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Endocrinology Vol. 143, No. 7 2664-2672
Copyright © 2002 by The Endocrine Society

RECEPTORS

A Targeted Thyroid Hormone Receptor {alpha} Gene Dominant-Negative Mutation (P398H) Selectively Impairs Gene Expression in Differentiated Embryonic Stem Cells

Yan-Yun Liu, Ken H. Tachiki and Gregory A. Brent

Molecular Endocrinology Laboratory and Research Service, Veterans Affairs Greater Los Angeles Healthcare System, Departments of Medicine and Physiology, University of California Los Angeles School of Medicine, Los Angeles, California 90073

Address all correspondence and requests for reprints to: Gregory A. Brent, Molecular Endocrinology Laboratory, Veterans Affairs Greater Los Angeles Healthcare System, Building 114, Room 230, 11301 Wilshire Boulevard, Los Angeles, California 90073. E-mail: . gbrent{at}ucla.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormone and retinoic acid (RA) are essential for normal neural development in vivo, yet all in vitro differentiation strategies of embryonic stem (ES) cells use only RA. We developed a novel differentiation strategy of mouse ES cells using T3. A dominant-negative knock-in point mutation (P398H) was introduced into the thyroid hormone receptor {alpha} gene to determine the influence of T3 on ES cell differentiation. Differentiation promoted by T3 (1 nM), RA (1 µM), or combined T3/RA was assessed in wild-type (wt) and mutant (m) ES cells on the basis of neuronal-specific gene expression and cell cycle. T3 alone stimulated neural differentiation in a similar fashion as that seen with RA in both wtES and mES cells. Expression of neurogranin and Ca2+/calmodulin-dependent kinase IV mRNA (identified in vivo as T3-regulated genes), however, was markedly reduced in mES, compared with wtES cells. RA treatment enhanced apoptosis, significantly greater than that seen with T3 stimulation. T3 treatment given with RA significantly reduced the apoptotic effects of RA, an effect not seen in mES cells. T3-induced ES cell neural differentiation of thyroid hormone {alpha} mutant and wtES cells provides an in vitro model to study T3-dependent gene regulation in neural development. This system could also be used to identify novel T3-regulated genes. The modulation of the apoptotic effects of RA by T3 may have implications for stem cell therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T3 IS ESSENTIAL FOR normal neural development as demonstrated by the severe neurological consequence of thyroid hormone deficiency (1). Iodine deficiency produces both maternal and fetal hypothyroidism and results in mental retardation, motor deficits, and growth retardation, which are largely irreversible after birth (2, 3). The consequences of congenital hypothyroidism, in which only the fetus is hypothyroid and the mother euthyroid, are considered reversible if adequate thyroid hormone replacement is started shortly after birth. Fetal protection is likely the result of maternal T4 crossing the placenta under conditions of reduced thyroid hormone levels in the fetus (4). Maternal hypothyroidism, during the second trimester of pregnancy, has recently been reported to be associated with modest intellectual deficits in the offspring (5).

Thyroid hormone action on the developing central nervous system is thought to be primarily mediated by nuclear thyroid hormone receptors (TRs) (6). The TR{alpha} gene is expressed in early brain development, whereas the TRß gene is expressed later in development (7). TRß knockout mice have reduced hearing because of a functional cochlear defect (8). Recent TR gene knockout studies in mice, however, have shown only modest abnormalities in the brain (9, 10, 11, 12, 13).

In the absence of ligand, the TR has been shown to repress positive gene transcription as well as antagonize retinoic acid (RA) action (14). The relatively modest neurologic abnormalities in TR-deficient mice, compared with the defects in iodine deficiency, suggest that the repressive action of the unligand receptor may be significant in vivo in neurological development. Thyroid hormone deficiency in the presence of TR, therefore, would have more significant consequences than TR deficiency. Additionally, critical actions of thyroid hormone in neural development may not be detected in analysis of the adult animal.

In contrast to the findings in TR gene knockout studies, a recent study of mice with a TRß gene dominant-negative point mutation showed severe neurological abnormalities (15). Significant abnormalities in cerebellar development and functions were noted. These findings fit with brain defects in the cerebellum previously associated with hypothyroidism (6). Mice with a different TRß gene point mutation, however, did not have gross brain abnormalities (16).

RA is required for normal development of the mammalian brain. Premature exposure to RA results in a high incidence of birth defects, especially in neural tube-derived structure, indicating the importance of the timing of RA exposure (17). However, because early embryogenesis is RA sensitive and the RA receptor is expressed along with TR{alpha}, it has been difficult to separate T3 effects from those of RA in neural differentiation (17, 18). Previous studies with cell lines including embryonic stem (ES) (19), PC12 (20) and neuro-2a (21), however, have shown an important role for TR in neural differentiation.

In vitro differentiation of ES cells provides a model to study the effects of RA and thyroid hormone on early neuronal development. Mouse totipotent ES cells undergo neural differentiation as a result of treatment with RA (22, 23, 24, 25). We used wild-type (wt) ES and ES cells in which we introduced a dominant-negative TR{alpha} mutant (P398H) to determine whether T3 alone can stimulate neural differentiation and the role of T3 in neural differentiation and cellular proliferation. Cells were treated with RA, T3, or RA/T3 to promote neural differentiation. Cell differentiation was assessed by the expression of neural-specific marker genes and cell proliferation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Gene targeting strategy
We adapted a hit-and-replace homologous recombination strategy to create a point mutant in the TR{alpha} gene. Briefly, a fragment spanning the TR{alpha} gene exons 6, 7, 8, and 9 was isolated from a genomic DNA library derived from the mouse strain BL129 and cloned into pBluescript SK+ (Fig. 1Go). The insert (~12 kb) was cut with EcoRI and subcloned to yield a 5.5-kb genomic clone containing exons 8 and 9 and adjacent intronic sequences. Two restriction sites, ClaI and NotI, were generated by PCR within exon 8 to insert the neomycin-thymidine kinase (neo-tk) cassette (4.0 kb). The first targeting vector contained the genomic fragment with the neo-tk insertion (Fig. 1Go). The second targeting vector contained a 5.5-kb genomic DNA with a single nucleotide mutation (cytosine to adenine) changing condon 398 from proline to histidine (Fig. 1Go). The first targeting vector was introduced into ES (J1) cells by electroporation (GenePulser, Bio-Rad Laboratories, Inc., Hercules, CA). After transfection, ES cells were plated on 0.2% gelatin-coated 10-cm plates and treated with G418 (0.8 mg/ml medium) for 7 d. Colonies were screened by PCR to identify the clones carrying the targeted insertion. Correctly targeted ES clones were identified and expanded. These cells were then transfected by electroporation with the second targeting vector. Cells were treated with ganciclovir (2 µM) for 10 d. Clones that retained the original vector neo-tk cassette were sensitive to ganciclovir and died. Clones in which the tk cassette was excised and replaced by the exon 9 mutation were resistant to ganciclovir treatment. The positive clones were confirmed by direct sequencing of isolated DNA from ES clones.



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  		Figure 1. Introduction of a point mutation in the TR{alpha} gene by hit-and-replace strategy. The hit-and-replace strategy involves two targeting vectors (1 and 2). In the hit step, targeting vector 1 containing selective markers was digested with KpnI and introduced into ES cells by electroporation. Note that the vector sequence is outside the recombined region. In the replace step, the second targeting vector containing the genomic DNA sequence with the desired mutation homologous to the targeting gene was used to replace the wt allele. R1, EcoRI; C, ClaI; B, BamHI; N, NotI; K, KpnI.

 
Cell culture and differentiation
ES (J1) cells and TR{alpha} mutant (P398H) ES cells were cultured in DMEM with 20% fetal bovine serum on a 0.2% gelatin coated tissue culture dish containing mouse embryonic fibroblast feeder cells (22). Cells were split at a 1:3 ratio every 3 d. The protocol for differentiation was modified from that previously reported by Ramirez-Solis et al. (22) and Lee et al. (19). ES cells were grown to confluence in a 10-cm dish containing mouse embryonic fibroblast feeder cells. Cells were then trypsinized and plated at a density of 4 x 106 cells/per 10-cm tissue culture dish coated with 0.2% gelatin, but without feeder cells, and grown for 3 d. Cells were gently trypsinized and split at a 1:3 ratio and plated on a 10-cm Petri-grade dish for an additional 2–3 d to form embryoid bodies (EBs). To initiate neural differentiation, EBs were plated on culture dish without gelatin coating and grown in DMEM/F-12 medium supplemented with insulin, transferrin, and sodium selenite and with addition of hormones (1µM RA, 1 nM T3, or combined 1 µM RA with 1 nM T3).

Fluorescence-activated cell sorter (FACS) for cell cycle analysis
Equipment. The FASC (immunocytometry system, model FACSVantage SE, Becton Dickinson Immunocytochemistry, San Jose, CA) is equipped with an INNOVA Enterprise argon ion laser with UV and 488-nm wavelength outputs as the primary light source. Operation of the flow cytometer was controlled with an Apple Macintosh computer (Cupertino, CA) with CELLQuest software (Beckton Dickinson Immunocytochemistry).

Sample preparation. EBs were plated in 10-cm dishes at a density of 4 x 105 cells/dish and differentiated in conditioned medium with 1 nM T3, 1 µM RA, or combined T3/RA for 5 d. Cells were trypsinized, dispersed into single-cell suspension, and centrifuged at 800 x g for 5 min. The cell pellets were resuspended in ice-cold PBS and counted. Cell density was adjusted to 3 x 106 cells/ml with ice-cold PBS. A 0.5-ml aliquot of cells was fixed by adding 1 ml cold methanol dropwise and mixing the cells on a vortex mixer. Fixed cells were stored at 4 C before use.

Cell cycle and apoptosis analysis. Methanol-fixed cells were treated with propidium iodide dye to stain DNA (26). Briefly, on the day of analysis, the cell suspension was centrifuged to remove the methanol layer. DNA was stained in 500 µl staining solution composed of propidium iodide (100 µg/ml), Triton X-100 (0.1%, vol/vol), and EDTA (37 µg/ml) in PBS followed by digestion of RNA with 500 µl ribonuclease I (2 mg/ml in PBS). The samples were incubated in the dark at room temperature for 30 min, placed on ice, and analyzed within 1 h by flow cytometry using 2.0 ModFit LT software (Verity Software House, Inc., Topsham, ME) with doublet correction through windowing. Apoptosis was assessed from the sub-G1 peak on DNA histograms.

Isolation of RNA and RT-PCR
Total RNA was isolated from ES cells using Trizole reagent (Invitrogen, Carlsbad, CA) and treated with DNaseI (Ambion, Inc., Austin, TX). Reverse transcription was performed in a 20-µl reaction volume containing 2 µg total RNA, 0.5 µg poly(dT)15 primer, and 2 U murine reverse transcriptase. Two microliters cDNA was PCR amplified with 33P-dATP labeling using Taq DNA polymerase in a thermal cycler for 25–30 cycles. The linear range of PCR cycle number was established individually for each primer pair. The primers used for PCR amplification included the following: mouse collagen IV{alpha}1 (27), 5'-ATGAATTCTCAGCGTCTGGCTTCTGCTG-3' (nt 155–174), and 5'-ATGGATCC GTTGCATCCTGGGATACCTG-3' (nt 500–519) mouse nestin (Gene Bank AA166324), 5'-GGTCAAGCGATTGACCATTT-3' (nt 262–281) and 5'-CTGTGC ATCTA GGCCCAA-3' (nt 496–513); mouse vimentin (Gene Bank AA637155), 5'-CCTCATTCCCTTGTTGCAGTT-3' (nt 48–69), and 5'-CTGGCCTGACGTGTAT CAA-3' (nt 372–399); mouse neurogranin RC3 (28), 5'-TGGACTGCTGCACGGAGAGCGC-3'(nt 256–278); and 5'-GGC GGCGGC CCCAGCGGAG-3' (nt 467–486); mouse CaMKIV (29), 5'- GGACAGCACAG ATCTTCTGGG-3' (nt 1130–1151), and 5'-GAGAAGCTGAAGAGTG TGGAGG-3' (nt 1348–1359). PCR products were analyzed by 5% polyacrylamide gel and quantified by PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The quantification of mRNA expression was normalized to the corresponding mouse ß-actin mRNA level.

Northern blot analysis
Total RNA (15 µg) was isolated from wt ES and mutant ES (mES) cells with or without hormone treatment (see Cell Culture and Differentiation). RNA was separated on a 1.2% agarose gel and transferred to a nylon membrane (Gene Screen Plus, NEN Life Science Products, Boston, MA) in 10x saline sodium citrate transfer buffer. The membrane was probed with 32P-labeled mouse RC3 cDNA in hybridization buffer (5x saline sodium citrate, 50% (wt/vol) deionized formamide, 1x Denhardt’ solution, 100 µg/ml denatured sheared salmon sperm DNA, and 1% SDS) at 42 C overnight. After washing the excess probe, the Northern blot was analyzed by PhosphorImager. The data were normalized to 18S RNA density.

Immunofluorescence staining
Cell aggregates were cultured on microscope cover slips in either the presence or absence of hormone treatment for 5 d. Cells were gently washed with PBS and fixed with 4% (wt/vol) paraformaldehyde in 0.1 M PBS overnight at 4 C. The cells were washed with 0.1 M PBS for 10 min, a total of three times, and then stained with monoclonal antibody Neurofilament 160 (diluted 1:200) (Sigma, St. Louis, MO) for 12–18 h at 4 C in a humidified chamber. The cover slips were washed 3 x 10 min with 0.1 M PBS before incubation with antimouse conjugate reagent fluorescein isothiocyanate (FITC) (1:300 dilution) (Sigma). The stained cells were photographed on a microscope at a magnification of 300x.

[125I]T3-binding assay
The preparation of nuclear extracts was modified from a previously described technique (30). The nuclear extract (15 µg) was incubated in buffer B (without glycerol) with increasing concentrations of [125I]T3 (0.01–0.08 pmol) either in the presence or absence of a 500-fold molar excess of cold T3. The incubation was carried out at 4 C for 4 h. When incubation was completed, mixtures were transferred to a 96-well multiscreen filtration system. The bound and free [125I]T3 was separated by filtration through a nitrocellulose membrane. The membrane was washed three times with buffer B (without glycerol), and then the membrane from each well was punched out and counted in a {gamma} counter.

Statistical analysis
Studies were performed in triplicate, unless otherwise noted, and the data are presented as mean ± SD. All studies with the mutant ES cells were verified with at least two of the three separately characterized mutant clonal lines. Data were analyzed by ANOVA with significance at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of TR{alpha}1 mutant ES cells
All thyroid hormone receptor mutations associated with the resistance to thyroid hormone syndrome have been identified in the TRß gene. TR{alpha}, however, is the principal receptor expressed in developing ES cells. A TR{alpha} mutation (P398H), based on the TRß mutant P449H associated with resistance to thyroid hormone, was previously shown to disrupt T3 binding and function as a dominant-negative receptor (31). In vitro studies showed that the dominant-negative activity of the TR{alpha} P398H was similar to TRß dominant-negative mutants (P449H and P448L) (31). We used a hit-and-replace strategy to generate a point mutation in the TR{alpha} gene, resulting in the P398H dominant-negative TR{alpha} gene mutation (Fig. 1Go). The hit-and-replace method is based on the classic hit-and-run technique, but the reciprocal homologous recombination does not leave any integrated vector sequence at the target locus (32, 33). Five correctly targeted clones of 10 clones tested were selected after transfection with the first vector and expanded. These cells were transfected with the second vector and grown with ganciclovir. The surviving clones were screened by PCR followed by direct DNA sequencing, and 22% of those tested (5 of 23 clones) were correctly targeted.

TR{alpha} P398H mES clones (three of five clones identified) were analyzed for functional TR content as reflected in T3 binding and compared with wtES cells. The binding assays were done simultaneously using nuclear extracts from both cell lines and a multiscreen filtration system (see Materials and Methods). Saturable hormone binding was detected at 0.45 pmol T3 for wtES cells and at 0.35 pmol for mES cell lines (Fig. 2AGo). The number of T3-binding sites in mES cells (35 fmol/100 µg protein) was reduced 50%, compared with wtES cells (70 fmol/100 µg protein). The dissociation constant for T3 binding in mES cells (0.6 nM) was significantly reduced, compared with wtES cells (1.8 nM) (Fig. 2BGo) as expected from previous in vitro studies (31).



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  		Figure 2. [125I]T3 binding to nuclear extracts of wtES and mES cells and Scatchard analysis. Nuclear extracts of ES cells (15 µg) (A) and mES cells (15 µg) (B) were incubated with increasing concentrations of [125I]T3 in either the presence or absence of a 500-fold molar excess of cold T3. After 4 h of incubation at 4 C, the bound and free T3 was separated and radioactivity counted (as described in Materials and Methods). The assays were done in triplicates at each concentration, and the mean of three determinations with SD is shown.

 
Morphology of hormone-induced neural differentiating wtES cells
Undifferentiated wtES cells were grown as colonies or patches in culture. To induce neural differentiation, cells were grown in conditioned media supplemented with RA, T3, or combined RA/T3. After 2–3 d of hormone treatment, wtES cells showed neural precursor morphology and cluster-forming cells (34, 35). After 5 d of treatment, neuronal morphology was observed with multiple neurite outgrowths (Fig. 3Go). A similar pattern of differentiation was seen with the mES cells (data not shown).



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  		Figure 3. Hormone-induced neural differentiation of wtES cells. wtES cells were differentiated for 5 d in conditioned media supplemented with 1 µM RA, 1 nM T3, or combined RA 1 µM/T3 1 nM. Control cells did not receive hormone treatment.

 
Neurofilament 160 (NF160) protein expression
To confirm the neural origin of the cells and determine whether other cell types were present, wtES and mES cells were differentiated on cover slips and stained with anti-NF160 antibody. In both wtES (Fig. 4AGo) and mES cells (Fig. 4BGo), T3, RA, or T3/RA treatment resulted in uniform NF160 antibody staining, indicating cells of neuronal lineage.



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  		Figure 4. Immunofluoresence staining of differentiated wtES and mES cells. Embryoid bodies were plated on cover slips and differentiated for 7 d in the presence of hormones as indicated. Cells were fixed with 4% (wt/vol) paraformaldehyde, stained with monoclonal antibody NF160 incubated with FITC. RA-, T3- or RA/T3-treated cells were incubated with NF160 and then conjugated with FITC. A, wtES cells; B, mES cells. Three groups of controls were used. C, Undifferentiated cells were stained with NF160 conjugated with FITC. wtES cells and mES cells were differentiated but only incubated with FITC.

 
Neuron marker gene expression in wtES and TR{alpha}-mutant ES cells
Differentiating ES cells have previously been shown to express genes sequentially in the same pattern as those seen in the in vivo developmental program (e.g. hematologic cell lines and skeletal muscle) (22, 24). To assess neural differentiation induced by T3 and RA treatment in detail, the expression of a number of neural cell-specific marker genes, reflecting stages of neural differentiation, were analyzed (Fig. 5AGo). Nestin is a neuron-specific gene expressed in proliferating neuronal precursor cells (34). Only a trace of nestin mRNA was detected in the undifferentiated wtES and mES cells by RT-PCR, and levels were significantly induced as a result of T3- or RA-stimulated neural differentiation (Fig. 5BGo). The overall expression of nestin, however, was much lower than subsequent markers, indicating that most neuronal cells had progressed to a later stage of differentiation.



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  		Figure 5. RT-PCR analysis of mRNA for specific markers of neural differentiation. Cells were induced with hormone to differentiate for 5 d. RNA was isolated and 5 µg was used for reverse transcription. cDNA (4 µl) was used for PCR amplification for 25 cycles with {alpha}-[33P]dATP labeling. The amplified bands were analyzed by 5% polyacrylamide gel and scanned. The error bars represent the SD of the mean from three separate experiments. In each panel the results of PCR analysis of mRNA as well as the quantitated density relative to ß actin are shown. Significant differences are shown for the comparison of wtES and mES cells. A, Diagram of neural differentiation process and the gene markers used in this study to determine the extent of neural differentiation in ES cells. B, RT-PCR of mRNA of nestin, vimentin, and collagen IV{alpha}1. C, Quantification of mRNA levels shown in B by PhosphorImager.

 
Vimentin is expressed along with nestin in proliferating neural precursor and later in astrocytes (34, 35). Vimentin is transiently expressed in all precursors of both neuronal and glial cells during development of the nervous system. As these cells differentiate, vimentin is replaced by neurofilament in neurons and glial fibrillar acidic protein in glial cells. Vimentin was expressed at a low level in undifferentiated cells and significantly induced in wtES cells by addition of T3 or RA (Fig. 5CGo). In mES cells, however, vimentin mRNA levels were significantly lower than in wtES cells. Recent studies in vimentin-deficient mice showed that Purkinje cell growth was stunted with necrosis and loss of spiny branchlets (36, 37). Because vimentin is also expressed in astrocytes, the induction of vimentin mRNA by hormone treatment may suggest both neurogenesis and astrogenesis in the ES cells. We measured the expression of glial fibrillar acidic protein mRNA as a marker of glial cells and did not detect glial fibrillar acidic protein signal by RT-PCR in cells from any of the treatment conditions.

Collagen IV{alpha}1 was previously reported as a marker of neural differentiation (38). Collagen IV{alpha}1 mRNA was not detectable by RT-PCR in undifferentiated wtES or mES cells (Fig. 5DGo). Both RA and T3 treatment induced collagen IV{alpha}1 mRNA significantly. The maximum induction of collagen IV{alpha}1 mRNA was seen in RA/T3-treated cells. In mES cells, RA or T3 treatment resulted in similar levels of collagen IV{alpha}1 mRNA levels as in wtES cells, but combined RA/T3 treatment resulted in significantly less induction of collagen IV{alpha}1 mRNA expression in mES cells, compared with wtES cells.

In general, T3-induced neural differentiation was similar to that seen with RA or RA/T3 treatment in wtES and mES cells. These findings are consistent with generally normal gross brain development in mice with TR receptor inactivations (9, 10, 11, 12). We postulated, however, that more subtle defects in gene expression could be identified.

Effect of TR{alpha} P398H mutation on T3-mediated gene expression
We wanted to determine whether specific T3-mediated gene expression was impaired in the TR{alpha} P398H mutant ES cells. We, therefore, examined the expression of previously recognized T3-responsive genes, RC3 and CaMKIV. RC3 is expressed in the neurons of the central nervous system and is especially enriched in neostriatum, neocortex, and hippocampus (28, 29). RC3 mRNA was detected at trace levels in untreated wtES and mES cells. RA treatment had no significant effect on RC3 mRNA expression in either wtES or mES cells (Fig. 6AGo). T3 treatment augmented RC3 mRNA 21-fold in wtES cells, but only very low expression was seen in mES cells (Fig. 6AGo). This demonstrates a clear T3-dependent gene in neural development, which is not expressed only as a consequence of differentiation but specifically requires TR.



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  		Figure 6. Effects of the TR{alpha} gene P398H mutation on RC3 and CaMKIV mRNA expression. ES cells (wtES and mES) were differentiated for 5 d under hormone treatment. Total RNA was isolated and analyzed for RC3 mRNA (A) for CaMKIV mRNA (B) by RT-PCR. PCR amplification was done in 25 cycles with 2 µl cDNA. Quantification was performed by PhosphorImager and normalized to the density of ß actin mRNA for RT-PCR. The error bars represent the SD of the mean from three separate experiments.

 
To confirm the RT-PCR results used for quantification, we additionally performed Northern analysis of RC3 mRNA (Fig. 7Go). Northern analysis showed a similar pattern of RC3 mRNA expression. A trace amount of RC3 mRNA was detected in both untreated and RA-treated wtES and mES cells. T3 treatment significantly induced RC3 mRNA in wtES cells but not in mES cells.



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  		Figure 7. Northern analysis of RC3 mRNA expression. Treatment conditions for wtES and mES cells are described in Fig. 6Go. Total RNA was isolated and an aliquot of 15 µg RNA was used for Northern blotting. The RNA was probed with 32P-labeled mouse RC3 cDNA. The hybridized bands were quantified using PhosphorImager and normalized to 18S RNA density.

 
Studies indicate that CaMKIV-deficient mice have defective synaptic plasticity and exhibit impaired neuronal cAMP response element-binding protein phosphorylation and Ca2+/cAMP response element-binding protein-dependent gene expression (40). The TR{alpha} P398H dominant-negative mutation also selectively impaired expression of CaMKIV. Although RA treatment modestly stimulated CaMKIV mRNA, T3 stimulated CaMKIV mRNA 4.5-fold in wtES cells but only 2.6-fold in mES cells (Fig. 6BGo).

Differential effects of RA and T3 on proliferation and cell cycle during cell differentiation
The influence of RA and T3 on differentiation and proliferation varies, depending on the system used and cell type studied. In general, cells treated with RA show a reduced proliferation rate and increased differentiation (38, 41). T3 treatment has been reported to stimulate cell proliferation or differentiation, depending on the type of cells or tissue studied (42, 43, 44, 45, 46).

We used a FACS to analyze the cell cycle of hormone-treated cells. The number of cells in G0-G1 phase, in hormone-treated cells, was significantly greater and in S phase significantly reduced, compared with untreated cells (Fig. 8AGo). There were similar patterns of the fraction of cells in G0-G1, G2-M, and S phase among the three hormone conditions. RA treatment induced apoptosis in 25% of cells, but T3 induced apoptosis in only 9% of cells (P < 0.05). Remarkably, combined T3/RA treatment significantly moderated the apoptotic effects of RA, inducing only 7% of cells into apoptosis, significantly less than RA alone (P < 0.05) and not significantly different from T3 alone (Fig. 8CGo).



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  		Figure 8. Cell cycle analysis. wtES cells were treated with/without hormones for 5 d. The cells were collected and fixed at a density of 1 million cells/ml. Cell cycle was analyzed by FACS. wtES cells (A) and mES cells (B) in G0-G1, G2-M, and S phase (*, P < 0.05 hormone treatment, compared with control). C, Apoptosis (*, significantly different, P < 0.05, from untreated, T3 and T3/RA treatment).

 
Mutant ES cells showed a similar pattern of cell cycle change in response to hormone treatment with a significant reduction in the fraction of cells in G0-G1 phase and an increase in the fraction of cells in S phase (Fig. 8BGo). The apoptosis rate in mES cells were significantly increased in T3- (14.2%) and T3/RA-treated (15.6%) cells, compared with wtES cells (Fig. 8CGo). These results indicate that during neural differentiation, apoptosis was not significantly reduced by T3 treatment in the presence of the dominant-negative TR{alpha} P398H mutation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Thyroid hormone alone can induce neural differentiation of ES cells, although the extent of differentiation as assessed by gene markers was similar to that seen with RA alone. In vitro differentiation of ES cells into neurons requires a hormonal signal but is also a consequence of the various culture conditions and plating techniques. The close regulation and coregulation of genes by RA and T3 make it especially difficult to separate out these mechanical effects from direct hormone signaling and specific gene regulation. The development of a TR{alpha} mutant ES cell line permits identification of T3-specific effects.

In early embryonic development, TR{alpha} is widely expressed and TRß is either at very low or undetectable levels (47, 48, 49, 50, 51, 52). Studies in rats have shown that TR{alpha}1 mRNA is expressed on embryonic d 14 in the developing brain and reaches a peak in the first postnatal week (7, 47). The level of TRß mRNA is very low before birth and increases 40-fold during the first postnatal week (6). During chick embryogenesis, the high levels of TR{alpha} expression are seen in the neural plate and neural tube (47). Our findings of a 50% reduction in nuclear T3 binding after mutating one TR{alpha} allele indicate that TR{alpha} is the only functional TR isoform in early ES cell development. It is also the first demonstration of functional TR in ES cells.

The most significant finding in the TR{alpha} mutant cells was the selective reduction in T3-induced gene expression. There are no reported T3-responsive genes in ES cells, although there are several known T3-responsive genes in the brain. Previous studies have shown that RC3 is a T3-responsive gene in the adult brain (53). RC3 expression was almost completely absent in the TR{alpha} mutant cells. The T3 induction of CaMKIV was also reduced, although some residual and RA-induced expression was seen. The CaMKIV hormone response element is stimulated by RA as well as T3 (Liu, Y.-Y., and G. A. Brent, unpublished observation).

Thyroid hormone has been known to stimulate growth and proliferation in a number of cell lines including neuroblastoma (44), neuroblast (45), chromaffin cells (54), and pituitary tumor cell line (46). In our studies, both T3 and RA stimulated differentiation. The cell cycle shift as a consequence of differentiation was similar among the hormone conditions. RA induced significantly more apoptosis, compared with T3, which was largely reversed by combined T3/RA treatment. In mES cells, T3 and T3/RA treatment significantly increased apoptotic effect, compared with wtES cells, suggesting that the T3 signaling pathway was affected by TR{alpha} mutation and lost its moderator role in blunting RA-induced apoptosis.

The findings from our model system of neural differentiation of ES cells demonstrate the importance of TR{alpha}1 in early neuronal development. Although selective TR{alpha} knockouts in mice do not have apparent abnormalities in brain development, the neurological function has not been examined in detail. Recent studies on knockin TR{alpha}1 (PV) mutant mice (55) showed that a dominant-negative mutation causes increased mortality and infertility. Mice with a TR{alpha} mutation showed markedly reduced glucose utilization in all brain regions, compared with TRß mutant, which implicates synaptic activity and related neurological development may be impaired in TR{alpha} mutant mice (56). In addition, it has been difficult to examine neural differentiation at stages of early embryogenesis. In the case of TRß, a dominant-negative mutation has much more significant effects on the brain than a TRß knockout (15). Finally, the modest manifestations of TR gene knockouts on brain development and function appear to contradict the many clinical manifestation of thyroid hormone deficiency on the brain. Part of the explanation may be the more significant repressive effects of unliganded compared with absent TR. Another feature is that abnormalities, such as mental retardation, with great clinical significance may be difficult to detect in a rodent model. Such abnormalities may relate to more subtle changes, especially the timing of T3-responsive gene expression (6). The studies presented here, using the model system of in vitro differentiation of ES cells, lead to the conclusion that TR{alpha}1 plays a role in ES cell neural differentiation in regulation of specific genes.

There are a range of applications of an in vitro system of T3-dependent neural differentiation of ES cells. The relative influence of T3 on the timing and magnitude of gene expression can be tested. Pharmacological agonists and antagonists of T3 action can be tested. The influence of putative toxins, or endocrine disrupters of T3 action, can be assessed (57). This system can facilitate the identification of novel genes regulated by thyroid hormone in early neural development. In addition to morphology and gene expression, functional measurements can be made. The applications of a similar system to study differentiation of ES cells into cardiac myocytes was recently described (58, 59). The recent development of human embryonic stem cells by two laboratories (60, 61) greatly expands the therapeutic possibilities of neuronal differentiation of embryonic stem cells.


   Acknowledgments
 
RC3 cDNA was kindly supplied by Dr. J. Bernal, Instituto de Investigaciones Biomedicas, Madrid, Spain.


   Footnotes
 
This work was supported by research funds from the American Thyroid Association, the Department of Veterans Affairs, and NIH Grant CA-89364.

Abbreviations: EB, Embryoid body; ES, embryonic stem; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; m, mutant; mES, mutant ES; neo-tk, neomycin-thymidine kinase; NF160, neurofilament 160; RA, retinoic acid; TR, thyroid hormone receptor; wt, wild-type; wtES, wild-type ES.

Received November 19, 2001.

Accepted for publication March 22, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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