©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Neurogenic Precursor Proliferation by Antisense Thyroid Hormone Receptor Oligonucleotides (*)

Frank Lezoualc'h , Isabelle Seugnet , Anne L. Monnier , Jacques Ghysdael (1), Jean-Paul Behr (2), Barbara A. Demeneix (§)

From the (1) Laboratoire de Physiologie Générale et Comparée, URA CNRS 90, Muséum National d'Histoire Naturelle, 7 rue Cuvier, F-75005 Paris, the Laboratoire d'Oncogenèse Retrovirale et Moléculaire, URA CNRS 1443, Institut Curie, F-91405, Orsay, and the (2) Laboratoire de Chimie Génétique, URA CNRS 1386, Université Louis Pasteur, B.P 24, F-67401, Strasbourg, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Thyroid hormone 3,5,3`-triiodo-L-thyronine (T) is required for normal brain development in vertebrates. T acts through two classes of nuclear receptors (TR and TR) that have distinct developmental spatial and temporal distributions suggesting different functions during neuronal development. One possibility is that TR, which is expressed early in embryogenesis, is involved in neuroblast proliferation. To test this hypothesis we used the embryonic chick optic lobe, as we found that T stimulates [H]thymidine incorporation in this tissue both in vivo and in vitro during embryonic days 6-9. We applied oligonucleotides (ODNs) against TR and TR to primary cultures of chick optic lobes. By employing a cationic lipid vector we could use very low ODN concentrations (<150 nM). Antisense ODNs against TR significantly inhibited [H]thymidine incorporation, whereas antisense TR had no significant effect. However, both ODNs inhibited expression of TRs, as they blocked transcription from a T-activated reporter gene. Random ODNs used as controls had no significant effect on [H]thymidine incorporation or on T-dependent transcription. These observations suggest that TR is implicated in neuroblast proliferation and add credence to the hypothesis that the multiplicity of nuclear receptors allows for specific actions of T during development.


INTRODUCTION

During vertebrate development thyroid hormones exert pleiotropic effects on various tissues including the central nervous system (1) . In the chick and the rat, experimental hypothyroidism causes impaired development of the central nervous system, and in man hypothyroidism results in cretinism, with retarded physical and mental growth (1) . Thyroid hormones and in particular the biologically most active form, 3,5,3`-triiodo-L-thyronine (T),() exert most of their diverse effects via nuclear receptors (TR) (2) , the c-erbA proto-oncogenes, belonging to the steroid hormone receptor superfamily (3-5). TRs are transcription factors that modulate gene expression by binding as dimers to specific DNA sequences known as T response elements, thereby regulating the transcription of target genes (6) .

In the chick, as in other species, two related genes encode TRs, TR and TR (3) . Both genes are expressed in the developing chick brain (7), but as in the rat (8, 9, 10) they have distinct temporal and spatial profiles of expression (11) , suggesting they have different developmental roles. Moreover, TRs bind to the TRE sequences in target genes as homodimers or heterodimers (12, 13) . Heterodimers can be composed of either TR and TR, or of a TR receptor with another class of nuclear factor, particularly the 9-cis-retinoic acid receptors, the RXRs (14, 15; for review, see Ref. 16). These multiple possibilities generate great potential for diversity of signal response. Cellular response depends on the expression of a given set of receptors at a given time and the presence or absence of the respective ligands. Interestingly, in the case of the T and the retinoic acid systems, expression of different receptor classes and production of ligands are both developmentally regulated processes.

Many morphological, biochemical, electrophysiological, and behavioral defects result from hypothyroidism during development, especially if it occurs during a hormone-sensitive period. Thyroid hormone requirements have been implicated in differentiation of neuronal and glial populations, affecting cell migration, myelination, and synaptogenesis (1, 17) . However, little is known about specific target cells for hormone action nor of the roles of the different receptors. In particular, there are virtually no data on the requirements for T and its actions during the proliferative stages of neurogenesis. Given that TR is expressed during the early mitotic phases of development, one hypothesis would be that this receptor has a specific role to play in regulating entry into the cell cycle.

To test the hypothesis we used the positive effect of thyroid hormones on division of neuroblasts from embryonic chick optic lobe as a model system to measure the actions of antisense oligodeoxynucleotides (ODNs) directed specifically against chick TR or TR. T increased proliferation of neuroblasts both in vivo and in vitro. However, we tested the actions of the TRs in vitro, since an in vivo approach would require homogenous, highly efficient delivery of the ODNs to the whole of the neuro-epithelium population, which is not yet technically feasible. Moreover, the use of ODNs to modify gene expression is limited by their low cellular uptake and their rapid degradation by extracellular nucleases. To protect the ODNs and enhance uptake, we complexed ODNs with a cationic lipid, dioctadecylamidoglycylspermine (Transfectam, DOGS). DOGS compacts the DNA and neutralizes the anionic charges carried by the ODN that interfere with its crossing of the plasma membrane (18) . Some recent reports have shown that cationic lipids do improve uptake of ODNs by cell lines (19, 20) , but no demonstration of their usefulness for introducing ODNs into primary neurons has appeared. By using this approach we found that we could introduce ODNs (at nanomolar concentrations) into primary cultures of embryonic neurons prepared during the proliferative phase and examine their biological effects. Our findings show that TR is implicated in neuronal proliferation, a result which conforms with the expression of this gene during the early stages of development.


EXPERIMENTAL PROCEDURES

[H]Thymidine Incorporation in Vivo

Fertilized white Gallus domestic eggs (Haas, Kaltenhouse, France) were incubated at 38 °C ± 0.5 °C. In one experimental series, eggs were injected at embryonic day 5 with 100 µl of NaCl (8.6‰) containing 0.9 MBq [H]thymidine ([5`-H]thymidine, specific activity 488 GBq, Amersham, Les Ulis, France) with or without T (3 µg). This amount of T increases embryonic tissue levels of T 5-fold, from 48 pg/g in saline-treated controls to 243 pg/g with T treatment (values determined by radioimmunoassay on pools of six embryos).() Injections were made through the shell into the yolk sac. On embryonic day 8 embryos were fixed in Carnoy's solution (1 h) dehydrated through alcohol then embedded in paraffin. Coronary sections (7 µm) mounted on gelatin-coated slides were left to dry (37 °C, 3 days) then deparaffinized in xylene (three times), followed by 95% alcohol (three times), and dried overnight at room temperature. Slides were placed in contact with H-sensitive film (Hyperfilm-H, Amersham, Les Ulis, France) for 6 weeks. The films were analyzed with a densitometer. In a second series of experiments eggs were injected at embryonic day 5 with 100 µl of saline (NaCl 8.6‰) ± T (3 µg). On embryonic day 8, [H]thymidine (0.9 M Bq in 100 µl of saline) was deposited on the extra-embryonic circulation. Embryos were sacrificed 2 h later, fixed, and sections cut as described above. Slides dipped in autoradiographic emulsion (NTB-2, Kodak, Les Ulis, France) were developed 6 days later. Labeled cells in the internal and external layers of the optic tectum were counted, using a 100 objective on a Leitz microscope. For standardization, a 90-µm wide strip within the ventrolateral area of a median section (i.e. at the middle of the rostrocaudal extent of the optic tectum) was counted for both layers.

Primary Neuroblast Cultures

Cells were prepared as described (21) . Briefly, optic lobes were dissected from chick embryos at embryonic day 6 (37.5 ± 0.5 °C) and transferred to Petri dishes containing a dissection medium composed of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12, supplemented with 25 mMD-glucose, 50 µg/ml penicillin, and 100 µg/ml streptomycin. After removing the meninges, tissue was minced and mechanically dissociated. Dissociated cells were suspended in serum-free medium (SFM, 21). For immunocytochemistry and ODN incorporation, cells were seeded on glass coverslips at 4.10 cells/well. Dishes were pretreated for 30 mn with gelatine (0.25 mgml), and overnight with poly-D-lysine (M 70,000, 10 mgml in 0.15 M sodium borate buffer, pH 8.0). After rinsing with 0.1 M phosphate-buffered saline (PBS) and dHO, dishes were coated with a mixture of Dulbecco's modified Eagle's medium-Ham's F12 (1:1) supplemented with 10% fetal calf serum (FCS) for 2 h. Cultures were grown at 37 °C in humid 93% air, 7% CO.

Oligodeoxynucleotide Uptake and Stability

[P]ODN Incorporation and Stability-[P]ODN incorporation and stability were measured according to Wickstrom et al.(22) . AS was 5`-labeled with [-P]ATP (Amersham, Les Ulis, France) by using T4 polymerase kinase (Amersham). For each point 2 10 counts/min of 5`-labeled ODN (1 pmol of labeled ODN and 10 µM carrier unlabeled ODN complexed with a three times charge excess of DOGS or 10 µM carrier unlabeled ODN) was added to 4.10 cells. After a further 12 h, labeled ODN was recovered from cells and culture medium and radioactivity determined by scintillation counting. To evaluate ODN stability, aliquots of cell extracts and culture medium were electrophoresed in a denaturing 20% polyacrylamide gel and exposed to X-OMAT film (Kodak, Les Ulis, France) for 24 h.

Intracellular Localization of Rhodamine-linked ODN

For fluorescence microscopy, 1 µM AS (final concentration) labeled in 3` with rhodamine was complexed with DOGS (at a three times charge excess, see below) and added to the cells. After 6 h, cells were fixed in 4% formaldehyde PBS for 30 mn. After rinsing twice with 0.1 M PBS and distilled water, coverslips were mounted in Moviol (Hoechst) and visualized by fluorescence, Nomarski optics, and confocal microscopy.

ODNs

The translation start regions of cTR and cTR mRNA were chosen as targets to block cTRs protein expression (see Refs. 3 and 7 for cDNA sequences). ODNs, purified on SDS-polyacrylamide gel electrophoresis, were purchased from Genosys (United Kingdom) and Eurogentec (Belgium). Control ODNs with an 18-mer randomized sequence of the same A, T, G, C content as antisens (AS) and antisens (AS) were, respectively, designated as random (RD) and random (RD). To follow ODN incorporation and intracellular distribution, AS was labeled in 3` with rhodamine (Eurogentec). Sequences were as follows: AS, 5`-GCTGGGCTTCTGTTCCAT-3`; RD, 5`-GTCGGCGTTCTGTTATCG-3`; AS, 5`-ATATACCCTGACATACTG-3`; RD, 5`-ATATACCTCCAGATAGTC-3`.

Plasmids

The T3RE-LUC construct contains a synthetic ODN sequence encoding a palidromic TRE in SV-luciferase (23) . It was kindly provided by Dr. C. K. Glass, San Diego.

Transfections

The lipid used was DOGS. DOGS (135 µM DOGS/ µM ODN) was incubated in 150 mM NaCl with ODN for 10 min at room temperature. The mixture was added directly to the cells and incubated together for a given time. For plasmid transfection, cultures were transfected according to published methodology (21) . DNA was complexed with DOGS (1 µl 4 mM DOGS/µg DNA) in 100 µl of 0.015 M NaCl, then diluted into the appropriate volume of SFM. After 1 h the transfection medium was replaced with fresh SFM. Transfection efficiency is high and very regular (21) . Thus, there has never been any need to normalize for transfection efficiency in any cells so far transfected by this method. [5`-H]Thymidine Incorporation Assay in Vitro

DNA synthesis was assayed at 2 days in vitro either by scintillation counting or by determining the number of labeled cells. For scintillation counting cells were seeded at 10 cells/well in 24-well plates. At 1 day in vitro, neuroblasts were labeled with 37 kBq of [5`-H]thymidine (specific activity 488 GBq, Amersham, Les Ulis, France) for 24 h. ODNs (25-150 nM, complexed with three time charge excess of DOGS, see above) were added at 24 h. At 48 h, cells were rinsed twice with SFM and lysed with 1% sodium dodecyl sulfate. Homogenates were counted in a scintillation counter.

To determine the number of labeled cells, 10 cells/well were plated in 6-well plates. The number of labeled cells represents the proportion of cells incorporating [H]thymidine in the nucleus, reflecting DNA synthesis. Cultures were incubated with 37 kBq of [5`-H]thymidine from 1 to 2 days in vitro. ODNs (150 nM, complexed with three times charge excess of DOGS, see above) were added at 24 h. At 2 days in vitro, cells were processed for autoradiography. Briefly, cells fixed in 4% formaldehyde PBS (30 mn), were dehydrated in ethanol, dipped into NTB2 emulsion (Kodak, Les Ulis, France) at 43 °C, dried, and exposed in the dark at 4 °C for 8 days. Slides were developed in Kodak DEKTOL, fixed in Kodak UNIFIX, and washed in water. Finally, slides were lightly stained for cell bodies and nucleus using eosin, hematoxylin, and coverslipped with DPX (Electron Microscopic Sciences). Positive and negative cells in seven or eight random areas were scored for silver granules at 100 magnification on each well until 500-1000 cells were counted. The percentage of positive cells was then calculated.

Luciferase Assay

Luciferase activity was measured using the Promega Luciferase assay system according to the manufacturer's instructions using a Tropix luminometer (MGM Instruments, Hamden, CT) calibrated against a tritium standard. Light emitted was measured over 10 s.

Immunocytochemistry

TR Labeling

Cultures were fixed at 3 days in vitro with ethanol, rinsed in 0.1 M PBS, supplemented with 5% FCS and 0.05% sodium azide (PBS/FCS/NaA), then incubated with a 1:100 dilution of a polyclonal antiserum against cTRs (24). Controls for the cTRs antibody were incubated in preimmune rabbit serum. After incubation overnight at room temperature, cells were washed (three times) in PBS/FCS/NaA and incubated (1 h, room temperature) with a 1:100 dilution of biotinylated anti-rabbit Ig (Amersham, Les Ulis, France). After three rinses cells were incubated (15 mn) with fluorescein-conjugated streptavidin (1:100, Amersham). Coverslips were mounted in Moviol.

Neurofilament Labeling

Cultures at 4 days in vitro were fixed and treated as above, then incubated with a 1:5 dilution of a monoclonal antibody against the 200-kDa neurofilament subunit (Boehringer Mannheim). After incubation overnight at 4 °C, cells were washed and incubated for 1 h (20 °C) with anti-mouse Ig linked to fluorescein (1:100, Amersham).

Statistical Analysis

Student's t test was used to calculate differences between means. Differences were considered significant when p 0.05.


RESULTS

T Increases Proliferation in the Optic Tectum of 8-Day-old Chick Embryos

To test the effect of increasing T levels on neuroblast proliferation, we injected 3 µg of T into the yolks of 5-day-old chick embryos and assessed the incorporation of [H]thymidine at embryonic day 8. [H]thymidine incorporation was found in the inner and external layers of the optic tectum of both control and treated embryos in both experiments (Fig. 1, a-c). Densitometry showed that T injection at day 5 significantly increased [H]thymidine incorporated into both inner and external layers of the optic tectum (p < 0.05 in each case).


Figure 1: T treatment increases [H]thymidine incorporation in vivo. a, optical densities of internal and external layers of the optic tectums of control and T-treated chick embryos sacrificed at embryonic day 8. At embryonic day 5, embryos were injected with physiological saline (100 µl containing 0.9 MBq [H]thymidine). The T-treated embryos also received 3 µg of T (dissolved in the 100 µl of saline with the thymidine). Means ± S. E. are given, n = 2 embryos/group. Six readings per embryo were made. b, coronary sections of optic lobes from control (b, left) and T-treated chick embryos (b, right) sacrificed at 8 days of incubation. At day 5 of incubation, control embryos were injected with saline (100 µl), and T-treated embryos received 3 µg of T (in 100 µl of saline). All embryos were injected with 0.9 MBq [H]thymidine 2 h before sacrifice. I, internal layer; E, external layer. Arrows show the few labeled in cells in the external layer. Bar = 500 µm. c, numbers of radiolabeled cells in the internal and external layers of optic lobes from control and T-treated chick embryos sacrificed at 8 days of incubation. Experimental methods as for b. Means ± S.E. are given, n = 6 embryos/group. ** = p < 0.01 (Student's t test).



Applying [H]thymidine just prior to sacrifice allowed us to assess the number of cells synthetizing DNA within a 2-h period. When the number of cells labeled with [H]thymidine was counted in the inner and external layers of optic tectum from saline- and T-treated embryos, we found similar results to those obtained by densitometry. As seen in Fig. 1, b and c, most labeled cells were in the inner zone. T significantly increased numbers of cells labeled in this inner zone as compared to the saline controls (p < 0.01).

T Increases Proliferation of Neurogenic Precursors in Vitro

Our aim in these experiments was to use an antisense approach to evaluate the contributions of different TRs to neuroblast proliferation. Given the wide distribution of the neurogenic precursors throughout the neuroepithelium it is apparent that efficient and homogenous delivery of ODNs to such an extensive area is currently technically unfeasible. Thus, we applied ODNs to neurogenic precursors maintained in primary culture, checking first that T increased [H]thymidine incorporation under these conditions (Fig. 2, a and b).


Figure 2: T increases [H]thymidine incorporation in neuroblasts in vitro.Primary cultures of neuroblasts from optic lobes of 6-day-old chick embryos were cultured in the presence of increasing concentrations of T (added immediately after plating) and [H]thymidine (37 kBq) added after 24 h. After a further 24 h, cells were either lysed and total radioactivity counted (a) or fixed and the percentage of labeled cells counted after autoradiography (b). Means ± S.E. are given, n = 6/group. * = p < 0.05 (Student's t test).



A dose-dependent response to T was found (Fig. 2a). A significant effect over controls (no T added) was seen at 1 nM, with maximum effect at 10 nM T. Increasing T concentrations to 1 µM, a pharmacological level, decreased [H]thymidine incorporation as compared to that seen at 100 nM T. Significant effects of T on neuroblast proliferation were found by assessing both the total amount of [H]thymidine incorporated into the cultures (Fig. 2a) and the percentage of cells labeled on histological examination (Fig. 2b). In both cases, the maximum T-dependent increase in proliferation was approximately 30%.

Use of Lipospermine Increases Efficiency of ODN Delivery to Embryonic Neuroblasts

A major problem with ODN delivery is their instability due to nucleases in the intra- and extracellular compartments. Compaction with a cationic lipid (DOGS) should increase uptake and reduce exposure to extracellular nucleases. Fig. 3b, shows that complexing ODNs with a three time charge excess of DOGS increased the percentage of ODNs found in the intracellular medium 12 h after application from <2% when applying ODNs alone, to >95% when complexed with lipid. Moreover, gel electrophoresis of intracellular and extracellular extracts showed that if ODNs were applied without DOGS, as the majority remained in the extracellular compartment, they were completely degraded 12 h after application and no signal was seen in the intracellular compartment (Fig. 3a, lane 4). In contrast, when ODNs are applied with DOGS, no signal is found in the extracellular compartment, and the signal from the intracellular medium shows a single band migrating at the level of an 18-mer (Fig. 3a, lane 3).


Figure 3: Uptake of oligonucleotides in the presence and absence of DOGS. a, autoradiogram of denaturing polyacrylamide gel of nucleic acids extracted from intra- and extracellular medium of cells treated with P-labeled oligonucleotide (10 µM) either alone or complexed with DOGS. B and X indicate, respectively, the migration levels of bromphenol blue and xylene (comigration with 28-mer ODNs and 8-mer ODNs, respectively). b, percentage of P-labeled oligonucleotide in the intracellular and extracellular compartments of cultures treated with oligonucleotide (10 µM), supplied either alone or complexed with DOGS. For both this experiment and that depicted in a, ODNs were applied 18 h after plating the cells and cells lysed 12 h later. Means ± S.E. are given, n = 6



To determine whether the ODNs applied with DOGS were actually within the cells and not just adhering to the membrane, we used a rhodamine-labeled ODN and examined its distribution in cultures exposed to ODN alone and ODN complexed with a three times charge excess of DOGS. Fluorescent optic microscopy (Fig. 4a) and confocal microscopy (data not shown) showed that when the ODNs were applied with the lipospermine vector, a strong signal was detected in the cytoplasm. However, in the cultures exposed to ODNs applied alone no signal could be detected.


Figure 4: a, intracellular distribution of a rhodamine-labeled oligonucleotide. ODNs were applied complexed (1 µM) with DOGS. Cells were exposed to ODNs 18 h after plating, then fixed after a further 6 h. Two representative fields are shown. Cells were examined with Nomarski optics (left, upper and lower) and appropriate filters for detecting rhodamine fluorescence (right, upper and lower). In cultures where ODNs were applied without DOGS, no fluorescent signal was found (data not shown). Bar = 2.5 µm. b, characterization of neuronal cultures. Cultures maintained for 4 days in vitro were fixed and immunocytochemistry carried out for neurofilament protein. A homogenous population of neurofilament protein-positive cells is revealed. Bar = 150 µm.



Antisense ODNs against TR Inhibit Neurogenic Precursor Proliferation

We next tested the effects of antisense ODNs against different TRs on the T-dependent proliferation of embryonic neuroblasts. First, we confirmed that the culture conditions gave rise to a wholly neuronal population. As shown in Fig. 4b, neurofilament immunocytochemistry carried out on cultures at 4 days in vitro showed a homogenous population of neuronal cells.

As we had previously determined that lipospermine in creases ODN uptake by neuroblasts and that when 1 µM ODNs are applied over 95% is within the cell, we tested the effects of the ODNs at concentrations <1 µM. ODNs with an 18-mer randomized sequence of the same A, T, G, C content as AS and AS were used as controls and were, respectively, designated as RD and RD. We found that 150 nM AS inhibited [H]thymidine incorporation by 46% (Fig. 5a). This inhibition was significant (p < 0.05). Application of the same amount of AS inhibited proliferation by approximately 30%, but this decrease was not significant. Both the random oligonucleotides had small, non-significant effects on neuroblast proliferation (Fig. 5a). In some experiments the sense controls were used instead of random sequences, and again these ODNs did not have any significant effect on proliferation (data not shown).


Figure 5: Effect of oligonucleotides directed against chick TR or TR on TH-dependent mitosis. a, cultures were incubated with 37 kBq of [5`-H]thymidine from day 1 to day 2 in vitro. ODNs (150 nM, complexed with three times charge excess of DOGS) were added at 24 h. At 48 h in vitro cells were processed for autoradiography. Labeled cells were counted with the [times 100 objective of a Leitz microscope. b, dose response: at 1 day in vitro, neuroblasts were labeled with [H]thymidine and varying concentrations of antisense TR (AS) or a control, random ODN containing the same proportion of bases as the antisense (RD). ODNs were complexed with a three times charge excess of DOGS (see ``Experimental Procedures''). After 24 h cells were rinsed twice with culture medium and lysed with 1% SDS. Homogenates were counted in a scintillation counter. Means ± S.E. are given, n = 6/group. Both experiments were repeated three times; representative results from one experiment are shown. * = p < 0.05 (Student's t test).



Antisense TR Inhibits Neuroblast Proliferation in a Dose-dependent Manner

Next we carried out a dose-response study using TR antisense ODN concentrations between 25 and 150 nM. AS significantly and maximally inhibited (p < 0.05) proliferation of the neurogenic precursors at 100 nM producing 30% inhibition of [H]thymidine incorporation. (Fig. 5b). The control ODN, RD, had no significant effect on proliferation at any of the concentrations used (Fig. 5b).

Oligonucleotides against Both TR and TR Inhibit T3-dependent Transcription

To examine whether both AS sequences (AS and AS) were functional, we used a cotransfection assay, where first we transfected in the cells a reporter construct containing a TRE linked to the luciferase reporter gene, and 1 h later transfected in the antisense ODNs or their respective controls. Cells were exposed to T (10 nM) for a further 24 h and then assayed for luciferase activity. As shown in Fig. 6, addition of T increased transcription 1.45-fold in controls, and both antisense ODNs inhibited this T-dependent transcription from the TRE construct (p < 0.05, in both cases as compared to controls). Transfection of the random controls had no significant effect on T-dependent transcription.


Figure 6: Both As and AS inhibit T-dependent transcription. Transcription from a TRE-luciferase was measured in the absence of ODN (controls, Ct) or presence of ODNs directed against chick TR (AS) or TR (AS) or random (RD) controls containing the same proportion of bases as AS (RD) or AS (RD). Means ± S.E. are shown, n = 9/group, The results are combined from three separate experiments. * = p < 0.05 (Student's t test).



Antisense TR Inhibit Expression of TR Receptor Protein

To examine whether the AS ODNs were functional in decreasing the levels of TR protein, we used immunocytochemistry as the levels of protein expression were insufficient for immunoblotting. Cultures were grown in the presence of T and treated with lipospermine-complexed ODNs 36 h after plating. After a further 24 h, cells were fixed and immunocytochemistry carried out employing an antibody specific for c-erbA- raised in rabbit (24) . We found expression of a nuclear-located protein in most of the cells in cultures exposed to 0.15 µM RD (Fig. 7a). A similar nuclear reaction was revealed in control cultures not treated with ODNs (data not shown). In contrast, in cultures treated with 0.15 µM AS, a nuclear signal was much reduced (Fig. 7b). Cultures treated with preimmune serum showed no nuclear reaction (data not shown).


Figure 7: Antisense ODNs against TR inhibit expression of thyroid hormone receptors. Immunocytochemistry against c-erb-A was performed on cultures treated with a random (RD) ODN (a) or an ODN against chick TR (b). Note reduced nuclear signal in b. Bar = 2,5 µm.



DISCUSSION

Our aim in these experiments was to analyze T effects on neuroblast proliferation and to determine the type of TR implicated in the response. We used neuroblasts from the chick embryo optic lobe, and our analysis was carried out during embryonic days 6-8 (or its equivalent in vitro). This window of time was chosen for four reasons. First, it is a period during which mitosis in the optic lobe is still occurring, albeit at a lower rate than during days 4-6 (25, 26, 27) . Second, it is the earliest stage when embryonic T levels can be experimentally increased by injecting T into the yolk without inducing embryonic mortality.() Third, it is the earliest stage at which pure neuronal cultures can be prepared (28) . Fourth, TR is first detected in the embryonic chick brain at this stage (7) . Indeed this observation of TR in a number of tissues, including the optic lobe (11) during early chick embryogenesis, coupled with the finding that TR is expressed in a more restricted fashion during later in development, led Forrest and co-workers (7, 11) to suggest that different TRs might have distinct functions in development. Similar patterns of TR expression, with TR appearing well before TR are found during development of the rat (10) and Xenopus(29, 30) . Taken together, these observations provide the basis for the hypothesis that T has a role to play in the early stages of embryogenesis, and that TR is implicated in T-dependent responses occurring at this time.

The data presented here show that T does affect neurogenesis. T-treated embryos showed increased proliferation of neuroblasts as assessed by [H]thymidine incorporation (Fig. 1, a-c), and this corroborates an early report (31) showing that treatment of premetamorphic Xenopus tadpoles with T increased the number of mitotic figures in the spinal cord.

The next question was that of the TR responsible for mediating these effects. We employed an antisense strategy to modulate differentially the expression of TR and TR. Even though TR mRNA has not been detected in the chick brain before embryonic day 16 (7) , we thought it relevant to test both TR and TR, since mRNA levels are not necessarily closely correlated with protein levels. Moreover, this seems to be particularly true for TR in neuronal context. Strait and co-workers (32) found that the molar concentrations of TR protein levels exceeded those of the corresponding mRNA 350-fold in the rat cerebellum. Thus, it remains possible that TR pro-tein may be present even when the mRNA is undetectable by hybridization.

We applied antisense ODNs to neuroblasts in primary cultures in defined SFM. We first verified that T stimulated proliferation of neuroblasts in these conditions (Fig. 2, a and b). We chose this approach, rather than an in vivo methodology, as the number of cells to be targeted in vivo is very large and it is currently technically unfeasible to deliver ODNs to the whole population of the neuro-epithelium germinal layer. Delivery of ODNs to a small part of the neuro-epithelium would result in ambiguous results and unreliable interpretations. However, the in vitro approach employing pure cultures of neuroblasts has advantages: it allows a large percentage of embryonic neurons to be transfected (up to 70%, 21) and allows one to determine if the effects are direct or indirect. The data presented here suggest that the effect of T is exerted directly on the neuronal precursor cells and that T does not require other cell types to affect neuronal proliferation. Two arguments bolster this hypothesis: first, it occurred in the primary cultures of optic lobe cells that develop into virtually 100% neuronal cultures as shown by neurofilament immunochemistry (Fig. 4b), and second, most of the cells in these cultures express TRs (Fig. 7a).

Our next step was to modulate the expression of TR and TR in these embryonic neuronal precursor cells. We used a cationic lipid to vectorize the ODNs, as this method resulted in high uptake and stability of the ODNs in the intracellular compartment for up to 12 h post-transfection ( Fig. 3and Fig. 4). This enabled us to use very low ODN concentrations (<150 nM) on the embryonic neuroblasts, thus reducing toxicity and nonspecific effects. Antisense ODNs (150 nM) directed against the chick TR significantly inhibited neuroblast proliferation by up to 45% (Fig. 5a), whereas AS and random ODNs based on the TR and the TR sequences used had no significant effect. Moreover, the inhibition of [H]thymidine incorporation seen with AS was dose dependent, with a maximal effect occurring at 100 nM (Fig. 5b). This demonstration provides a physiological role for TR in early neurogenesis that correlates with expression of the mRNA for this receptor in the brain of the 6-8-day-old chick embryo (7) .

The question arose as to whether the antisense TR chosen was functional in inhibiting the production of TR protein. To address this problem we used a cotransfection approach where we cotransfected a TRE-reporter construct with the different ODNs to be tested, AS, AS, and their respective randomized-sequence controls. Application of AS and AS reduced T-dependent transcription (Fig. 6) showing both AS constructions to have a biological effect. However, in the case of AS, T-dependent transcription was completely abolished, whereas with AS a residual stimulation of transcription in the presence of T was seen, possibly due to the action of TR, most probably the more abundant receptor. Thus, even though blocking expression of TR did not affect neuroblast proliferation, it did diminish T3-dependent transcription from a consensus TRE.

These observations suggest that if low levels of TR are present they are not required for the T-dependent stimulation of the mitotic response observed in vitro and that the response is mediated through TR. Different TRs have been shown to play distinct roles in regulating target gene transcription in defined cellular contexts (21, 33) . Indeed, structural differences between the and the forms provide a biochemical basis for differential transcriptional activities. Such structural differences in the chick TR and TR include variations in their N termini sequences (7) that engender various possibilities for phosphorylation by Ser/Thr kinases of this part of TR (34) but not TR. Other differences lie in the DNA-binding domain in a region of the second zinc finger that could be involved both in determining target gene specificity (35) and in dimer formation on TREs (36) .

Taken together our results indicate that T does have a role to play in stimulating proliferation of neurogenic precursors during neurogenesis and that TR, which is expressed during this period, is implicated in mediating this response. These observations broaden the perspective in which TRs have previously been thought to function so as to include the early formation of brain regions, as well as the later maturation phases, well known to be T-dependent (37) . They also demonstrate that TR function in early embryogenesis is independent of the embryonic thyroid, as neuronal proliferation occurs before the onset of endogenous thyroid function (at about embryonic day 11 in the chick, 38). This implies that the T present in the yolk and embryo is sufficient to activate the receptors. Significant amounts of ligand are available at this stage (39 and this report). It is probable that in other species where TR is expressed before endogenous thyroid activity, maternal supplies of hormone are sufficient for activating TR-dependent transcription during early development.

Certain fundamental questions on TR roles during development remain: what factors determine the distinctive expressions of the and genes and what will be the consequences on development if these genes are blocked in the whole animal? An important area for future work will be the manipulation of these genes, first through mutations introduced by homologous recombination and the production of transgenic animals, and second, through modulation of expression during development in species (such as amphibians) where T has dramatic effects and in which it is now possible to introduce exogenous genes (40) . More specifically concerning the role of TR, it will be interesting to see if this proto-oncogene is also implicated in other phenomena occurring during neurogenesis. Since certain proto-oncogenes, particularly c-myc(41) , affect both proliferation and apoptosis, it will pertinent to examine if TR is implicated in both these intimately related processes during neurogenesis.

In conclusion, these data suggest that TR provides a specific contribution to transducing the mitotic promoting effects of T during neurogenesis. They add credence to the hypothesis that multiplicity of nuclear receptors allows for specific actions of T in defined cellular contexts. Differential control of TR expression during development could provide a mechanism through which one extracellular signal can be linked to a variety of specific cellular responses.


FOOTNOTES

*
This work was funded by the CNRS and by grants from the Association pour la Recherche contre le Cancer (ARC), and the Association Franaise contre les Myopathies (AFM). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-1-40793607; Fax: 33-1-40793618; E mail: demeneix@cimrs1.mnhn.fr.

The abbreviations used are: T, 3,5,3`-triiodo-L-thyronine; TRE, thyroid hormone response element; ODNs, oligodeoxynucleotides; DOGS, dioctadecylamidoglycylspermine; SFM, serum-free medium; PBS, phosphate-buffered saline; FCS, fetal calf serum.

I. Seugnet, unpublished observations.

B. A. Demeneix, unpublished observations.


ACKNOWLEDGEMENTS

We thank Anne Marie Ableitner for densitometry readings and Hanem Abdel Tawab for histology.


REFERENCES
  1. Dussault, J. H., and Ruel, J.(1987) Annu. Rev. Physiol. 49, 321-334 [CrossRef][Medline] [Order article via Infotrieve]
  2. Oppenheimer, J. H., Schwartz, H. L., Surks, M. I., Koerner, D., and Dillman, W. H.(1976) Recent Progr. Hormone Res. 32, 529-565 [Medline] [Order article via Infotrieve]
  3. Sap, J., Munoz, A., Damm, K., Goldberg, Y., Ghysdael, J., Leutz, A., Beug, H., and Vennström, B.(1986) Nature 324, 635-640 [Medline] [Order article via Infotrieve]
  4. Weinberger, C., Thompson, C. C., Ong, E. S., Lebo, R., Gruol, D. J., and Evans, R. M.(1986) Nature 324, 641-646 [Medline] [Order article via Infotrieve]
  5. Laudet, V., Hanni, C., Coll, J., Catzeflis, F., and Stéhelin, D. (1992) EMBO J. 11, 1003-1013 [Abstract]
  6. Glass, C. K., Franco, R., Weinberger, C., Albert, V. R., Evans, R. M., and Rosenfeld, M. G.(1987) Nature 329, 738-741 [CrossRef][Medline] [Order article via Infotrieve]
  7. Forrest, D., Sjöberg, M., and Vennström, B.(1990) EMBO J. 9, 1516-1528
  8. Bradley, D. J., Young, W. S., III, and Weinberger, C.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7250-7254 [Abstract]
  9. Bradley, D. J., Towle, H. C., and Young, W. S.(1992) J. Neurosci. 6, 2288-2302
  10. Strait, K. A., Schwartz, H. L., Perez, C. A., and Oppenheimer, J. H. (1990) J. Biol. Chem. 265, 10514-10521 [Abstract/Free Full Text]
  11. Forrest, D., Hallbök, F., Persson, H., and Vennström, B. (1991) EMBO J. 10, 269-275 [Abstract]
  12. Andersson, M. L., Nordström, K., Demczuk, S., Harbers, M., and Vennström, B.(1992) Nucleic Acids Res. 20, 4803-4810 [Abstract]
  13. Forman, B. M., Casanova, J., Raaka, B. M., Ghysdael, J., and Samuels, H. H.(1992) Mol. Endocrinol. 6, 429-442 [Abstract]
  14. Bugge, T. H., Pohl, J., Lonnoy, O., and Stunnenberg, H. G.(1992) EMBO J. 11, 1409-1418 [Abstract]
  15. Yen, P. M., Darling, D. S., Carter, R. L., Frogione, M., Umeda, P. K., and Chin, W. W.(1992) J. Biol. Chem. 267, 3565-3568 [Abstract/Free Full Text]
  16. Stunnenberg, H. G.(1993) Bioessays l15, 309-315 [Medline] [Order article via Infotrieve]
  17. Nicholson, J. L., and Altman, J.(1972) Brain Res. 44, 13-23 [CrossRef][Medline] [Order article via Infotrieve]
  18. Behr, J. P., Demeneix, B. A., Loeffler, J. P., and Perez, J.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6982-6986 [Abstract]
  19. Cappaccioli, S., Pasquale, G. P., Mini, E., Mazzei, T., and Quattrone, A.(1993) Biochem. Biophys. Res. Commun. 197, 818-825 [CrossRef][Medline] [Order article via Infotrieve]
  20. Betram, J., Killian, M., Brysch, W., Schlingensiepen, K. H., and Kneba, M.(1994) Biochem. Biophys. Res. Commun. 200, 661-667 [CrossRef][Medline] [Order article via Infotrieve]
  21. Lezoualc'h, F., Hassan, A. H. S., Giraud, P., Loeffler, J. P., Lee, S. L., and Demeneix, B. A.(1992) Mol. Endocrinol. 6, 1797-1804 [Abstract]
  22. Wickstrom, E., Bacon, T. A., Gonzales, A., Freeman, D. L., Lyman, G. H., and Wickstrom, E.(1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1028-1032 [Abstract]
  23. Glass, C. K., Lipkin, S. M., Devary, O. L., and Rosenfeld, M. G.(1989) Cell 59, 697-708 [Medline] [Order article via Infotrieve]
  24. Goldberg, Y., Glineur, C., Gesquière, J. C., Ricouart, A., Sap, J., Vennström, B., and Ghysdal, J.(1988) EMBO J. 7, 2425-2433 [Abstract]
  25. Hart Lavail, J., and Maxwell Cowan, W.(1971) Brain Res. 28, 391-419 [CrossRef][Medline] [Order article via Infotrieve]
  26. Hart Lavail, J., and Maxwell Cowan, W.(1971) Brain Res. 28, 421-441 [CrossRef][Medline] [Order article via Infotrieve]
  27. Jacobson, M.(1991) Developmental Neurobiology, 3rd Ed. pp. 282-283, Plenum Press, New York
  28. Louis, J. C., Pettman, B., Courageot, J., Rumigny, J. F., Mandel, P., and Sensenbrenner, M.(1981) Exp. Brain Res. 42, 63-72 [Medline] [Order article via Infotrieve]
  29. Yaoita, Y., and Brown, D. D.(1990) Genes & Dev. 4, 1917-1924
  30. Kawahara, A., Baker, B. S., and Tata, J. R.(1991) Development 112, 933-943 [Abstract]
  31. Baffoni, G. M.(1960) Red. Accad. Nazl. Lincei. 28, 102-108
  32. Strait, K. A., Schwartz, H. L., Seybold, V. S., Ling, N. C., and Oppenheimer, J. H.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3887-3391 [Abstract]
  33. Strait, K. A., Zou, L., and Oppenheimer, J. H.(1992) Mol. Endocrinol. 6, 1874-1880 [Abstract]
  34. Glineur, C., Zenke, M., Beug, H., and Ghysdael, J.(1990) Genes & Dev. 4, 1663-1676
  35. Umesono, K., Murakami, K. K., Thompson, C. C., and Evans, R. M.(1989) Cell 57, 1139-1146 [Medline] [Order article via Infotrieve]
  36. Hirst, M. A., Hinck, L., Danielson, M., and Ringold, G. M.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5527-5531 [Abstract]
  37. Balazs, R., Kovacs, S., Teichgraber, P., Cocks, W. A., and Eayrs, J. T. (1968) J. Neurochem. 15, 1335-1349 [Medline] [Order article via Infotrieve]
  38. Thommes, R. C.(1987) J. Expl. Zool. Suppl. 1, 273-279
  39. Prati, M., Calvo, R., and Morreale de Escobar, G.(1992) Endocrinology 130, 2651-2659 [Abstract]
  40. De Luze, A., Sachs, L., and Demeneix, B. A.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7322-7326 [Abstract]
  41. Harrington, E. A., Fanidi, A., and Evan, G. I.(1994) Curr. Opin. Genet. Dev. 4, 120-129

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