers of the visual cortex. Moreover, by P10, there was a marked increase in the number of radial glial processes in hypothyroid animals in superficial and deep zones of the visual cortex with respect to control animals. Our data indicate an important delay in the formation of the radial glial scaffold during the embryonic stage in hypothyroid animals that was interestingly accompanied by the later presence of abundant nestin immunoreactive fibers at P10. This impairment in the evolution of radial glia during development might be affecting the normal neuronal migratory pattern in the neocortex of hypothyroid rats. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Hormones and development Keywords: Radial glia; Methylmercaptoimidazole; Thyroid hormone; Nestin; Neocortex; Development
1. Introduction The mammalian neocortex is a highly ordered structure. Neurons are generated in an ordered progression, with cells of the deepest layers being born first, followed by cells of the middle and finally the upper layers. Each successively generated postmitotic neuron must bypass predecessors which had migrated along the same glial fiber, before ultimately settling at the outermost level of the cortical plate, just below the marginal zone [3,7,19,37,6]. In addition to * Corresponding author. Tel.: +34 967 59 92 00x2932; fax: +34 967 59 93 27. E-mail address: [email protected] (J.R. Martinez-Galan). 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2004.08.002
this inside-out gradient for the cortical plate neurons, cells in the ganglionic eminence are able to transgress the corticostriatal boundary and migrate tangentially into the developing neocortex [14,2,40,24]. Radial glial-guided migration is adopted predominantly by excitatory projecting pyramidal cells, whereas tangential migration is used by inhibitory non-pyramidal neurons (for review, see Refs. [33,29]). After neuronal migration, radial glial cells lack vimentin and nestin immunoreactivity, lose their pial or ependymal attachments and differentiate into adult glial cells [36,39,30]. Recently, it has been shown a novel role for radial glial cells, namely that these cells are also able to generate newborn neurons [28,34]. Multiple signals are known to maintain radial glia identity and to regulate radial glia interaction with
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postmitotic migratory neurons [4,5,16,17]. However, little is known about the interaction of hormones with radial glia cells and their possible influence on neuronal migration. In the present study, we have assessed the effect of thyroid hormones (TH) on the normal development of the radial glia scaffold in the rat neocortex. Most of the multiple biological effects of TH are mediated by the binding of 3, 3V,5-triiodothyronine (T3) to the nuclear receptors that share structural similarities with the steroid receptor superfamily (see Ref. [15] for a review). These receptors are encoded by two genes: alpha and beta c-erbA [38,42]. In the rat, these two genes produce three functional receptors: alpha-1, beta-1 and beta-2, and a protein (alpha-2) which is unable to bind T3 [41,22,25]. We are not aware of the data in the literature regarding the type of thyroid hormone receptor (TR) isoform, which is present in radial glial cells. However, in situ hybridization studies in the embryonic rat neocortex show TR-beta1 expression in the ventricular layer [8]. To elucidate whether TH elicit any effect on radial glial cells in the neocortex or not, we have employed embryonic and postnatal rats that were made hypothyroid by treating their mothers with methylmercaptoimidazole (MMI) in the drinking water. Our results show that radial glia scaffold is severely affected in the hypothyroid fetus near term (E21). As a consequence of the delay in the timing of development of radial glial cells, an abnormal increase in the number of radial glial fibers is detected from P5 to P10, a period during which neuronal migration has finished in the control neonates. This situation could disrupt the radialguided neuronal migration and contribute to the altered histogenesis found in hypothyroid animals.
2. Materials and methods 2.1. Animals Experimental procedures involving live animals were carried out in accordance with National and European Union regulations and guidelines for animal handling and welfare. Wistar rats were housed in temperature-controlled animal quarters, with automatic light and darkness cycles of 14 and 10 h, respectively. There were two groups of four dams each one. One of the groups received the standard laboratory diet and served as controls. The second group was fed on the standard laboratory diet, with 0.02% MMI in the drinking water. This MMI treatment started 10 days before mating, as their circulating TH were very low compared with the control group by this time. T4 was 4.5F1.3 ng/ml in MMI animals and 49.3F5.9 ng/ml in control rats. T3 was undetectable in MMI animals and 0.72F0.1 ng/ml in control rats. The day of pregnancy was assessed by vaginal smears and microscopic visualization of spermatozoa, and designated as day 0 of pregnancy (E0), when hysterectomy was performed on day 21 of gestation (E21). For postnatal brains, the day of birth was considered as postnatal day 0 (P0).
Pregnant mothers and P10 animals were perfused through the left ventricle with 0.1 M phosphate-buffered saline, pH 7.4 (PBS), their brains were dissected out and rapidly frozen on dry ice and stored at 70 8C until the day the tissues were extracted and purified for the RIAs. 2.2. Radioimmunoassay The degree of thyroid hormone deficiency attained by the animals exposed to the different experimental conditions was assessed measuring the levels of T4 and T3 in fetal brain by specific radioimmunoassays after extensive extraction and purification as detailed elsewhere [32]. 2.3. Immunohistochemistry Animals at E21, P5 and P10 were perfused through the left ventricle with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were dissected out, postfixed in fresh fixative at 4 8C for 4 h and soaked overnight in 30% sucrose for cryoprotection. Coronal sections, 50 Am thick, of the visual cortex were cut using a freezing microtome. Free floating sections were incubated first in PBS containing 3% hydrogen peroxide (H2O2) for 30 min, at room temperature. After washing three times in PBS for 5 min, sections were then incubated in normal horse serum for 30 min, at room temperature, followed by incubation in the primary antisera at full strength, a monoclonal antibody against the intermediate filament nestin, known as RAT-401 [21], commercially available from the Developmental Studies Hybridoma Bank (University of Iowa, IA). The sections were left for 36 h in a shaker at 4 8C. After washing three times in PBS for 5 min each, incubation with biotinylated goat anti-mouse (Vectastain, Vector) at 1:100 in PBS was carried out for 60 min at room temperature. After several washes in PBS, sections were incubated with the biotin–avidin complex (Vectastain, ABC-kit, Vector) in PBS for 60 min. After washing in PBS, the bound peroxidase was reacted with 0.05% 3,3V-diaminobenzidine as chromogen in PBS containing 0.03% H2O2. After further rinsing in PBS the sections were mounted on gelatin-coated slides and allowed to air-dry, were dehydrated and coverslipped. In all experiments control sections were processed without the primary antibody. No staining was found in control preparations. 2.4. Quantification of radial glial processes The density of radial glia was determined by counting the number of intersections of immunostained fibers with a horizontally oriented 100 Am wide bar. This bar was placed at the border between the marginal zone and the cortical plate (superficial cortex) or at the border between cortical plate and intermediate zone (deep cortex) at the E21 stage. In P5 and P10 rats, the bar was placed either at the border between layer I and layer II (superficial cortex) or at the
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border between layer V and VI (deep cortex). The location of the borders was determined with Nissl stained sections. For quantitative analysis, we studied 10 animals for MMI condition (four embryos at E21, three pups at P5 and three pups at P10) and 10 normal animals (four embryos at E21, three pups at P5 and three pups at P10). Pups were obtained from two different litters per age. 2.5. Statistical analysis of the data The differences between control and experimental data has been assessed by means of the two-way analysis of variance (ANOVA) age (E21, P5, P10)treatment (control and MMI) applied independently to the superficial glial and the deep glia processes. Both analyses were followed by the post-ANOVA analysis of Q in order to assess for the individual differences between C and MMI mean values at each age. The statistical significance among the mean values of body weight, thickness of neocortex and the levels of T3 and T4, corresponding to the control and the experimental condition, was assessed using a Student’s t-test.
3. Results 3.1. Body weight, thickness of neocortex and cerebral thyroid hormone level The reduction of the body weight in the MMI animals with respect to controls was significant in all the studied ages (Table 1). At E21, the body weight of the MMI embryos was 71% of that found in control embryos. At P5 and P10 pups, the reduction was 56.5% and 45.1%, respectively, of those found in control pups. The differences in the thickness of neocortex measured in the visual cortex between control and MMI groups (Table 1) were also significant in E21 and P5. At E21 and P5, the thickness of neocortex in MMI was 92% and 74%, respectively, from those found in control rats. At P10, the thickness of neocortex of MMI animals was 96% of that found in control pups. MMI caused a significant decrease in T3 and T4 at the prenatal and postnatal stages. Fig. 1 shows the concenTable 1 Mean (FS.E.M.) body weights and thickness of E21 embryos and P5 and P10 pups from dams fed with a goitrogen (MMI) or with standard diet (control) Age
Weight (g) Control
E21 P5 P10
5.03F0.12 11.66F0.44 19.40F0.58
Thickness of neocortex (Am) MMI a
3.62F0.13 6.36F0.44a 8.75F2.22a
Control
MMI
871.0F15.42 1180F29.88 1258F11.95
802.3F15.20a 878.6F16.64a 1215F32.08
a Differences between the control and MMI rats are statistically significant. Student’s t-test, Pb0.001.
Fig. 1. Mean valuesFS.E.M. of the concentrations of T3 and T4 in the brain of MMI rats (gray bars) at E21 and P10, as a percentage of the values for control rats (black bars). The concentrations of T3 and T4 in the brain of fetuses at E21 from control dams were 1242F46 and 1505F163 pg/g, respectively. The concentrations of T3 and T4 in the brain of control animals at P10 were 3216F163 and 2350F157 pg/g, respectively. a Difference significant versus control with Pb0.05; bDifference significant versus control with Pb0.01; cDifference significant versus C with Pb0.001.
trations in the brain of T3 and T4 expressed as a percentage of control values. At E21, the concentrations of T3 and T4 in MMI rats were 29% and 56%, respectively, from those found in control rats. At P10 the reduction of the concentrations of T3 and T4 was still highly significant with respect to the controls: The concentration of T3 in MMI rats was 25% of that found in controls. The concentration of T4 in MMI rats was 24% of that found in controls rats. 3.2. Radial glial fibers in control and MMI rats Fig. 2 shows the evolution of the nestin staining from E21 to P10 in the superficial visual cortex of control and MMI rats. Fig 3 shows the number of intersections of nestin immunostained fibers with a 100 Am wide bar horizontally oriented (see materials and methods). At E21, abundant fibers are labeled in the superficial cortex of control animals (arrows in Fig. 2A) as compared with the scarce labeling corresponding to the neocortex of MMI fetus (Fig. 2B). The arrow in Fig. 2B points to a nestin immunoreactive fiber in the marginal zone of a MMI fetus. At this age, the number of radial glia fibers in MMI animals is 49% of controls in the superficial cortex and 68% of controls in the deep cortex. At P5, there is not apparent difference in the amount of nestin inmunorreactive fibers between control and MMI animals (Fig. 2C and D). However, in Fig. 3 we can observe that in P5 the number of the fibers of MMI animals is 129% of controls in the superficial cortex. This difference was not noticed in the deep cortex. At P10, the scarce labeling corresponds to the control situation. The arrows in Fig. 2E point to two nestin immunoreactive fibers
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which is reflected by the non-significant value of F obtained corresponding to the effect of treatment ( F(1,114)=2.11). Nevertheless, the post-ANOVA test of Q performed to assess the differences between the pairs of control and MMI mean values corresponding to E21, P5 and P10 ages, has shown the existince of significant differences with Pb0.001 among the three pairs of considered values. The two-way ANOVA performed with the data shown in lower part of Fig. 3 (deep cortex) has shown that these values also show a significant interaction between age and treatment ( F(2,114)=25.94, Pb0.0005) and a significant value of F due to treatments. ( F(1,114)=11.28; Pb0.005). The post-ANOVA test of Q performed to assess the differences among the three pairs of control and MMI values corresponding to E21, P5 and P10 has given as a result that, while these values are significantly different at E21 ( Q=10.28, Pb0.001) and P10 ( Q=4.09, Pb0.05), control and MMI mean values are not significantly different at P5.
Fig. 2. Microphotographs of coronal sections of the rat visual cortex at E21, P5 and P10 showing radial glial fibers immunoreactive to nestin in the neocortical upper layers. A, C and E correspond to control rats; B, D and F to the MMI group. Arrows in A point to different radial glial fibers at E21. A low amount of fibers is present in the MMI group at this age. The arrow in B points to a nestin immunoreactive fiber in the marginal zone. At P5, C and D, no differences could be detected qualitatively between control and MMI groups. At P10, the neuronal migration in the neocortex has finished and the scarcity of fibers in the controls (arrows in E) is in contrast with the abnormally high number of nestin immunoreactive fibers in the MMI animals (arrows in F). The horizontal bar represents 50 Am.
found in layers I and II/III, respectively. By contrast, the number of fibers labeled in MMI animals is increased with respect to controls in the superficial and deep cortex. The arrows in Fig. 2F point to several fibers visible in MMI animals in layers II/III. The number of nestin immunoreactive fibers in MMI animals is 228% in the superficial cortex with respect to controls, and 156% in the deep cortex. The two-way ANOVA applied to the data shown in the upper part of Fig. 3 (superficial cortex) has shown the existence of a great interaction between age and treatment ( F(2,114)=47.01; Pb0.0001). As a consequence of this interaction, the overall mean values corresponding to control and MMI animals are very close, something
Fig. 3. Quantitative measures of nestin immunoreactive fibers show a delay in the evolution of the radial glia scaffold in the rat neocortex. Mean valuesFS.E.M. of nestin immunoreactive intersections with a horizontally oriented 100 Am wide bar at E21, P5 and P10 in the superficial and deep cortex are represented as black bars (control) and gray bars (MMI). a Difference significant versus control with Pb0.05; bdifference significant versus control with Pb0.001.
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4. Discussion The experimental model we have used in this study (MMI treatment starting 10 days before mating) produced in the dams and in their progeny the following effects: (1) The circulating levels of T3 and T4 in MMI dams with respect to controls the day of pregnancy were very low; (2) the body weight of the embryos and pups was severely affected by the MMI treatment; (3) the brain levels of T3 and T4 in the MMI rats as compared with controls were significant lower in all the studied ages. Although we do not know the values of circulating TH in embryos and pups, our data are indicating that MMI produce severe hypothyroidism and neurohistological alterations could be attributable to the deficit of TH in the brain of MMI animals. In this study, we show that the pattern of distribution of nestin immunoreactive fibers is severely altered in the embryonic and postnatal neocortex of MMI-treated animals. Before birth, at E21, the number of nestin immunoreactive fibers has decreased in hypothyroid animals. However, an increase in the number of radial glial fibers is observed in the hypothyroid animals with respect to controls during postnatal development, indicating a delay in the temporal development of the radial glia scaffold. We have previously shown that maternal hypothyroxinemia, caused by iodine deficiency, resulted in impaired maturation of the radial glial cells in the embryonic rat hippocampus [31]. Hypothyroidism and maternal hypothyroxinemia also affect the normal migratory pattern in auditory [27] and somatosensory cortex and in hippocampus [23]. Moreover, maternal hypothyroxinemia produces an abnormal pattern in the distribution of [3H]thymidine labeled neurons in the visual cortex after injections of the tracer on the embryonic days during which the major part of pyramidal layer V are born: E16 and E17 (J.R. MartinezGalan, G. Morreale de Escobar and A. Ruiz-Marcos, unpublished data). Present data do not allow us to clarify whether T3 could exert its regulatory action by controlling directly gene expression in radial glial cells or by an indirect effect through growth factors or extracellular matrix proteins that could act on radial glial cell differentiation. TR are expressed mainly in neurons and oligodendrocytes and with low levels in astrocytes [26,10,11]. Although the radial glial cell is a precursor of astrocytes, we cannot confirm that they share the same abundance and type of TR. Oligodendrocytes (see Ref. [9] for a review), and in some cases neurons [28,34], have their origin in radial glial cells. Another possibility to explain the action of TH on radial glial cells is that the effect might be mediated through signalling molecules released by neurons that would act modulating radial glia differentiation. A candidate for this role is the extracellular matrix protein reelin. This molecule is released by Cajal-Retzius cells in the layer 1 and it is crucial for laminar organization and the maintenance of the neocortical inside-out gradient
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[35,12,20,13]. Moreover, Reelin affects the biochemical maturation and radial glia morphology [18]. A possible link between radial glia, reelin and T3 is the fact that TH regulate reelin expression during brain development [1]. These authors showed that the number of reelin immunoreactive cells is decreased in the layer I of the hypothyroid neocortex at E18 and P0. This is an intense period of neuronal migration and these data are in conceptual agreement with our results that show a severe reduction in the number of radial glial fibers at E21. From P5 to P15, no significant differences were found between control and hypothyroid animals by Alvarez-Dolado et al. This could explain the postnatal recovery of the number of radial glia fibers in hypothyroid animals we found. Our results are consistent with the idea that TH are able to act on radial glial cells in the rat neocortex and promote their differentiation. This could be responsible, at least in part, for the altered histogenesis found in hypothyroid animals. Further experiments to elucidate the exact molecular mechanisms by which TH control neurogenesis and neuronal migration need to be addressed.
Acknowledgements This investigation has been supported by grants to Dr. A. Ruiz-Marcos from the bFondo para Investigaciones Sanitarias de la Seguridad SocialQ (Grant No. 93/0160) and from the bComisio´n Interministerial de Ciencia y TecnologiaQ (Grant No. PM95-0011).
References [1] M. Alvarez-Dolado, M. Ruiz, J.A. Del Rio, S. Alcantara, F. Burgaya, M. Sheldon, K. Nakajima, J. Bernal, B.W. Howell, T. Curran, E. Soriano, A. Munoz, Thyroid hormone regulates reelin and dab1 expression during brain development, J. Neurosci. 19 (1999) 6979 – 6993. [2] S.A. Anderson, D.D. Eisenstat, L. Shi, J.L. Rubenstein, Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes, Science 278 (1997) 474 – 476. [3] J.B. Angevine, R.L. Sidman, Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse, Nature 192 (1961) 766 – 768. [4] E.S. Anton, M.A. Marchionni, K.F. Lee, P. Rakic, Role of GGF/ neuregulin signaling in interactions between migrating neurons and radial glia in the developing cerebral cortex, Development 124 (1997) 3501 – 3510. [5] E.S. Anton, J.A. Kreidberg, P. Rakic, Distinct functions of alpha3 and alpha(v) integrin receptors in neuronal migration and laminar organization of the cerebral cortex, Neuron 22 (1999) 277 – 289. [6] S.A. Bayer, J. Altman, Neocortical Development, Raven Press, New York, 1991. [7] M. Berry, A.W. Rogers, The migration of neuroblasts in the developing cerebral cortex, J. Anat. 99 (1965) 691 – 709. [8] D.J. Bradley, H.C. Towle, W.S. Young, Spatial and temporal expression of alpha- and beta-thyroid hormone receptor mRNAs, including the beta 2-subtype, in the developing mammalian nervous system, J. Neurosci. 12 (1992) 2288 – 2302.
114
J.R. Martinez-Galan et al. / Developmental Brain Research 153 (2004) 109–114
[9] R.S. Cameron, P. Rakic, Glial cell lineage in the cerebral cortex: a review and synthesis, Glia 4 (1991) 124 – 137. [10] D.J. Carlson, K.A. Strait, H.L. Schwartz, J.H. Oppenheimer, Immunofluorescent localization of thyroid hormone receptor isoforms in glial cells of rat brain, Endocrinology 135 (1994) 1831 – 1836. [11] D.J. Carlson, K.A. Strait, H.L. Schwartz, J.H. Oppenheimer, Thyroid hormone receptor isoform content in cultured type 1 and type 2 astrocytes, Endocrinology 137 (1996) 911 – 917. [12] G. D’Arcangelo, G.G. Miao, S.C. Chen, H.D. Soares, J.I. Morgan, T. Curran, A protein related to extracellular matrix proteins deleted in the mouse mutant reeler, Nature 374 (1995) 719 – 723. [13] G. D’Arcangelo, K. Nakajima, T. Miyata, M. Ogawa, K. Mikoshiba, T. Curran, Reelin is a secreted glycoprotein recognized by the cr-50 monoclonal antibody, J. Neurosci. 17 (1997) 23 – 31. [14] J.A. De Carlos, L. Lopez-Mascaraque, F. Valverde, Dynamics of cell migration from the lateral ganglionic eminence in the rat, J. Neurosci. 16 (1996) 6146 – 6156. [15] R.M. Evans, The steroid and thyroid hormone receptor superfamily, Science 240 (1988) 889 – 895. [16] E. Forster, A. Tielsch, B. Saum, K.H. Weiss, C. Johanssen, D. Graus-Porta, U. Muller, M. Frotscher, Reelin, Disabled 1, and beta 1 integrins are required for the formation of the radial glial scaffold in the hippocampus, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 13178 – 13183. [17] W. Halfter, S. Dong, Y.P. Yip, M. Willem, U. Mayer, A critical function of the pial basement membrane in cortical histogenesis, J. Neurosci. 22 (2002) 6029 – 6040. [18] E. Hartfuss, E. Forster, H.H. Bock, M.A. Hack, P. Leprince, J.M. Luque, J. Herz, M. Frotscher, M. Gotz, Reelin signaling directly affects radial glia morphology and biochemical maturation, Development 130 (2003) 4597 – 4609. [19] S.P. Hicks, C.J. D’Amato, Cell migrations to the isocortex in the rat, Anat. Rec. 160 (1968) 619 – 634. [20] S. Hirotsune, T. Takahara, N. Sasaki, K. Hirose, A. Yoshiki, T. Ohashi, M. Kusakabe, Y. Murakami, M. Muramatsu, S. Watanabe, The reeler gene encodes a protein with an EGF-like motif expressed by pioneer neurons, Nat. Genet. 10 (1995) 77 – 83. [21] S. Hockfield, R.D. McKay, Identification of major cell classes in the developing mammalian nervous system, J. Neurosci. 5 (1985) 3310 – 3328. [22] S. Izumo, V. Madavi, Thyroid hormone receptor alpha isoforms generated by alternative splicing differentially activate myosin HC gen transcription, Nature 334 (1988) 539 – 542. [23] R. Lavado-Autric, E. Auso, J.V. Garcia-Velasco, C. Arufe Mdel, F. Escobar del Rey, P. Berbel, G. Morreale de Escobar, Early maternal hypothyroxinemia alters histogenesis and cerebral cortex cytoarchitecture of the progeny, J. Clin. Invest. 111 (2003) 1073 – 1082. [24] A.A. Lavdas, M. Grigoriou, V. Pachnis, J.G. Parnavelas, The medial ganglionic eminence gives rise to a population of early neurons in the developing cerebral cortex, J. Neurosci. 19 (1999) 7881 – 7888. [25] M.A. Lazar, R.A. Hodin, D.S. Darling, W. Chin, Identification of a rat c-erbA alpha-related protein which binds deoxyribonucleic acid but does not bind thyroid hormone, Mol. Endocrinol. 2 (1988) 893 – 901.
[26] J.L. Leonard, A.P. Farwell, P.M. Yen, W.W. Chin, M. Stula, Differential expression of thyroid hormone receptor isoforms in neurons and astroglial cells, Endocrinology 135 (1994) 548 – 555. [27] R.A. Lucio, J.V. Garcia, J.R. Cerezo, P. Pacheco, G.M. Innocenti, P. Berbel, The development of auditory callosal connections in normal and hypothyroid rats, Cereb. Cortex 7 (1997) 303 – 316. [28] P. Malatesta, E. Hartfuss, M. Gotz, Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage, Development 127 (2000) 5253 – 5263. [29] O. Marin, J.L. Rubenstein, Cell migration in the forebrain, Annu. Rev. Neurosci. 26 (2003) 441 – 483. [30] M. Marin-Padilla, Prenatal development of fibrous (white matter), protoplasmic (gray matter), and layer I astrocytes in the human cerebral cortex: a Golgi study, J. Comp. Neurol. 357 (1995) 554 – 572. [31] J.R. Martı´nez-Gala´n, P. Pedraza, M. Santacana, F. Escobar del Rey, G. Morreale de Escobar y, A. Ruiz-Marcos, Early effects of iodine deficiency on radial glial cells of the hippocampus of the rat fetus: a model of neurological cretinism, J. Clin. Invest. 99 (1997) 2701 – 2709. [32] G. Morreale de Escobar, R. Pastor, M.J. Obrego´n, F. Escobar del Rey, Effects of maternal hypothyroidism on the weight and thyroid hormone content of rat embryonic tissues, Endocrinology 117 (1985) 1890 – 1896. [33] B. Nadarajah, J.G. Parnavelas, Modes of neuronal migration in the developing cerebral cortex, Nat. Rev., Neurosci. 3 (2002) 423 – 432. [34] S.C. Noctor, A.C. Flint, T.A. Weissman, R.S. Dammerman, A.R. Kriegstein, Neurons derived from radial glial cells establish radial units in neocortex, Nature 409 (2001) 714 – 720. [35] M. Ogawa, T. Miyata, K. Nakajima, K. Yagyu, M. Seike, K. Ikenaka, H. Yamamoto, K. Mikoshiba, The reeler gene-associated antigen on Cajal-Retzius neurons is a crucial molecule for laminar organization of cortical neurons, Neuron 14 (1995) 899 – 912. [36] S.K. Pixley, J. de Vellis, Transition between immature radial glia and mature astrocytes studied with a monoclonal antibody to vimentin, Brain Res. 317 (1984) 201 – 209. [37] P. Rakic, Mode of cell migration to the superficial layers of fetal monkey neocortex, J. Comp. Neurol. 145 (1972) 61 – 84. [38] J. Sap, A. Munoz, K. Damm, Y. Goldberg, J. Ghysdael, A. Leutz, H. Beug, B. Vennstrom, The c-erb-A protein is a high-affinity receptor for thyroid hormone, Nature (1986) 635 – 640. [39] C.C. Stichel, C.M. Muller, K. Zilles, Distribution of glial fibrillary acidic protein and vimentin immunoreactivity during rat visual cortex development, J. Neurocytol. 20 (1991) 97 – 108. [40] N. Tamamaki, K.E. Fujimori, R. Takauji, Origin and route of tangentially migrating neurons in the developing neocortical intermediate zone, J. Neurosci. 17 (1997) 8313 – 8323. [41] C.C. Thompson, C. Weinberger, R. Lebo, R.M. Evans, Identification of a novel thyroid hormone receptor expressed in the mammalian central nervous system, Science 237 (1987) 1610 – 1614. [42] C. Weinberger, C.C. Thompson, E.S. Ong, R. Lebo, D.J. Grujold, R.M Evans, The c-erb-A gene encodes a thyroid hormone receptor, Nature 324 (1986) 641 – 646.
Research report
Hypothyroidsm alters the development of radial glial cells in the term fetal and postnatal neocortex of the rat Juan Ramon Martinez-Galana,*, Francisco Escobar del Reyb, Gabriela Morreale de Escobarb, Maria Santacanac, Antonio Ruiz-Marcosc a
Facultad de Medicina and Centro Regional de Investigacio´n Biome´dica, Universidad de Castilla-La Mancha, Avenida de Almansa s/n, 02071 Albacete, Spain b Unidad de Neuroanatomia, Instituto Cajal, Doctor Arce 37, Madrid, Spain c Unidad de Endocrinologia Experimental, Instituto de Investigaciones Biome´dicas Alberto Sols. CSIC and UAM, Arturo Duperier 4, Madrid, Spain Accepted 5 August 2004 Available online 11 September 2004
Abstract Alterations of thyroid function during human development are known to produce extensive damage to the central nervous system including severe mental retardation. Using immunohistochemistry to identify the intermediate filament nestin, we have studied the possible influence of fetal and neonatal hypothyroidism on neocortical neuronal migration by arresting the normal development of the radial glial scaffold. By embryonic day 21 (E21), hypothyroid animals had a significant decrease in the number of nestin immunoreactive processes in the presumptive visual cortex. By postnatal day 5 (P5), hypothyroid animals showed a significant increase in the number of glial processes in relation with controls, although only in the upper layers of the visual cortex. Moreover, by P10, there was a marked increase in the number of radial glial processes in hypothyroid animals in superficial and deep zones of the visual cortex with respect to control animals. Our data indicate an important delay in the formation of the radial glial scaffold during the embryonic stage in hypothyroid animals that was interestingly accompanied by the later presence of abundant nestin immunoreactive fibers at P10. This impairment in the evolution of radial glia during development might be affecting the normal neuronal migratory pattern in the neocortex of hypothyroid rats. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Hormones and development Keywords: Radial glia; Methylmercaptoimidazole; Thyroid hormone; Nestin; Neocortex; Development
1. Introduction The mammalian neocortex is a highly ordered structure. Neurons are generated in an ordered progression, with cells of the deepest layers being born first, followed by cells of the middle and finally the upper layers. Each successively generated postmitotic neuron must bypass predecessors which had migrated along the same glial fiber, before ultimately settling at the outermost level of the cortical plate, just below the marginal zone [3,7,19,37,6]. In addition to * Corresponding author. Tel.: +34 967 59 92 00x2932; fax: +34 967 59 93 27. E-mail address: [email protected] (J.R. Martinez-Galan). 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2004.08.002
this inside-out gradient for the cortical plate neurons, cells in the ganglionic eminence are able to transgress the corticostriatal boundary and migrate tangentially into the developing neocortex [14,2,40,24]. Radial glial-guided migration is adopted predominantly by excitatory projecting pyramidal cells, whereas tangential migration is used by inhibitory non-pyramidal neurons (for review, see Refs. [33,29]). After neuronal migration, radial glial cells lack vimentin and nestin immunoreactivity, lose their pial or ependymal attachments and differentiate into adult glial cells [36,39,30]. Recently, it has been shown a novel role for radial glial cells, namely that these cells are also able to generate newborn neurons [28,34]. Multiple signals are known to maintain radial glia identity and to regulate radial glia interaction with
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postmitotic migratory neurons [4,5,16,17]. However, little is known about the interaction of hormones with radial glia cells and their possible influence on neuronal migration. In the present study, we have assessed the effect of thyroid hormones (TH) on the normal development of the radial glia scaffold in the rat neocortex. Most of the multiple biological effects of TH are mediated by the binding of 3, 3V,5-triiodothyronine (T3) to the nuclear receptors that share structural similarities with the steroid receptor superfamily (see Ref. [15] for a review). These receptors are encoded by two genes: alpha and beta c-erbA [38,42]. In the rat, these two genes produce three functional receptors: alpha-1, beta-1 and beta-2, and a protein (alpha-2) which is unable to bind T3 [41,22,25]. We are not aware of the data in the literature regarding the type of thyroid hormone receptor (TR) isoform, which is present in radial glial cells. However, in situ hybridization studies in the embryonic rat neocortex show TR-beta1 expression in the ventricular layer [8]. To elucidate whether TH elicit any effect on radial glial cells in the neocortex or not, we have employed embryonic and postnatal rats that were made hypothyroid by treating their mothers with methylmercaptoimidazole (MMI) in the drinking water. Our results show that radial glia scaffold is severely affected in the hypothyroid fetus near term (E21). As a consequence of the delay in the timing of development of radial glial cells, an abnormal increase in the number of radial glial fibers is detected from P5 to P10, a period during which neuronal migration has finished in the control neonates. This situation could disrupt the radialguided neuronal migration and contribute to the altered histogenesis found in hypothyroid animals.
2. Materials and methods 2.1. Animals Experimental procedures involving live animals were carried out in accordance with National and European Union regulations and guidelines for animal handling and welfare. Wistar rats were housed in temperature-controlled animal quarters, with automatic light and darkness cycles of 14 and 10 h, respectively. There were two groups of four dams each one. One of the groups received the standard laboratory diet and served as controls. The second group was fed on the standard laboratory diet, with 0.02% MMI in the drinking water. This MMI treatment started 10 days before mating, as their circulating TH were very low compared with the control group by this time. T4 was 4.5F1.3 ng/ml in MMI animals and 49.3F5.9 ng/ml in control rats. T3 was undetectable in MMI animals and 0.72F0.1 ng/ml in control rats. The day of pregnancy was assessed by vaginal smears and microscopic visualization of spermatozoa, and designated as day 0 of pregnancy (E0), when hysterectomy was performed on day 21 of gestation (E21). For postnatal brains, the day of birth was considered as postnatal day 0 (P0).
Pregnant mothers and P10 animals were perfused through the left ventricle with 0.1 M phosphate-buffered saline, pH 7.4 (PBS), their brains were dissected out and rapidly frozen on dry ice and stored at 70 8C until the day the tissues were extracted and purified for the RIAs. 2.2. Radioimmunoassay The degree of thyroid hormone deficiency attained by the animals exposed to the different experimental conditions was assessed measuring the levels of T4 and T3 in fetal brain by specific radioimmunoassays after extensive extraction and purification as detailed elsewhere [32]. 2.3. Immunohistochemistry Animals at E21, P5 and P10 were perfused through the left ventricle with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were dissected out, postfixed in fresh fixative at 4 8C for 4 h and soaked overnight in 30% sucrose for cryoprotection. Coronal sections, 50 Am thick, of the visual cortex were cut using a freezing microtome. Free floating sections were incubated first in PBS containing 3% hydrogen peroxide (H2O2) for 30 min, at room temperature. After washing three times in PBS for 5 min, sections were then incubated in normal horse serum for 30 min, at room temperature, followed by incubation in the primary antisera at full strength, a monoclonal antibody against the intermediate filament nestin, known as RAT-401 [21], commercially available from the Developmental Studies Hybridoma Bank (University of Iowa, IA). The sections were left for 36 h in a shaker at 4 8C. After washing three times in PBS for 5 min each, incubation with biotinylated goat anti-mouse (Vectastain, Vector) at 1:100 in PBS was carried out for 60 min at room temperature. After several washes in PBS, sections were incubated with the biotin–avidin complex (Vectastain, ABC-kit, Vector) in PBS for 60 min. After washing in PBS, the bound peroxidase was reacted with 0.05% 3,3V-diaminobenzidine as chromogen in PBS containing 0.03% H2O2. After further rinsing in PBS the sections were mounted on gelatin-coated slides and allowed to air-dry, were dehydrated and coverslipped. In all experiments control sections were processed without the primary antibody. No staining was found in control preparations. 2.4. Quantification of radial glial processes The density of radial glia was determined by counting the number of intersections of immunostained fibers with a horizontally oriented 100 Am wide bar. This bar was placed at the border between the marginal zone and the cortical plate (superficial cortex) or at the border between cortical plate and intermediate zone (deep cortex) at the E21 stage. In P5 and P10 rats, the bar was placed either at the border between layer I and layer II (superficial cortex) or at the
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border between layer V and VI (deep cortex). The location of the borders was determined with Nissl stained sections. For quantitative analysis, we studied 10 animals for MMI condition (four embryos at E21, three pups at P5 and three pups at P10) and 10 normal animals (four embryos at E21, three pups at P5 and three pups at P10). Pups were obtained from two different litters per age. 2.5. Statistical analysis of the data The differences between control and experimental data has been assessed by means of the two-way analysis of variance (ANOVA) age (E21, P5, P10)treatment (control and MMI) applied independently to the superficial glial and the deep glia processes. Both analyses were followed by the post-ANOVA analysis of Q in order to assess for the individual differences between C and MMI mean values at each age. The statistical significance among the mean values of body weight, thickness of neocortex and the levels of T3 and T4, corresponding to the control and the experimental condition, was assessed using a Student’s t-test.
3. Results 3.1. Body weight, thickness of neocortex and cerebral thyroid hormone level The reduction of the body weight in the MMI animals with respect to controls was significant in all the studied ages (Table 1). At E21, the body weight of the MMI embryos was 71% of that found in control embryos. At P5 and P10 pups, the reduction was 56.5% and 45.1%, respectively, of those found in control pups. The differences in the thickness of neocortex measured in the visual cortex between control and MMI groups (Table 1) were also significant in E21 and P5. At E21 and P5, the thickness of neocortex in MMI was 92% and 74%, respectively, from those found in control rats. At P10, the thickness of neocortex of MMI animals was 96% of that found in control pups. MMI caused a significant decrease in T3 and T4 at the prenatal and postnatal stages. Fig. 1 shows the concenTable 1 Mean (FS.E.M.) body weights and thickness of E21 embryos and P5 and P10 pups from dams fed with a goitrogen (MMI) or with standard diet (control) Age
Weight (g) Control
E21 P5 P10
5.03F0.12 11.66F0.44 19.40F0.58
Thickness of neocortex (Am) MMI a
3.62F0.13 6.36F0.44a 8.75F2.22a
Control
MMI
871.0F15.42 1180F29.88 1258F11.95
802.3F15.20a 878.6F16.64a 1215F32.08
a Differences between the control and MMI rats are statistically significant. Student’s t-test, Pb0.001.
Fig. 1. Mean valuesFS.E.M. of the concentrations of T3 and T4 in the brain of MMI rats (gray bars) at E21 and P10, as a percentage of the values for control rats (black bars). The concentrations of T3 and T4 in the brain of fetuses at E21 from control dams were 1242F46 and 1505F163 pg/g, respectively. The concentrations of T3 and T4 in the brain of control animals at P10 were 3216F163 and 2350F157 pg/g, respectively. a Difference significant versus control with Pb0.05; bDifference significant versus control with Pb0.01; cDifference significant versus C with Pb0.001.
trations in the brain of T3 and T4 expressed as a percentage of control values. At E21, the concentrations of T3 and T4 in MMI rats were 29% and 56%, respectively, from those found in control rats. At P10 the reduction of the concentrations of T3 and T4 was still highly significant with respect to the controls: The concentration of T3 in MMI rats was 25% of that found in controls. The concentration of T4 in MMI rats was 24% of that found in controls rats. 3.2. Radial glial fibers in control and MMI rats Fig. 2 shows the evolution of the nestin staining from E21 to P10 in the superficial visual cortex of control and MMI rats. Fig 3 shows the number of intersections of nestin immunostained fibers with a 100 Am wide bar horizontally oriented (see materials and methods). At E21, abundant fibers are labeled in the superficial cortex of control animals (arrows in Fig. 2A) as compared with the scarce labeling corresponding to the neocortex of MMI fetus (Fig. 2B). The arrow in Fig. 2B points to a nestin immunoreactive fiber in the marginal zone of a MMI fetus. At this age, the number of radial glia fibers in MMI animals is 49% of controls in the superficial cortex and 68% of controls in the deep cortex. At P5, there is not apparent difference in the amount of nestin inmunorreactive fibers between control and MMI animals (Fig. 2C and D). However, in Fig. 3 we can observe that in P5 the number of the fibers of MMI animals is 129% of controls in the superficial cortex. This difference was not noticed in the deep cortex. At P10, the scarce labeling corresponds to the control situation. The arrows in Fig. 2E point to two nestin immunoreactive fibers
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which is reflected by the non-significant value of F obtained corresponding to the effect of treatment ( F(1,114)=2.11). Nevertheless, the post-ANOVA test of Q performed to assess the differences between the pairs of control and MMI mean values corresponding to E21, P5 and P10 ages, has shown the existince of significant differences with Pb0.001 among the three pairs of considered values. The two-way ANOVA performed with the data shown in lower part of Fig. 3 (deep cortex) has shown that these values also show a significant interaction between age and treatment ( F(2,114)=25.94, Pb0.0005) and a significant value of F due to treatments. ( F(1,114)=11.28; Pb0.005). The post-ANOVA test of Q performed to assess the differences among the three pairs of control and MMI values corresponding to E21, P5 and P10 has given as a result that, while these values are significantly different at E21 ( Q=10.28, Pb0.001) and P10 ( Q=4.09, Pb0.05), control and MMI mean values are not significantly different at P5.
Fig. 2. Microphotographs of coronal sections of the rat visual cortex at E21, P5 and P10 showing radial glial fibers immunoreactive to nestin in the neocortical upper layers. A, C and E correspond to control rats; B, D and F to the MMI group. Arrows in A point to different radial glial fibers at E21. A low amount of fibers is present in the MMI group at this age. The arrow in B points to a nestin immunoreactive fiber in the marginal zone. At P5, C and D, no differences could be detected qualitatively between control and MMI groups. At P10, the neuronal migration in the neocortex has finished and the scarcity of fibers in the controls (arrows in E) is in contrast with the abnormally high number of nestin immunoreactive fibers in the MMI animals (arrows in F). The horizontal bar represents 50 Am.
found in layers I and II/III, respectively. By contrast, the number of fibers labeled in MMI animals is increased with respect to controls in the superficial and deep cortex. The arrows in Fig. 2F point to several fibers visible in MMI animals in layers II/III. The number of nestin immunoreactive fibers in MMI animals is 228% in the superficial cortex with respect to controls, and 156% in the deep cortex. The two-way ANOVA applied to the data shown in the upper part of Fig. 3 (superficial cortex) has shown the existence of a great interaction between age and treatment ( F(2,114)=47.01; Pb0.0001). As a consequence of this interaction, the overall mean values corresponding to control and MMI animals are very close, something
Fig. 3. Quantitative measures of nestin immunoreactive fibers show a delay in the evolution of the radial glia scaffold in the rat neocortex. Mean valuesFS.E.M. of nestin immunoreactive intersections with a horizontally oriented 100 Am wide bar at E21, P5 and P10 in the superficial and deep cortex are represented as black bars (control) and gray bars (MMI). a Difference significant versus control with Pb0.05; bdifference significant versus control with Pb0.001.
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4. Discussion The experimental model we have used in this study (MMI treatment starting 10 days before mating) produced in the dams and in their progeny the following effects: (1) The circulating levels of T3 and T4 in MMI dams with respect to controls the day of pregnancy were very low; (2) the body weight of the embryos and pups was severely affected by the MMI treatment; (3) the brain levels of T3 and T4 in the MMI rats as compared with controls were significant lower in all the studied ages. Although we do not know the values of circulating TH in embryos and pups, our data are indicating that MMI produce severe hypothyroidism and neurohistological alterations could be attributable to the deficit of TH in the brain of MMI animals. In this study, we show that the pattern of distribution of nestin immunoreactive fibers is severely altered in the embryonic and postnatal neocortex of MMI-treated animals. Before birth, at E21, the number of nestin immunoreactive fibers has decreased in hypothyroid animals. However, an increase in the number of radial glial fibers is observed in the hypothyroid animals with respect to controls during postnatal development, indicating a delay in the temporal development of the radial glia scaffold. We have previously shown that maternal hypothyroxinemia, caused by iodine deficiency, resulted in impaired maturation of the radial glial cells in the embryonic rat hippocampus [31]. Hypothyroidism and maternal hypothyroxinemia also affect the normal migratory pattern in auditory [27] and somatosensory cortex and in hippocampus [23]. Moreover, maternal hypothyroxinemia produces an abnormal pattern in the distribution of [3H]thymidine labeled neurons in the visual cortex after injections of the tracer on the embryonic days during which the major part of pyramidal layer V are born: E16 and E17 (J.R. MartinezGalan, G. Morreale de Escobar and A. Ruiz-Marcos, unpublished data). Present data do not allow us to clarify whether T3 could exert its regulatory action by controlling directly gene expression in radial glial cells or by an indirect effect through growth factors or extracellular matrix proteins that could act on radial glial cell differentiation. TR are expressed mainly in neurons and oligodendrocytes and with low levels in astrocytes [26,10,11]. Although the radial glial cell is a precursor of astrocytes, we cannot confirm that they share the same abundance and type of TR. Oligodendrocytes (see Ref. [9] for a review), and in some cases neurons [28,34], have their origin in radial glial cells. Another possibility to explain the action of TH on radial glial cells is that the effect might be mediated through signalling molecules released by neurons that would act modulating radial glia differentiation. A candidate for this role is the extracellular matrix protein reelin. This molecule is released by Cajal-Retzius cells in the layer 1 and it is crucial for laminar organization and the maintenance of the neocortical inside-out gradient
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[35,12,20,13]. Moreover, Reelin affects the biochemical maturation and radial glia morphology [18]. A possible link between radial glia, reelin and T3 is the fact that TH regulate reelin expression during brain development [1]. These authors showed that the number of reelin immunoreactive cells is decreased in the layer I of the hypothyroid neocortex at E18 and P0. This is an intense period of neuronal migration and these data are in conceptual agreement with our results that show a severe reduction in the number of radial glial fibers at E21. From P5 to P15, no significant differences were found between control and hypothyroid animals by Alvarez-Dolado et al. This could explain the postnatal recovery of the number of radial glia fibers in hypothyroid animals we found. Our results are consistent with the idea that TH are able to act on radial glial cells in the rat neocortex and promote their differentiation. This could be responsible, at least in part, for the altered histogenesis found in hypothyroid animals. Further experiments to elucidate the exact molecular mechanisms by which TH control neurogenesis and neuronal migration need to be addressed.
Acknowledgements This investigation has been supported by grants to Dr. A. Ruiz-Marcos from the bFondo para Investigaciones Sanitarias de la Seguridad SocialQ (Grant No. 93/0160) and from the bComisio´n Interministerial de Ciencia y TecnologiaQ (Grant No. PM95-0011).
References [1] M. Alvarez-Dolado, M. Ruiz, J.A. Del Rio, S. Alcantara, F. Burgaya, M. Sheldon, K. Nakajima, J. Bernal, B.W. Howell, T. Curran, E. Soriano, A. Munoz, Thyroid hormone regulates reelin and dab1 expression during brain development, J. Neurosci. 19 (1999) 6979 – 6993. [2] S.A. Anderson, D.D. Eisenstat, L. Shi, J.L. Rubenstein, Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes, Science 278 (1997) 474 – 476. [3] J.B. Angevine, R.L. Sidman, Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse, Nature 192 (1961) 766 – 768. [4] E.S. Anton, M.A. Marchionni, K.F. Lee, P. Rakic, Role of GGF/ neuregulin signaling in interactions between migrating neurons and radial glia in the developing cerebral cortex, Development 124 (1997) 3501 – 3510. [5] E.S. Anton, J.A. Kreidberg, P. Rakic, Distinct functions of alpha3 and alpha(v) integrin receptors in neuronal migration and laminar organization of the cerebral cortex, Neuron 22 (1999) 277 – 289. [6] S.A. Bayer, J. Altman, Neocortical Development, Raven Press, New York, 1991. [7] M. Berry, A.W. Rogers, The migration of neuroblasts in the developing cerebral cortex, J. Anat. 99 (1965) 691 – 709. [8] D.J. Bradley, H.C. Towle, W.S. Young, Spatial and temporal expression of alpha- and beta-thyroid hormone receptor mRNAs, including the beta 2-subtype, in the developing mammalian nervous system, J. Neurosci. 12 (1992) 2288 – 2302.
114
J.R. Martinez-Galan et al. / Developmental Brain Research 153 (2004) 109–114
[9] R.S. Cameron, P. Rakic, Glial cell lineage in the cerebral cortex: a review and synthesis, Glia 4 (1991) 124 – 137. [10] D.J. Carlson, K.A. Strait, H.L. Schwartz, J.H. Oppenheimer, Immunofluorescent localization of thyroid hormone receptor isoforms in glial cells of rat brain, Endocrinology 135 (1994) 1831 – 1836. [11] D.J. Carlson, K.A. Strait, H.L. Schwartz, J.H. Oppenheimer, Thyroid hormone receptor isoform content in cultured type 1 and type 2 astrocytes, Endocrinology 137 (1996) 911 – 917. [12] G. D’Arcangelo, G.G. Miao, S.C. Chen, H.D. Soares, J.I. Morgan, T. Curran, A protein related to extracellular matrix proteins deleted in the mouse mutant reeler, Nature 374 (1995) 719 – 723. [13] G. D’Arcangelo, K. Nakajima, T. Miyata, M. Ogawa, K. Mikoshiba, T. Curran, Reelin is a secreted glycoprotein recognized by the cr-50 monoclonal antibody, J. Neurosci. 17 (1997) 23 – 31. [14] J.A. De Carlos, L. Lopez-Mascaraque, F. Valverde, Dynamics of cell migration from the lateral ganglionic eminence in the rat, J. Neurosci. 16 (1996) 6146 – 6156. [15] R.M. Evans, The steroid and thyroid hormone receptor superfamily, Science 240 (1988) 889 – 895. [16] E. Forster, A. Tielsch, B. Saum, K.H. Weiss, C. Johanssen, D. Graus-Porta, U. Muller, M. Frotscher, Reelin, Disabled 1, and beta 1 integrins are required for the formation of the radial glial scaffold in the hippocampus, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 13178 – 13183. [17] W. Halfter, S. Dong, Y.P. Yip, M. Willem, U. Mayer, A critical function of the pial basement membrane in cortical histogenesis, J. Neurosci. 22 (2002) 6029 – 6040. [18] E. Hartfuss, E. Forster, H.H. Bock, M.A. Hack, P. Leprince, J.M. Luque, J. Herz, M. Frotscher, M. Gotz, Reelin signaling directly affects radial glia morphology and biochemical maturation, Development 130 (2003) 4597 – 4609. [19] S.P. Hicks, C.J. D’Amato, Cell migrations to the isocortex in the rat, Anat. Rec. 160 (1968) 619 – 634. [20] S. Hirotsune, T. Takahara, N. Sasaki, K. Hirose, A. Yoshiki, T. Ohashi, M. Kusakabe, Y. Murakami, M. Muramatsu, S. Watanabe, The reeler gene encodes a protein with an EGF-like motif expressed by pioneer neurons, Nat. Genet. 10 (1995) 77 – 83. [21] S. Hockfield, R.D. McKay, Identification of major cell classes in the developing mammalian nervous system, J. Neurosci. 5 (1985) 3310 – 3328. [22] S. Izumo, V. Madavi, Thyroid hormone receptor alpha isoforms generated by alternative splicing differentially activate myosin HC gen transcription, Nature 334 (1988) 539 – 542. [23] R. Lavado-Autric, E. Auso, J.V. Garcia-Velasco, C. Arufe Mdel, F. Escobar del Rey, P. Berbel, G. Morreale de Escobar, Early maternal hypothyroxinemia alters histogenesis and cerebral cortex cytoarchitecture of the progeny, J. Clin. Invest. 111 (2003) 1073 – 1082. [24] A.A. Lavdas, M. Grigoriou, V. Pachnis, J.G. Parnavelas, The medial ganglionic eminence gives rise to a population of early neurons in the developing cerebral cortex, J. Neurosci. 19 (1999) 7881 – 7888. [25] M.A. Lazar, R.A. Hodin, D.S. Darling, W. Chin, Identification of a rat c-erbA alpha-related protein which binds deoxyribonucleic acid but does not bind thyroid hormone, Mol. Endocrinol. 2 (1988) 893 – 901.
[26] J.L. Leonard, A.P. Farwell, P.M. Yen, W.W. Chin, M. Stula, Differential expression of thyroid hormone receptor isoforms in neurons and astroglial cells, Endocrinology 135 (1994) 548 – 555. [27] R.A. Lucio, J.V. Garcia, J.R. Cerezo, P. Pacheco, G.M. Innocenti, P. Berbel, The development of auditory callosal connections in normal and hypothyroid rats, Cereb. Cortex 7 (1997) 303 – 316. [28] P. Malatesta, E. Hartfuss, M. Gotz, Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage, Development 127 (2000) 5253 – 5263. [29] O. Marin, J.L. Rubenstein, Cell migration in the forebrain, Annu. Rev. Neurosci. 26 (2003) 441 – 483. [30] M. Marin-Padilla, Prenatal development of fibrous (white matter), protoplasmic (gray matter), and layer I astrocytes in the human cerebral cortex: a Golgi study, J. Comp. Neurol. 357 (1995) 554 – 572. [31] J.R. Martı´nez-Gala´n, P. Pedraza, M. Santacana, F. Escobar del Rey, G. Morreale de Escobar y, A. Ruiz-Marcos, Early effects of iodine deficiency on radial glial cells of the hippocampus of the rat fetus: a model of neurological cretinism, J. Clin. Invest. 99 (1997) 2701 – 2709. [32] G. Morreale de Escobar, R. Pastor, M.J. Obrego´n, F. Escobar del Rey, Effects of maternal hypothyroidism on the weight and thyroid hormone content of rat embryonic tissues, Endocrinology 117 (1985) 1890 – 1896. [33] B. Nadarajah, J.G. Parnavelas, Modes of neuronal migration in the developing cerebral cortex, Nat. Rev., Neurosci. 3 (2002) 423 – 432. [34] S.C. Noctor, A.C. Flint, T.A. Weissman, R.S. Dammerman, A.R. Kriegstein, Neurons derived from radial glial cells establish radial units in neocortex, Nature 409 (2001) 714 – 720. [35] M. Ogawa, T. Miyata, K. Nakajima, K. Yagyu, M. Seike, K. Ikenaka, H. Yamamoto, K. Mikoshiba, The reeler gene-associated antigen on Cajal-Retzius neurons is a crucial molecule for laminar organization of cortical neurons, Neuron 14 (1995) 899 – 912. [36] S.K. Pixley, J. de Vellis, Transition between immature radial glia and mature astrocytes studied with a monoclonal antibody to vimentin, Brain Res. 317 (1984) 201 – 209. [37] P. Rakic, Mode of cell migration to the superficial layers of fetal monkey neocortex, J. Comp. Neurol. 145 (1972) 61 – 84. [38] J. Sap, A. Munoz, K. Damm, Y. Goldberg, J. Ghysdael, A. Leutz, H. Beug, B. Vennstrom, The c-erb-A protein is a high-affinity receptor for thyroid hormone, Nature (1986) 635 – 640. [39] C.C. Stichel, C.M. Muller, K. Zilles, Distribution of glial fibrillary acidic protein and vimentin immunoreactivity during rat visual cortex development, J. Neurocytol. 20 (1991) 97 – 108. [40] N. Tamamaki, K.E. Fujimori, R. Takauji, Origin and route of tangentially migrating neurons in the developing neocortical intermediate zone, J. Neurosci. 17 (1997) 8313 – 8323. [41] C.C. Thompson, C. Weinberger, R. Lebo, R.M. Evans, Identification of a novel thyroid hormone receptor expressed in the mammalian central nervous system, Science 237 (1987) 1610 – 1614. [42] C. Weinberger, C.C. Thompson, E.S. Ong, R. Lebo, D.J. Grujold, R.M Evans, The c-erb-A gene encodes a thyroid hormone receptor, Nature 324 (1986) 641 – 646.