thyroid hormone regulates tag-1 expression in the developing rat brain

Thyroid hormone regulates TAG-1 expression in thedeveloping rat brain

Manuel Alvarez-Dolado,1 AngeÂ lica Figueroa,1 Serguei Kozlov,2,* Peter Sonderegger,2 Andrew J. Furley3 andAlberto MunÄoz1

1Instituto de Investigaciones BiomeÂdicas `Alberto Sols', Consejo Superior de Investigaciones CientõÂ®cas (CSIC) ± Universidad

AutoÂnoma de Madrid (UAM), Arturo Duperier 4, 28029 Madrid, Spain2Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland3Developmental Genetics Programme, University of Shef®eld, Shef®eld S10 2TN, UK

Keywords: adhesion, axon, gene expression, hypothyroidism, neural differentiation

Abstract

TAG-1 is a member of the immunoglobulin superfamily of cell adhesion molecules thought to play important roles in neuronal

differentiation and the establishment of connectivity during brain development. Because these are processes also affected byhypothyroidism, we studied the effects of thyroid hormone deprivation and administration on TAG-1 expression in the developing

rat brain. By in situ hybridization, immunohistochemistry and Western blotting we found that TAG-1 RNA and protein levels are

upregulated in the hypothyroid brain. From embryonic day 20 to postnatal day (P) 15, elevated TAG-1 RNA was found in severalareas including the cerebral cortex, hippocampus and olfactory bulb. In agreement with this, TAG-1 protein was overexpressed in

the major ®bre tracts arising from these structures, including the corpus callosum, anterior and hippocampal commissures and

lateral olfactory tract. A similar overexpression of TAG-1 by hypothyroidism was detected in the cerebellum, but starting only atP15. In all cases, elevation of TAG-1 RNA and protein expression could be reversed by thyroid hormone treatment. These results

show that the deregulation of TAG-1 might contribute to the alterations caused by the lack of thyroid hormone during brain

development.

Introduction

The neural cell adhesion molecule (CAM) TAG-1, a member of the

immunoglobulin (Ig) superfamily (axonin-1 in chick; TAX-1/CNTN2

in human; Furley et al., 1990; Zuellig et al., 1992; Hasler et al., 1993),

is thought to play an important role in axonal growth and guidance,

neurite outgrowth and the formation of highly ordered neural

connections (Ruegg et al., 1989; Furley et al., 1990; Stoeckli et al.,

1991; Sonderegger & Rathjen, 1992; Stoeckli, 1997; Fitzli et al.,

2000). It is present as a surface component of the axonal "pathway

sensor" apparatus (Vogt et al., 1996), and has been implicated in the

migration and local differentiation of cerebellar granule cells (Fishell

& Hatten, 1991; Wolfer et al., 1994; Bailly et al., 1996; Buttiglione

et al., 1998). Although capable of homophilic binding, it is the

heterophilic interactions of TAG-1, particularly with L1/Ng-CAM and

NrCAM, that appear to be important in these processes (Kuhn et al.,

1991; Felsenfeld et al., 1994; Stoeckli et al., 1997).

TAG-1 is expressed on many classes of neurons from the earliest

stages of axonogenesis, when it can ®rst be found on cell bodies as

axons begin to form and later on growing axons and their growth

cones (Yamamoto et al., 1986; Dodd et al., 1988; Wolfer et al., 1994;

Yoshihara et al., 1995; Karagogeos et al., 1997). On several classes

of axon its expression is transient, notably on spinal commissural and

motor axons, where disappearance of TAG-1 coincides with axons

reaching intermediate or ®nal targets (Dodd et al., 1988).

Nonetheless, high levels of TAG-1 expression continue through to

the third postnatal week, most notably in the cerebellum and

neocortex where TAG-1 protein is found on differentiating granule

cells in the inner external granule cell layer (iEGL) and on

corticothalamic projections (Wolfer et al., 1994; Yoshihara et al.,

1995). However, even in these regions TAG-1 expression is precisely

controlled with the protein disappearing at speci®c stages of

axonogenesis.

Very little is known about how the expression of TAG-1 is

controlled, although in vitro promoter analysis studies have identi®ed

regions upstream of the ®rst exon that will direct expression in

transfected cultured lines (Giger et al., 1995; Kozlov et al., 1995).

Thyroid hormone (triiodothyronine, T3) and the prohormone

thyroxine (T4) plays a crucial role in CNS development

(Oppenheimer & Schwartz, 1997; Bernal & GuadanÄo, 1998), and is

known to regulate the expression of a number of proteins involved in

axonogenesis, including the IgCAMs MAG, NCAM and L1

(RodrõÂguez-PenÄa et al., 1993; Iglesias et al., 1996; Alvarez-Dolado

et al., 2000). Severe thyroid hormone de®ciency in humans leads to

cretinism, a syndrome associated with mental retardation and

neurological de®cits (DeLong, 1990). Hypothyroidism in rodents

causes a complex array of alterations which include abnormal

arborization and distribution of dendritic spines, de®cient neurite

outgrowth, aberrant patterns of connections, myelination defects,

abnormal cortical lamination, and a delay in the migration of

Correspondence: Dr A. MunÄoz, as above.Email: [email protected]

*Present address: Cancer and Developmental Biology Laboratory, ABL-BasicResearch Program, NCI-FCRDC, Bldg. 539, Room 121 A, PO Box B,Frederick, MD 21702±1201, USA

Received 30 March 2001, revised 14 July 2001, accepted 20 August 2001

European Journal of Neuroscience, Vol. 14, pp. 1209±1218, 2001 ã Federation of European Neuroscience Societies

cerebellar granule cells (Legrand, 1984; Dussault & Ruel, 1987). The

involvement of TAG-1 in many of the processes altered by

hypothyroidism, the fact that other IgCAMs are regulated by T3,

and the coincidence of the peaks of TAG-1 expression with T3 effects

in the CNS, led us to investigate the effects of thyroid status on

TAG-1. To our knowledge, this is the ®rst time that an epigenetic

factor, T3, has been shown to regulate TAG-1 expression.

Materials and methods

Preparation of hypothyroid animals

Wistar rats raised in our animal facilities were used. The maintenance

and handling of the animals were as recommended by the European

Communities Council Directive of November 24th, 1986 (86/609/

EEC) following the guidelines and approval of the Committee for

Animal Research of our Institute. All efforts were made to reduce the

number of animals used, and to use alternatives to in vivo techniques.

To induce embryonal and neonatal hypothyroidism, 2-mercapto-1-

methylimidazole (0.02% MMI, Sigma, St. Louis, MO, USA) and

potassium perchlorate (1% KClO4, Merck, Germany) were adminis-

tered in the drinking water of the dams from the ninth day after

conception until the animals were killed, always under adequate

anaesthesia (1 mL/100 mg body weight of a solution containing

2 mg/mL diazepam, 25 mg/mL ketamine and 0.1 mg/mL atropine).

Postnatal day (P) 0 animals were killed 8±12 h after birth. T4

treatment (Sigma) consisted of daily single intraperitoneal injections

of 1.8 mg/100 g body weight starting 4 days before death. Rats were

killed 24 h after the last T4 injection. For treatment of embryos

(5 mL/h, 3 mg T4/100 g body weight per day) an osmotic pump

(2ML2 model; Alzet, CA, USA) was subcutaneously implanted into

the dams (using the anaesthetic solution above). T3 treatments

(Sigma) consisted of a single intraperitoneal injection (1 mg/g body

weight) 8 h before the animals were killed. The results obtained in

the animal studies referred to consistent ®ndings based on the

analysis of sections from at least three animals per experimental

group. Hypothyroid animals showed an arrest of body weight

increase (24% on P15, 55% on P25) and lower levels of both T4

(from 1.46 to 0.022 ng/g, 66.3-fold) and T3 (from 4.4 to 0.2 ng/g; 22-

fold) in brain tissue as measured by a speci®c radioimmunoassay

similar to those described previously Morreale de Escobar et al.,

1985; GarcõÂa-FernaÂndez et al., 1997; Alvarez-Dolado et al., 2000).

Samples for in situ hybridization and immunohistochemistry

Under profound pentobarbitone anaesthesia (4 mg/100 g, Abbott,

Chicago, IL, USA), animals were perfused transcardially with saline

and then with cold 4% p-formaldehyde in 0.1 M sodium phosphate

(pH 7.4). The brains were removed quickly and cryoprotected in 4%

p-formaldehyde +30% sucrose (w/v) in phosphate-buffered saline

(PBS) at 4 °C. Subsequently, parallel serial 40 mm [for embryonal

day (E)20] or 25 mm (P0±P30) thick coronal sections were cut using a

cryostat (Leica Instruments, Germany).

Preparation of riboprobes

To obtain antisense or sense TAG-1 riboprobes a 1.6 kb fragment of

the rat TAG-1 cDNA subcloned into the Bluescript vector was

transcribed in vitro using T7 or T3 RNA polymerases, respectively, in

the presence of 50 mCi of 35S-UTP. Non-radioactive riboprobes were

prepared using a digoxigenin RNA labelling kit (Boehringer

Mannheim, Germany). Riboprobes were cleaned by centrifugation

through Chroma Spin-100 DEPC-H2O columns (Clontech, Palo Alto,

CA, USA).

In situ hybridization

In situ hybridization on ¯oating sections followed the procedure of

Gall & Isackson (1989). Sections were thawed, washed with PBS for

5 min and treated for an additional 10 min at room temperature under

free-¯oating conditions with 0.1% Triton X-100, 0.2 M HCl, 0.25%

acetic anhydride in 0.1 M triethanolamine, and post®xed with 4%

p-formaldehyde. The sections were then preincubated in hybridiz-

ation solution (0.6 M NaCl, 20 mM PIPES-Na, 10 mM EDTA, 50%

formamide, 0.2% SDS, 5 3 Denhardt's solution, 10% dextran

sulphate, 50 mM DTT, 250 mg/mL of sheared salmon sperm DNA

and 250 mg/mL of yeast tRNA) for 3±5 h at 55 °C, and later

incubated overnight at 55 °C in the same solution containing 250 ng/

mL of the digoxigenin-labelled riboprobe or the 35S-UTP-labelled

riboprobe (15 3 106 c.p.m./mL). Sections were consecutively

washed once in 2 3 standard saline citrate (1 3 SSC: 0.15 M

NaCl, 0.015 M sodium citrate) + 10 mM b-mercaptoethanol at room

temperature for 30 min, once in 5 3 TEN (50 mM Tris pH 7.5, 5 mM

EDTA, 0.5 M NaCl) supplemented with 4 mg/mL mRNAse at 37 °C

for 1 h, twice in 0.5 3 SSC + 50% formamide + 10 mM b-mer-

captoethanol at 55 °C for 1 h, once in 0.1 3 SSC + 10 mM b-

mercaptoethanol at 68 °C for 1 h, and ®nally once in PBS at room

temperature for 5 min. To develop the digoxigenin staining we used

the nucleic acid detection kit from Boehringer Mannheim (Germany).

For radioactive in situ hybridization, sections were mounted onto

slides, dehydrated by ethanol series (containing 0.3 M NH4Ac) and

exposed for 5 days to Hyper®lm b-MAX ®lms (Amersham-

Pharmacia Biotech, UK), developed with Kodak D19, and ®xed.

For anatomical abbreviations we followed those in Paxinos & Watson

(1998).

Immunohistochemistry

Tissue sections were incubated overnight with a 1 : 10 000±5000

dilution of a rabbit polyclonal anti-TAG-1 antibody (Dodd et al.,

1988; a gift from Dr T. Jessell) or with the 1C12 monoclonal anti-rat

TAG-1 antibody (Dodd et al., 1988; a gift from Dr D. Karagogeos) at

4 °C under free-¯oating conditions. Sections were incubated with a

goat biotinylated anti-rabbit or anti-mouse Ig (Vector Laboratories,

UK) at a dilution of 1 : 200 in 0.1 M PBS + 0.1% Triton X-100 + 3%

goat serum. Sections were then washed three times in 0.1 M

PBS + 0.1 M Triton X-100 and incubated for a further 30 min with

avidin-biotinylated horseradish peroxidase macromolecular complex

(ABC, Vector Laboratories, UK) at a dilution of 1 : 100 in 0.1 M

PBS. The IgG fraction obtained from nonimmunized rabbits was used

for control staining. The peroxidase activity was visualized using a

solution containing 0.5 mg/mL diaminobenzidine (Sigma) and 0.3%

H202 in 0.1 M PBS. Sections were washed with 0.1 M PBS, and

mounted onto slides. After air-drying, they were dehydrated in graded

alcohol solutions, cleared in xylene and ®nally coverslipped with

DePeX mounting medium (Sigma).

Western blotting

Membrane-enriched protein extracts for immunoblot analysis of L1

expression were obtained as described (Rettig et al., 1992). Brie¯y,

brain tissue (cerebral cortex or cerebellum) was homogenized in 7

volumes of chilled, high pH buffer (30 mM diethylamine, 1 mM

EDTA, pH 11.5), cleared by centrifugation at 900 3 g for 15 min at

4 °C, and ®nally centrifuged at 100 000 3 g for 30 min at the same

temperature. Aliquots of the pellets containing 25±50 mg protein

were electrophoresed in 6% polyacrylamide gels and transferred to

nylon PVDF membranes overnight using a vertical transfer apparatus

(Bio-Rad Laboratories, CA, USA) at 1 mA/cm2. Care was taken to

1210 M. Alvarez-Dolado et al.

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1209±1218

ensure equal loading of individual extracts by measuring the protein

concentration. The ®lters were washed, blocked with Blotto (5%

skimmed milk in PBS), and incubated with the TG3 rabbit polyclonal

anti-TAG-1 antibody (generously donated by Dr T. Jessel) at a

1 : 10 000 dilution. The blots were developed by a peroxidase

reaction using the ECL detection system (Amersham-Pharmacia

Biotech, UK).

Results

In situ hybridization and immunohistochemical studies

To determine whether TAG-1 expression is regulated by thyroid

hormone in the developing rat brain, we performed in situ

hybridization and immunohistochemical studies of embryonal and

postnatal control and hypothyroid rats.

E20

In agreement with previous reports (Yoshihara et al., 1995), in situ

hybridization analysis showed that in control animals TAG-1

transcripts were prominent in the cortical plate (CP), intermediate

zone (IZ) of the neocortex, hippocampus, olfactory bulb, and

habenula (Fig. 1A, D and E). Lower levels of TAG-1 expression

were detected in the hypothalamic nuclei and the prospective EGL of

the cerebellum. Hypothyroidism did not change the regional pattern

of expression, but caused an increase in TAG-1 RNA expression in

the neocortex, hippocampus, olfactory bulb, and habenula (compare

Fig. 1B, F and G with Fig. A, D and E). In contrast, no differences

were observed in other brain regions, particularly the hypothalamus

and prospective cerebellum (not shown). The effect of in vivo

hormonal treatment was also investigated. As shown in Fig. 1C, H

and I, T4 administration to hypothyroid dams diminished TAG-1

RNA expression almost to normal levels in affected regions.

TAG-1 immunoreactivity was detected almost exclusively in

axonal compartments (Fig. 1J and K). As described previously

(Wolfer et al., 1994), at this age TAG-1 protein was found in the

white matter of the neocortex, internal capsule, corpus callosum,

anterior commissure, hippocampus, ®mbria, and lateral olfactory tract

of control rats. Cell bodies in the cortical plate were TAG-1-negative,

but stained axons entering this zone from the subplate were found. In

hypothyroid animals, TAG-1 immunostaining was increased substan-

tially in the corpus callosum, white mater of the neocortex, internal

capsule, hippocampus, and olfactory tract (compare Fig. 1L and M

with Fig. J and K). No signi®cant differences were observed in other

brain regions. In concordance with RNA data, T4 treatment of

hypothyroid dams led to a partial normalization of TAG-1 protein

expression (Fig. 1N and O). These results indicated that alterations in

the thyroid status modulate TAG-1 RNA and protein expression at

this embryonal age.

P0

At P0, TAG-1 RNA expression in control rats followed the same

pattern as at E20 in all brain regions (Fig. 2A and D). In hypothyroid

rats a clear upregulation of TAG-1 transcripts was apparent in the

prospective layer V of all areas of the neocortex, dentate gyrus, CA2±

3 of the hippocampus, and anterior olfactory bulb (Fig. 2B and F). In

contrast, no obvious differences were observed in other regions such

as the habenula (not shown) or the external granular layer (EGL) of

the cerebellum (Fig. 2C and E).

Both the pattern and intensity of TAG-1 immunoreactivity were

similar to those found at E20 (Fig. 2G, H and L), with the addition

that it was also detected strongly in the inner rim of the EGL of the

cerebellum (Fig. 2K). In hypothyroid rats, in agreement with RNA

results, there was an signi®cant increase in TAG-1 protein levels in

the corpus callosum, anterior and hippocampal commissures, ®mbria,

lateral olfactory tract, and, to a lesser extent, in the forceps major and

white matter of the cerebral cortex of hypothyroid rats (compare

Fig. 2G, H and L with Fig. I, J and N). However, once again, no

obvious differences were observed in the cerebellum (Fig. 2K and

M), although by this time, as noted above, TAG-1 expression levels

are very high in the EGL anyway.

P5

At this age, TAG-1 RNA expression was restricted to layer V of the

cerebral cortex, layers II±III of the retrosplenial cortex, hippocampus,

and the EGL of the cerebellum of euthyroid animals (Fig. 3A, D, G

and H). Hypothyroidism induced an abnormal high level of TAG-1

transcripts in the retrosplenial cortex and, also but to a lesser degree,

in layer V of the cerebral cortex and hippocampus (compare Fig. 3B,

E and I with Fig. A, D and G). Daily treatment of hypothyroid

animals with T4 from P1 to P5 led to a normalization of TAG-1 RNA

levels in these regions (Fig. 3C, F and K). At this age, the normal

high level of expression of TAG-1 in the EGL of the cerebellum

remained apparently unresponsive to changes in thyroid status

(Fig. 3H, J and L), a result also found at P10 (not shown).

In control euthyroid rats TAG-1 immunoreactivity diminished

substantially with respect to earlier ages except in the cerebellum,

where by contrast, expression increased (as has been reported by

Wolfer et al., 1994). TAG-1 protein was detected in the corpus

callosum, anterior and dorsal hippocampal commissures, and the

iEGL of the cerebellum (Fig. 3M, P and Q). In accordance with RNA

results, TAG-1 levels in hypothyroid rats were higher than in controls

in the corpus callosum and the anterior and hippocampal commis-

sures (compare Fig. 3N and R with Fig. M and P) but remained

unchanged in the cerebellum (Fig. 3Q). Also in agreement with in

situ hybridization data, T4 treatment substantially reduced TAG-1

protein levels in responsive areas (Fig. 3O, T and U).

P15±30

By digoxigenin in situ hybridization, a high level of TAG-1

expression at P15 was found to be con®ned to the cerebellum as in

previous studies (Furley et al., 1990; Yoshihara et al., 1995; data not

shown). In comparison to earlier ages, TAG RNA levels were slightly

higher in both the iEGL and IGL. Unexpectedly, considering the

apparent lack of effect in these regions at earlier ages and in contrast

to results obtained at E20 and P0±P5, hypothyroidism caused an

increase in TAG-1 RNA expression. A relative increase in expression

of TAG-1 in the hypothyroid cerebellum was also found by

conventional radioactive in situ (not shown). Using this technique,

TAG-1 RNA was detected in other brain regions but, in contrast to

earlier ages, its expression levels in these regions were not obviously

affected by thyroid status (not shown).

We extended our analysis of the cerebellum to P20 and P25. At

P20, TAG-1 RNA was detected in the EGL and IGL (Fig. 4, left),

whereas TAG-1 immunostaining was restricted to short processes

only in the EGL (Fig. 4, right). TAG-1 RNA and protein expression

were substantially higher in hypothyroid rats as compared to control.

At these ages it is well documented that the number of cells in the

EGL is higher in hypothyroid rats than in control due to a marked

delay in the migration of granular cells to the IGL (Clos et al., 1980;

Legrand, 1984; Dussault & Ruel, 1987). To avoid the complication

that this altered migration might be affected by prolonged T4

treatment (normally 4 days) such that changes in cell localization

Hypothyroidism alters brain TAG-1 expression 1211


could alter the interpretation of the results, hypothyroid animals were

instead subjected to an acute (8 h) treatment with T3 to investigate

hormonal effects on TAG-1 expression. Remarkably, T3 treatment

diminished profoundly the levels of TAG-1 RNA and protein in both

hypothyroid and control animals. This rapid action did not noticeably

affect cell number or localization, and therefore it is likely to be a

consequence of a direct hormonal effect on TAG-1 gene expression.

Identical results were obtained at P25, although TAG-1 expression

levels were lower than at P20 (not shown).

At P30, TAG-1 expression has already declined to the low levels

found in adult life. Consistent with previous reports (Furley et al.,

1990; Wolfer et al., 1994; Yoshihara et al., 1995), although TAG-1

RNA was found in the cerebellum, and to a lesser degree in the

hippocampus and cortex layer V, TAG-1 protein was not detectable.

No signi®cant effects of the thyroid status on TAG-1 expression were

observed at this age.

Developmental study of TAG-1 expression by immunoblotanalysis

To further assess and also quantify the effects of hypothyroidism on

TAG-1 protein expression during development, we performed

Western blot analysis using axonal membrane-enriched extracts of

cerebral cortex (including white matter and corpus callosum) and

cerebellum of control and hypothyroid animals at different ages. In

agreement with previous studies (Furley et al., 1990; Wolfer et al.,

1994), a single band of approximately 135 kDa was detected (Fig. 5).

In line with the asynchronous maturation of these two brain regions,

TAG-1 protein content in the cerebral cortex was maximal at P5 and

diminished later, whereas in cerebellum it progressively increased to

peak at P15. Hypothyroidism did not change the temporal pattern of

expression in the cerebral cortex, but caused a statistically signi®cant

increase (2±3-fold) of TAG-1 protein levels at P0 and P5. This is in

agreement with the stronger immunostaining found in the white

matter and the corpus callosum of hypothyroid rats at these ages

(Figs 2 and 3). The difference between control and hypothyroid rats

was much reduced at P15 and completely disappeared at P25. In line

also with immunohistochemical data, hypothyroidism increased

(around twofold) TAG-1 content in the cerebellum at P15±25, but

not obviously at early ages.

Discussion

The TAG-1 gene encodes an immunoglobulin-like cell adhesion

molecule that is thought to play a crucial role in axonal growth and

guidance, migratory/differentiation processes of the cerebellum, and

FIG. 1. Effect of hypothyroidism on TAG-1 expression at E20. Upper panels (A±I) show an in situ hybridization analysis of TAG-1 mRNA expression.Lower panels (J±O) show TAG-1 protein immunoreactivity. TAG-1 expression was analysed in coronal sections of control (A, D±E, J and K), hypothyroid(B, F±G, L and M), and T4-treated hypothyroid (C, H, I, N and O) rat brains. Note the higher TAG-1 RNA expression found in the neocortex (Nc),hippocampus (Hi), habenula (Hb) and olfactory tract (OT) of hypothyroid animals. D, F, and H are magni®cations of the area marked in (A, B and C),respectively. Note that TAG-1 RNA expression was higher in the cortical plate (CP) and intermediate zone (IZ). Higher immunoreactivity was found in whitematter (wm), hippocampus, and olfactory tract of hypothyroid brains. Processing was standard for all experimental groups. The developing reaction wasstopped when differences between control and hypothyroid animals were most clear. M, molecular layer; NSZ, neocortical subventricular zone; S, striatum;SP, subplate. Scale bar 1 mm (A), 250 mm (D), 1 mm (E), 2 mm (K).



the formation of highly ordered neural connections (Ruegg et al.,

1989; Furley et al., 1990; Stoeckli, 1997; Sonderegger, 1998). Here

we show that thyroid hormone, a major epigenetic factor during

development, regulates TAG-1 expression during brain maturation in

the rat. To our knowledge, this is the ®rst identi®cation of a systemic

factor that can regulate TAG-1.

As in the case of other T3 target genes (GarcõÂa-FernaÂndez et al.,

1997; Alvarez-Dolado et al., 1998, 1999, 2000), TAG-1 is regulated

FIG. 2. Upregulation of TAG-1 expression by hypothyroidism in newborn rats. Upper panels (A±F) show an in situ hybridization analysis of TAG-1 RNAexpression. Lower panels (G±N) show TAG-1 protein immunoreactivity. Note the higher TAG-1 RNA expression in layer V of the neocortex (arrow in B),dentate gyrus (DG) and CA regions of the hippocampus, and anterior olfactory nuclei (AON) of hypothyroid rats. Immunoreactivity was found elevated incorpus callosum (cc), forceps major (fmj), ®mbria (®), lateral olfactory tract (lo), and anterior commissure (aca) of hypothyroid rats. EGL, external granularlayer; RC, retrosplenial cortex. Scale bar 1 mm (A and C), 1 mm (C±F), 2 mm (G); 1 mm (H), 600 mm (K±N).



FIG. 3. Upregulation of TAG-1 expression by hypothyroidism at P5. Upper panels (A±L) show an in situ hybridization analysis of TAG-1 RNA expression.Lower panels (M±U) show TAG-1 protein immunoreactivity. Note the elevated TAG-1 RNA expression in layers II±III of the retrosplenial cortex (RC), andlayer V (arrows in G, Y and K) of the cortex in hypothyroid rats. Immunoreactivity was found elevated in the dorsal hippocampal commissure (dhc), corpuscallosum, and anterior commissure (ac). T4 treatment of hypothyroid animals was as described in the methods section and normalized the TAG-1 expressionlevels. Scale bar 1 mm (A), 250 mm (D), 1 mm (G), 500 mm (H), 1 mm (P).



in a region- and temporal-speci®c fashion. The pattern of regulation

found supports a role for TAG-1 in mediating the effects of T3 on

neuronal differentiation. In the cortex, TAG-1 expression is regulated

by T3 during the ®rst two postnatal weeks, when neuronal maturation

is maximal in this area. Similarly, TAG-1 is regulated by T3 in the

postnatal cerebellum most obviously so after P10, coincident with the

main period of neuronal maturation. However, the strongest regula-

tion of TAG-1 in the cerebellum, most evident from P15±P25,

contrasts with a lack of effect of T3 on NCAM, L1 and SWAP gene

expression during the same period (Iglesias et al., 1996; Cuadrado

et al., 1999; Alvarez-Dolado et al., 2000), indicating that T3-

responsive genes do not act as a cohort. The differential regulation of

T3-responsive genes might re¯ect the spatio-temporal pattern of

expression of the different thyroid receptors, the interplay with other

regulatory agents such as region-speci®c coactivators or corepressors

that modulate their activity, and/or the local regulation of T3

concentrations by variable expression of the iodothyronine deiodases

family (EscaÂmez et al., 1999; GuadanÄo-Ferraz et al., 1999). Our

FIG. 5. Immunoblotting analysis of TAG-1 protein levels in the cortex and cerebellum during the early postnatal period in control (C) and hypothyroid (H)rats. Quanti®cation of protein levels in control (white squares) and hypothyroid (black circles) animals at each developmental age is shown. Data shown arethe mean 6 SD of three independent experiments with three animals per experimental group (P < 0.001).

FIG. 4. Effects of T3 deprivation and administration on TAG-1 expression in the cerebellum at P20. Hypothyroidism caused an upregulation of TAG-1 RNAand protein. By contrast, T3 treatment (8 h) diminished TAG-1 expression in both control and hypothyroid animals. Left panels show an in situ hybridizationanalysis of TAG-1 RNA expression. Right panels show TAG-1 immunoreactivity. EGL, external granular layer; IGL, internal granular layer; ML, molecularlayer; PCL, Purkinje cell layer; wm, white matter. Scale bar (A), 200 mm.



results show an abnormally high expression of TAG-1 induced by

hypothyroidism in several brain regions. Whether the regulation of

TAG-1 by T3 is direct or indirect remains unclear, although the tight

correlation of RNA and protein levels in response to thyroid status,

and the rapid response to acute T3 treatment in the cerebellum

suggests a simple regulatory mechanism.

It is known that TAG-1 is expressed during the formation of callosal

and commissural projections, playing an important role in axon

guidance and fasciculation (Dodd et al., 1988; Wolfer et al., 1994;

Stoeckli, 1997; Sonderegger, 1998). The injection of soluble TAG-1 or

anti-TAG-1 antibodies into the developing spinal cord has dramatic

effects on spinal commissural axon fasciculation and path®nding, most

likely due to alterations in interactions with the IgCAMs L1/NgCAM

and Nr-CAM (Stoeckli & Landmesser, 1995), to which TAG-1 is

known to bind heterophilically (Kuhn et al., 1991; Felsenfeld et al.,

1994; BruÈmmendorf & Rathjen, 1998; Volkmer et al., 1998).

Interestingly, hypothyroidism impairs the maturation of axons in

both the corpus callosum and the anterior commissure (Gravel et al.,

1990; Berbel et al., 1994; GuadanÄo-Ferraz et al., 1994). Hypothyroid

rats display inappropriate callosal connections from cortical layers II±

III, with a widespread tangential distribution of callosally projecting

neurons in the visual, parietal and auditory areas (Gravel & Hawkes,

1990; Berbel et al., 1993; Lucio et al., 1997). Although the major

elevation in TAG-1 mRNA expression that we see in the cortex at E20

is in layer V and not in II/III, and the majority of axons in the corpus

callosum are thought to derive from layer II/III, both our protein data,

and that of others (Wolfer et al., 1994), clearly demonstrate signi®cant

levels of TAG-1 protein on ®bres in the corpus callosum (and anterior

commissure) from as early as E16 and remaining until postnatal stages

(Figs 2 and 3). Whether this protein is derived from layer II/III neurons

in the retrosplenial cortex (where TAG-1 mRNA is detected), from the

subset of layer V neurons known to project in the corpus callosum

(Kasper et al., 1994a, 1994b), or perhaps from a transient population of

callosal axons that also might contribute to the path®nding of layer II/

III axons (Ding & Elberger, 2001) is unclear. However, our

immunohistochemistry clearly demonstrates that levels of TAG-1 on

axons in the corpus callosum and anterior commissure increase in

hypothyroid animals (Figs 2 and 3). This increase might directly affect

the path®nding of the overexpressing axons, be they layer II/III axons

or an another axon subset on which layer II/III are dependent.

Additionally, because TAG-1 is naturally produced in two isoforms,

membrane-bound and secreted (Ruegg et al., 1989; Furley et al.,

1990), it is conceivable that the TAG-1 overexpression during

hypothyroidism causes an abnormal accumulation of the soluble

TAG-1 isoform which might alter the interactions with other CAMs

necessary for the formation of this major ®bre tract. We have also

found that L1/Ng-CAM expression is abnormally high in the main

commissural processes of hypothyroid rats (Alvarez-Dolado et al.,

2000). Together, the alteration of TAG-1 and L1/Ng-CAM levels in

these ®bre tracts might contribute to the anomalies found in the

development of commissural and callosal projections during hypo-

thyroidism.

In the hypothyroid brain, many neurons are inappropriately located

for their birthdate causing an abnormal laminar distribution (Lucio

et al., 1997 and references therein). The extracellular environment of

the intermediate zone and subplate during cortical development is

critical for adequate neuronal migration and guidance of thalamic

axons (Rauch, 1997). The expression of the extracellular matrix

proteins Reelin and Tenascin-C have been reported to be altered by

hypothyroidism during cortical development (Alvarez-Dolado et al.,

1998, 1999). The expression of TAG-1 is coincident with that of

Reelin and Tenascin-C, which in turn are expressed coincident with

the maturation of thalamocortical axon pathways and cortical plate

neurons (D'Arcangelo et al., 1995; Fukuda et al., 1997; GoÈtz et al.,

1997). Because TAG-1 is secreted from neurons and is a possible

target for integrin binding (Ruegg et al., 1989; Furley et al., 1990;

Felsenfeld et al., 1994), the overexpression of these molecules in

hypothyroid animals might alter the balance of neuronal interactions

with the extracellular matrix, leading to altered migration and

guidance.

Both T3 and TAG-1 have also been implicated in neuronal

migration and neurite outgrowth of cerebellar granule cells in rodents

(Legrand, 1984; DeLong, 1990; Bailly et al., 1996; Buttiglione et al.,

1998). Expression of TAG-1 begins at the earliest stages of granule

cell differentiation, as these cells become postmitotic in the iEGL, but

seems to disappear shortly after the cells begin to migrate inward

(Furley et al., 1990; Kuhar et al., 1993; Bailly et al., 1996), at which

time F3/contactin becomes expressed (AJF and G. Gennarini,

unpublished observations). A functional interaction between TAG-1

and F3 has been proposed to be necessary for these differentiation

steps (Buttiglione et al., 1998), although in vitro experiments suggest

that TAG-1 is not necessary for the migration process itself (Fishell &

Hatten, 1991). Indeed, one possibility is that downregulation of TAG-

1 might be necessary to allow full inward migration to the IGL to

occur. In this respect it is interesting to note that F3 expression, which

seems to mark the next stage in differentiation, is not affected by

thyroid status (C. Navarro-Yubero, MA-D, G. Gennarini and AM,

unpublished data), supporting the idea that T3 speci®cally affects

granule cells at the stage in differentiation in which they normally

express TAG-1. Thus the delay in granule cell migration seen in

hypothyroid animals (Clos et al., 1980; Legrand, 1984; Dussault &

Ruel, 1987) could be due, in part, to a failure of granule cells to turn

off TAG-1 at an appropriate point in differentiation.

Finally, it has long been suggested that T3 exerts its effects on

brain development during the postnatal period (Schwartz et al.,

1997). However, our data show that TAG-1 expression is also

affected by hypothyroidism during late embryonic stages. Similar

results were recently obtained for reelin, a gene which is down-

regulated by thyroid hormone deprivation before birth (Alvarez-

Dolado et al., 1999) and NSP and Oct-1 genes (Dowling et al., 2000).

Together, these results demonstrate that in contrast to what was

previously thought, the rat brain is responsive to thyroid hormone

during the late gestational period.

In summary, our results show that thyroid hormone de®ciency

causes an asynchronous abnormal upregulation of TAG-1 gene

expression in several areas of the developing rat brain. Changes in

TAG-1 protein levels may contribute to explain at least partially the

alterations in neuronal maturation and connectivity and the delayed

cerebellar cell migration found in the hypothyroid brain.

Acknowledgements

We are grateful to Fernando NuÂnÄez and Pablo SenÄor for the care of animalsand to Margarita GonzaÂlez and Teresa MartõÂnez for their expert technical help.This work was supported by grants from European Union (Biomed-2, BMH4-CT97-2653) to A.M., P.S., and A.J.F., and Plan Nacional de Salud, Ministeriode Ciencia y TecnologõÂa of Spain (SAF98-0060) to A.M. M.A.-D. wassupported by the abovementioned EU project, and A.F. by a fellowship fromPlan Nacional.

Abbreviations

CAM, cell adhesion molecule; CNS, central nervous system; E, embryonicday; EGL, external granule layer; iEGL, inner external granule layer; GPI,glycosyl-phosphatidylinositol; IGL, internal granular layer; IgCAM, immuno-globulin cell adhesion molecules; P, postnatal day; T3, triiodothyronine; T4,thyroxine.



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thyroid hormone regulates tag-1 expression in the developing rat brain

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