Abstract
In the rat cerebellum, migration of neurons from the external granular layer to the internal granular layer occurs postnatally and is dependent upon the presence of thyroid hormone. In hypothyroidism, many neurons fail to complete their migration and die. Key guidance signals to these migrating neurons are provided by laminin, an extracellular matrix protein that is fixed to the surface of astrocytes. Expression of laminin in the brain is developmentally timed to coincide with neuronal growth spurts. In this study, we examined the role of thyroid hormone on the expresssion and distribution of laminin in the rat cerebellum. We show that laminin content steadily increased 2- to 3-fold from birth to maximal levels on postnatal day 8–10 then steadily decreased to a plateau by postnatal day 12 in the euthyroid cerebellum. Immunoreactive laminin appeared in the molecular layer of the euthyroid cerebellum by postnatal day 4, reached maximal intensity by postnatal day 8–10, and was gone by postnatal day 14. In contrast, laminin content in the hypothyroid cerebellum remained unchanged from birth until postnatal day 10 and then increased to maximal levels over the next two days; maximal levels were approximately 35% less than those levels in the euthyroid cerebellum. Laminin staining did not appear in the molecular layer of the hypothyroid rat cerebellum until postnatal day 10, reached maximal intensity by postnatal day 15 and disappeared by postnatal day 18, despite the continued presence granular neurons in the external granular layer. These data indicate that the disruption of the timing of the appearance and regional distribution of laminin in the absence of thyroid hormone may play a major role in the profound derangement of neuronal migration observed in the cretinous brain.
THYROID HORMONE IS an essential regulatory factor in the brain developmental program. The absence of thyroid hormone during the critical period of neurogenesis and neuronal migration results in multiple irreversible morphological abnormalities (1–5). While the consequences of hypothyroidism on brain development are well characterized, the biochemical and molecular bases for the effects of this morphogenic hormone on neuronal integration remain elusive.
Fundamental to the morphological abnormalities in the hypothyroid brain are disordered neuronal migration and blunted axonal projection (5–12). Neurons require multiple guidance cues to migrate down specific paths to their target destinations (13–16). Key guidance signals are produced by the extracellular matrix protein (ECM)1, laminin (17–23). Laminin is secreted into the ECM by the astrocytes and fixed to the astrocyte surface by binding to specific transmembrane receptors known as integrins (17, 24–26). The integrins are clustered together into structures known as focal contacts by binding to the F-actin microfilaments (27–29), and this clustering results in the presentation of the laminin bound to the integrins in a very specific pattern. The migrating neurons recognize these laminin-derived guidance signals through integrins located in the migrating neurite. In this fashion, the neuron is able to follow the laminin-derived path to its target destination. Once neuronal migration is complete, laminin disappears from the brain paranchyma and is restricted to the vasculature (19, 20, 30, 31). Any factor that disrupts the formation of the laminin derived pathways would disrupt neuronal migration.
The postnatal development of the rat cerebellum is an excellent model to study the effects of thyroid hormone on brain development (5, 8–12, 32, 33). There is extensive proliferation of granular neurons for the first few days after birth to form the external granular layer. This is followed by migration of the granular neurons from the external granular layer to the internal granular layer, where they form connections with interneurons in the inner layer and with the Purkinje cells. By postnatal day 10, extensive migration is occurring and the purkinje cells are elaborating processes. By postnatal day 21, the external granular layer is gone and the extensive wiring network of the cerebellum is established.
In this study, we examined the effects of thyroid hormone on the expression and distribution of laminin in the developing rat cerebellum.
Materials and Methods
Animals and reagents
Pregnant (16–17 day gestation) Sprague Dawley rats were obtained from Charles River Laboratories, Inc. (Kingston, NY). The study was approved by the Animal Research Committee and complies with the institutional assurance certificate of the University of Massachusetts Medical Center. Neonatal hypothyroidism was induced by adding PTU (200 mg/liter) to the drinking water of pregnant dams beginning at day 17 and continuing throughout the neonatal period.
EHS laminin, rabbit polyclonal antilaminin IgG, and BSA were purchased from Sigma (St. Louis, MO). Antirabbit IgG-horseradish peroxidase conjugate was purchased from Promega Corp. (Madison, WI), rabbit polyclonal anti-GFAP IgG was purchased from Biomedical Technologies (Stoughton, MA) and Hybond ECL nitrocellulose was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). The Lumiglo chemiluminescent kit, True Blue Peroxidase Substrate kit and HRP Blocking Solution was obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD). All other reagents were of the highest grade available.
Tissue harvest and hormone assays
Animals were killed every other day from birth to postnatal day 20. In all experiments, animals were weighed, killed by decapitation, and their blood collected for hormone assays. The cerebellum was rapidly isolated, and the vermis fixed by quick freezing the tissue in OTC compound with liquid nitrogen. The tissue blocks were kept at −70 C until sectioning. The remaining cerebellar hemispheres were homogenized in 10 volumes of 50 mm Tris (pH 8.0), 0.4 m NaCl, 5 mm EDTA and 1% Nonidet-40. The homogenates were then sonicated and kept at −70 C until use.
Serum TSH was measured in duplicate by RIA using materials obtained from the National Pituitary Agency, National Institutes of Health (Bethesda, MD). Serum T4, T3 and rT3 were determined in duplicate by species-adapted specific RIAs.
Immunocytochemistry
Tissue blocks were cut in 6-μm sections from similar orientations focusing on the midline of the vermis and three sections were mounted per slide. Sections were thaw-mounted and then fixed at− 20 C in iced acetone for 15 min, followed by iced methanol for 5 min. Sections were rehydrated in 150 mm NaCl, 10 nm Na2HPO4, pH 7.4 (PBS), endogenous peroxidases were blocked by incubation with HRP Blocking Solution and nonspecific binding sites were blocked by incubation with BSA blocker (1 mg/ml BSA in PBS). Laminin was identified by incubating sections with antilaminin IgG (1:100 dilution in BSA blocker) or BSA blocker alone (nonspecific binding control) for 1 h and immune complexes were visualized by incubation with an antirabbit IgG conjugated with HRP (1:2500 dilution in BSA blocker) followed by development with True Blue Peroxidase Substrate (antilaminin sections) or an antirabbit IgG. Sections were then dehydrated through alcohols to xylene and mounted with Permount (Fisher, Fairlawn, NJ). Astrocytes were identified by incubating sections with antibodies to the astrocyte-specific marker, glial fibrillary acidic protein (GFAP) (1:500 dilution in BSA blocker) for 1 h. Immune complexes were visualized by immunofluorescence after incubation with an antirabbit IgG conjugated with Texas Red (1:50 dilution in BSA blocker). A Carl Zeiss Axioskop microscope equipped with an Olympus Corp. OM4 camera and Kodak TMAX ASA 100 (laminin) or 400 (GFAP) film (Eastman Kodak Co., Rochester, NY) was used for image acquisition.
Slot-blot immunoanalysis
Cerebellar homogenates were thawed, 10 μl aliquots were obtained for DNA analysis, and the remaining samples spun in a microfuge for 5 min at room temperature. DNA content was determined by measuring the binding of DNA to 33258 Hoechst dye (34, 35) with a TKO 100 Mini Fluorometer (Hoefer Scientific Instruments, San Francisco, CA), and sample aliquots were normalized as to their DNA content to ensure analysis of equivalent samples of cells. Cerebellar homogenates containing 200 ng DNA were diluted in 20 mm Tris-HCl, 137 mm NaCl, pH 7.5, and proteins were denatured in a boiling water bath for 5 min then applied to nitrocellulose via slotblot under vacuum. Blots were blocked for 1 h with milk blocker (20 mm Tris-HCl, 0.1%(vol/vol) Tween-20, 5 g/ml powdered milk, 137 mm NaCl, pH 7.5) then probed for 1 h at room temperature with anti-laminin IgG (1:1000 dilution in milk blocker). Nonspecific signal was determined by incubating parallel blots with antilaminin IgG preabsorbed with excess laminin and was subtracted from the signal obtained with the antisera alone. Immune complexes were then visualized by incubation with an antirabbit IgG conjugated to horseradish peroxidase (1:2500 in blocker) and developed with the Lumiglo chemiluminescent kit. Blots were analyzed by scanning densitometry and laminin was quantified by comparison with results obtained with a standard curve of EHS laminin (0–5 ng) that was run on each blot.
Statistical methods
Where indicated, results are reported as mean ± se. Statistical analysis was performed by single-factor ANOVA. When necessary, justification for performance of single-factor ANOVA was determined by two-factor ANOVA with replication. Statistical significance was determined to be achieved at the P < 0.05 level.
Results
Neonatal hypothyroidism
As shown previously (10, 11, 32), PTU administration to the pregnant dam is an excellent method to induce neonatal hypothyroidism. Shown in Fig. 1 are the hormone levels achieved in the euthyroid and PTU-treated neonatal rats. Serum T4 and TSH concentrations showed the most marked changes between the treatment groups. In the euthyroid animals, serum T4 concentrations steadily increased from birth to a plateau at postnatal day 14–16, while remaining undetectable in the PTU-treated animals throughout the treatment period. Euthyroid serum T4 concentrations became significantly different from those of the PTU-treated animals by postnatal day 2 and remained so for the entire treatment period. Serum TSH concentrations remained stable in both sets of animals, with the TSH concentrations in the PTU-treated rats 2- to 3-fold higher than those in the euthyroid animals. Serum T3 concentrations steadily increased after birth in the euthyroid animals and were higher at all time points than in the PTU-treated animals, reaching statistical significance at postnatal day 4. Serum T3 concentrations in the PTU-treated animals remained stable throughout the treatment period. For the first few days after birth, serum rT3 concentrations were 2- to 3-fold greater in the PTU-treated rats than in the euthyroid animals. From postnatal day 6 and throughout the remainder of the treatment period, serum rT3 concentrations remained similar in both treatment groups. These results are similar to those reported by others (4, 10, 11, 32, 36).
Iodothyronine levels in euthyroid and PTU-treated developing rats. Animals were killed from birth to postnatal day 20 and blood collected for hormone assays as described in Materials and Methods. Points are mean ± se of at least five animals in at least two experiments.
The euthyroid animals steadily gained weight throughout the treatment period (Fig. 2). Body weight also increased in the PTU-treated rats, but at a slower rate and reached a significant plateau after postnatal day 12. Again, these observations are similar to those made by others (10, 11, 32) and provide further evidence that the PTU-treated animals were indeed hypothyroid.
Body weight in euthyroid and PTU-treated developing rats. Animals were weighed from birth to postnatal day 20 before they were killed. Points are mean ± se of at least five animals in at least two experiments. *, P < 0.05 compared with PTU-treated animals.
Time course of laminin expression in the developing cerebellum
Shown in Fig. 3 is the biochemical analysis of the time course of laminin expression in the neonatal rat cerebellum. At birth, laminin content in the cerebellum was similar in both the euthyroid and PTU-treated animals (Fig. 3, Table 1). In the euthyroid animal, the cerebellar content of laminin steadily increased after birth, becoming significantly greater than that observed in the PTU-treated rats by postnatal day 4 and reaching peak levels by postnatal day 8. Cerebellar laminin content then decreased to plateau levels after postnatal day 10 and remained stable throught the rest of the treatment period. In contrast, there was little change in cerebellar laminin content from birth through postnatal day 12 in the PTU-treated animal (Fig. 3). Cerebellar laminin content then increased to levels equal to the euthyroid rats by postnatal day 14 and remained stable throughout the rest of the treatment period. The maximal laminin content in the cerebellum of the PTU-treated rats was approximately 35% less than that found in the euthyroid cerebellum (Table 1). Importantly, the pattern of laminin expression in the cerebellum of the PTU-treated rat did not show a peak, as was observed in the euthyroid rats; rather, levels steadily increased from low levels to plateau levels. These data represent changes in the total laminin content of the cerebellum, which includes laminin deposited in the vasculature as well as that deposited in the cerebellum paranchyma. The regional expression of laminin in the neonatal rat cerebellum was examined next by immunocytochemistry.
Laminin content in the cerebellum of euthyroid and PTU-treated developing rats. Animals were killed from birth to postnatal day (P) 20 and the cerebella were homogenized and analyzed for laminin by Western blot as described in Materials and Methods. Points are mean ± se of triplicate values from at least five animals in at least two experiments. *, P < 0.05 compared with PTU-treated animals.
Maximal cerebellar content in developing euthyroid and PTU-treated developing rats
| Condition | Laminin content at birth | Maximal laminin content | Postnatal day maximal content achieved |
|---|---|---|---|
| (ng/μg DNA) | (ng/μg DNA) | ||
| Euthyroid | 1.19± 0.23 | 3.58± 0.69a | 8 |
| Hypothyroid | 1.01± 0.2 | 2.34± 0.38 | 14 |
| Condition | Laminin content at birth | Maximal laminin content | Postnatal day maximal content achieved |
|---|---|---|---|
| (ng/μg DNA) | (ng/μg DNA) | ||
| Euthyroid | 1.19± 0.23 | 3.58± 0.69a | 8 |
| Hypothyroid | 1.01± 0.2 | 2.34± 0.38 | 14 |
P < 0.05 as compared with PTU-treated animals.
Animals were killed from birth to postnatal day 20, and the cerebella were homogenized and analyzed for laminin by Western blot as described in Materials and Methods. Points are mean ± se of triplicate values from at least five animals in at least two experiments.
Maximal cerebellar content in developing euthyroid and PTU-treated developing rats
| Condition | Laminin content at birth | Maximal laminin content | Postnatal day maximal content achieved |
|---|---|---|---|
| (ng/μg DNA) | (ng/μg DNA) | ||
| Euthyroid | 1.19± 0.23 | 3.58± 0.69a | 8 |
| Hypothyroid | 1.01± 0.2 | 2.34± 0.38 | 14 |
| Condition | Laminin content at birth | Maximal laminin content | Postnatal day maximal content achieved |
|---|---|---|---|
| (ng/μg DNA) | (ng/μg DNA) | ||
| Euthyroid | 1.19± 0.23 | 3.58± 0.69a | 8 |
| Hypothyroid | 1.01± 0.2 | 2.34± 0.38 | 14 |
P < 0.05 as compared with PTU-treated animals.
Animals were killed from birth to postnatal day 20, and the cerebella were homogenized and analyzed for laminin by Western blot as described in Materials and Methods. Points are mean ± se of triplicate values from at least five animals in at least two experiments.
Distribution of laminin in the developing cerebellum
Shown in Fig. 4 are representative sections of the euthyroid rat cerebellum stained for laminin. Control sections incubated with either nonimmune serum or laminin antisera preincubated with excess laminin showed no significant staining at any time point (data not shown). At birth, the specific cerebellar layers have not yet formed (11, 32, 33). Because laminin is a major component of the basement membrane of the blood vessels (37), the only structure stained for laminin at this time point is the vasculature. By postnatal day 5, definite layers of cerebellar cells have formed. Laminin staining is readily apparent in the molecular layer and is distinct from the vasculature. By postnatal day 10, there is intense laminin staining diffusely throughout the molecular layer in punctate clusters and clumps (Fig. 4). This punctate pattern of laminin has been previously described in the neonatal cerebellum (18–20, 30, 38). By postnatal day 15, laminin staining in the euthyroid cerebellum has disappeared from the molecular layer and is again restricted to the vasculature (Fig. 4). In addition, there are few. If any, cells remaining in the EGL, indicating that granular neuron migration has been completed (8–11, 32, 39).
Immunocytochemical analysis of the distribution of laminin in the cerebellum of euthyroid developing rats. Animals were killed from birth to postnatal day 20, and frozen sections of the cerebella fixed and stained for laminin as described in Materials and Methods. Shown are representative sections from cerebella obtained from at least five animals in at least two experiments. ELM, External limiting membrane; V, blood vessel; EGL, external granular layer; ML, molecular layer; IGL, internal granular layer. Magnification, 400×.
Representative sections of the PTU-treated rat cerebellum stained for laminin are shown in Fig. 5. Similar to what was observed in the euthyroid animal, laminin is restricted to the vasculature at birth in the PTU-treated cerebellum. However, in contrast to the euthyroid cerebellum, no significant staining for laminin is observed in the molecular layer until postnatal day 10 in the PTU-treated cerebellum, and the most extensive laminin staining observed in these animals is found at postnatal day 15 (Fig. 5). Importantly, this pattern of laminin staining in the molecular layer of the PTU-treated cerebellum at postnatal day 15 is clearly less diffuse, with smaller punctate collections of stain, than the pattern of laminin staining in the molecular layer of the euthyroid animal observed at postnatal day 10 (Fig. 4). By postnatal day 18, laminin staining has disappeared from the molecular layer in the PTU-treated cerebellum. The EGL still contains several rows of cells on postnatal day 18, consistent with the observation by others that granular neuron migration continues up until postnatal day 21 in the hypothyroid rat (8–11, 32, 39).
Immunocytochemical analysis of the distribution of laminin in the cerebellum of PTU-treated developing rats. Animals were killed from birth to postnatal day 20 and frozen sections of the cerebella fixed and stained for laminin as described in Materials and Methods. Shown are representative sections from cerebella obtained from at least five animals in at least two experiments. ELM, External limiting membrane; V, blood vessel; EGL, external granular layer; ML, molecular layer; IGL, internal granular layer. Magnification, 400×.
Astrocytes are the source of secreted laminin in the cerebellum (18, 24, 25). One possibility for the delayed appearance of laminin in the PTU-treated cerebellum is a generalized delay in the astrocyte formation in the molecular layer. To examine this possibility, we used the astrocyte-specific marker, glial fibrillary acidic protein (GFAP) to identify astrocytes in the developing rat cerebellum. Shown in Fig. 6 are representative sections from the cerebellum obtained from euthyroid and PTU-treated rats on postnatal day 10. The top sections are stained for laminin, again showing extensive staining in the molecular layer in the euthyroid cerebellum and minimal staining in the molecular layer of the PTU-treated cerebellum. The bottom sections show extensive GFAP staining of cells in the molecular layer of both the euthyroid and PTU-treated animals, consistent with the presence of the radial Bergman glial fibers (14, 40). If anything, GFAP staining in the PTU-treated cerebellum is more extensive, extending into the EGL. Similarly, there is extensive GFAP staining in the cerebellum of both animals at postnatal days 14–15 (Fig. 7), indicating the presence of abundant astrocytes. By postnatal days 14–15, laminin has disappeared from the molecular layer of the euthyroid cerebellum while still being expressed in the molecular layer of the PTU-treated cerebellum (Fig. 7). These data show that the differential appearance and disappearance of immunoreactive laminin in the molecular layer of the cerebellum between the euthyroid and PTU-treated developing rats is not due to differential astrocyte formation in these animals.
Immunocytochemical analysis of laminin and GFAP in the cerebellum of euthyroid and PTU-treated rat pups on postnatal day 10. Euthyroid and PTU-treated rat pups were killed on postnatal day 10 and frozen sections of the cerebella fixed and stained for laminin or GFAP as described in Materials and Methods. Shown are representative sections from cerebella obtained from at least 5 animals in at least two experiments. LM, Laminin; GFAP, glial fibrillary acidic protein; EGL, external granular layer; ML, molecular layer; IGL, internal granular layer. Magnification, 400×.
Immunocytochemical analysis of laminin and GFAP in the cerebellum of euthyroid and PTU-treated rat pups on postnatal day 14–15. Euthyroid and PTU-treated rat pups were killed on postnatal day 14 or 15 and frozen sections of the cerebella fixed and stained for laminin or GFAP as described in Materials and Methods. Shown are representative sections from cerebella obtained from at least five animals in at least two experiments. LM, Laminin; GFAP, glial fibrillary acidic protein; EGL, external granular layer; ML, molecular layer; IGL, internal granular layer. Magnification, 400×.
Discussion
Laminin, a major component of the basement membrane in the vasculature (41), is a key guidance molecule in the developing brain (17–23, 41). The evidence supporting such a key role for laminin is 3-fold: 1) laminin transiently appears in the brain parenchyma around the routes of migrating neurons in the embryonic brain and is restricted to the vasculature after neuronal migration is completed (19, 20, 42–44); 2) blocking antibodies to the β1 integrin subunit of an integrin receptor complex disrupts neural crest cell migration in vivo (45); and 3) neurons attach and spread preferentially on laminin in vitro (46–49). Laminin exists in four patterns in the developing brain, two of which (small and large punctate forms) are developmentally expressed (30).
In this paper, we show that the punctate forms of laminin are transiently expressed in the molecular layer of the rat cerebellum at times coinciding with migration of the granular neurons from the external granular layer to the inner granular layer. In the euthyroid rat, small and large punctates of laminin first appear around postnatal day 3–5 when the molecular layer is formed (Fig. 4). Maximum expression of laminin in the cerebellum is found between postnatal days 8–10, and the punctate laminin disappears from the molecular layer of the cerebellum by postnatal day 15 (Fig. 4). This pattern is markedly different from that observed in the hypothyroid rat, where the punctate laminin does not begin to appear in the molecular layer until postnatal day 10, peaks on postnatal days 14–15, and disappears by postnatal day 18 (Fig. 5). The only previous study that examined laminin expression in the developing cerebellum identified punctate laminin in the molecular layer of the cerebellum at postnatal day 10, which then was not present on postnatal day 24 (20). This the first study to document, in detail, the developmental expression of laminin in the rat cerebellum.
The delayed pattern of laminin expression in the hypothyroid brain is likely to represent a pathological process rather than one due to the general delay in brain development that occurs in the cretinous brain. Essentially, all of the granular neurons have left the external granular layer by the time punctate laminin disappears in the euthyroid rat (Fig. 4), indicating that granular cell migration is complete. In the hypothyroid rat cerebellum, many granular neurons remain in the external granular layer when the punctate laminin disappears, suggesting that granular neuron migration through the molecular layer is still ongoing when punctate laminin disappears in the hypothyroid cerebellum (Fig. 5). Further, maximal expression of laminin in the euthyroid cerebellum is significantly greater than that observed in the hypothyroid cerebellum (Table 1). Thus, the expression of laminin is both delayed and diminished in the hypothyroid cerebellum. These data suggest that the regulation of laminin expression in the cerebellum by thyroid hormone may play a major role in the profound derangements of neuronal migration observed in the cretinous brain.
The mechanism whereby thyroid hormone regulates the expression and distribution of laminin in the developing rat cerebellum remains to be established. Many actions of thyroid hormone are mediated by specific nuclear receptor proteins that act as ligand-activated transcription factors (50–52). Nuclear receptors for thyroid hormone are developmentally expressed in the brain (53–55), and T3-regulated genes have been identified in the cerebellum (for review see Refs. 2, 56). Interestingly, many of these T3-regulated genes eventually achieve euthyroid expression levels even in the absence of T3 (57) or in the absence of specific thyroid hormone receptor isoforms in transgenic mice (58, 59). In contrast to the steady increase in expression observed with these previously identified T3-regulated genes, peak laminin expression in the euthyroid rat occurs early in cerebellar development, followed by a fall to a lower plateau level in the euthyroid rat. Laminin content in the PTU-treated cerebellum, which eventually achieves the lower plateau levels observed in the euthyroid rat, never reaches the peak levels observed in the euthyroid animal (Table 1). Finally, few of the genes that have been identified to be altered in the hypothyroid cerebellum have shown to also play a major role in neuronal migration. If laminin gene expression in the cerebellum is discovered to be under direct regulation by thyroid hormone, it would provide the first evidence of thyroid hormone-dependent transcriptional regulation of a protein directly involved with neuronal migration.
Laminin is synthesized and secreted by astrocytes (24–26, 42, 60); thus, any action of thyroid hormone on laminin gene expression would have to be targeted to the astrocyte. We (61) and others (62) have shown that astrocytes lack significant numbers of functional nuclear thyroid hormone receptors (TRs). The predominant (>95%) thyroid hormone receptor in astrocytes is the nonT3-binding isoform c-erbAα2 (61). Small quantities of TRα1 (61) and TRβ2 (63) have been identified in cultured astrocytes, although the dominant negative activity of the c-erbAα2 (64–66) is likely to render these TR isoforms transcriptionally inert. These studies suggest that actions of thyroid hormone in astrocytes are not mediated by thyroid hormone receptors (62).
How might thyroid hormone influence laminin expression in astrocytes, if not by nuclear receptor-mediated transcriptional regulation? We have shown that thyroid hormone, primarily T4, regulates microfilament organization (67–69) and vesicle recycling (70, 71) in cultured astrocytes via an extranuclear mechanism (26). T4-dependent alterations in the microfilament organization in astrocytes also results in altered integrin-laminin interactions (72) and altered organization of laminin bound to the astrocyte surface (26). If similar events occur in vivo, abnormal deposition of laminin into the extracellular matrix in the absence of thyroid hormone would likely activate extracellular matrix-degrading proteases that function to maintain the integrity of the extracellular matrix (73) and alter laminin protein expression without alterations in gene expression. Studies are ongoing in our lab to examine this potential paradox.
In summary, we have shown that the appearance of laminin in the molecular layer of the hypothyroid cerebellum is markedly delayed and less abundant than in the euthyroid cerebellum. These data indicate that the disruption of the timing of appearance and regional distribution of laminin in the absence of thyroid hormone may play a major role in the profound derangements of neuronal migration observed in the cretinous brain.
This work was supported by NIH Grant DK-49998 (to A.P.F). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.
Acknowledgments
The authors would like to acknowledge the technical assistance of Scott Stone, who performed the serum hormone assays.
References
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