Thyroid hormone-responsive genes in developing cerebellum include a novel synaptotagmin and a hairless homolog

Abstract

Proper development of the mammalian CNS requires sufficient thyroid hormone; thyroid hormone deficiency during a brief perinatal period produces severe neurological defects in humans and experimental animals. Thyroid hormone exerts its effects through nuclear receptors, which modulate the transcription of downstream genes in response to hormone binding. Surprisingly, few genes that are regulated by thyroid hormone receptors in the CNS have been described. Here, I report the isolation and characterization of genes that are expressed in response to thyroid hormone in developing rat brain. One such gene (Srg1) encodes a novel protein related to synaptotagmin, a protein involved in regulating neurotransmitter release; another (hr) encodes a putative zinc finger protein related to the product of a recently identified mouse gene, hairless. Both Srg1 and hr are induced rapidly (<4 hr), suggesting that they are regulated directly by thyroid hormone. The temporal and spatial expression of both Srg1 and hr is characteristic of genes important to nervous system development. Srg1 and hr are likely part of a cascade of gene activation induced by thyroid hormone that is critical for CNS organization and development.

Fig. 1.

Isolation of novel thyroid hormone-responsive genes from developing rat brain. A, Influence of thyroid hormone on expression of rat growth hormone (GH) mRNA in methimazole-treated rats. RNA prepared from the pituitaries of 12-d-old rats was used for Northern analysis with a radiolabeled rGH cDNA probe. hypothyroid, Methimazole-treated;euthyroid, untreated; control, hypothyroid animals injected with saline; TH-treated, hypothyroid animals injected with thyroid hormone. Thyroid hormone treatment was for 24 hr. Top panel, Autoradiograph of Northern blot; bottom panel, ethidium bromide-stained gel. B, Northern analysis for two novel thyroid hormone-responsive genes (TRG16 andTRG37). A cDNA fragment isolated from the subtracted library is the probe; total RNA (15 μg per lane) prepared from cerebellum of hypothyroid P12 rats injected with saline or thyroid hormone for 48 hr was used. Right panel, Ethidium bromide-stained gel indicates equivalent loading and positions of 18S and 28S RNAs.

Fig. 2.

TRG37 encodes a putative zinc finger protein related to hairless; TRG16encodes a novel synaptotagmin. A, Amino acid sequence ofTRG37 (94% amino acid identity with ORF from mousehairless gene). Cysteine residues potentially involved in formation of a zinc finger are underlined.B, Amino acid sequence of TRG16(Srg1). Protein kinase C-related (C2) domains areunderlined; putative transmembrane domain is indicated by dashed underline. C, Schematic representation of Srg1 structure.Cross-hatched boxes represent C2 domains; Srg1 shows between 26 and 37% amino acid identity in the region encompassing the C2 domains with synaptotagmins I–VIII (19–31% in C2-A; 33–42% inC2-B). Black box(TM) at the N terminus indicates putative transmembrane domain. The complete nucleotide sequences have been submitted to GenBank under accession numbers U71293 (hr) and U71294 (Srg1).

Fig. 3.

Srg1 and hr respond rapidly to thyroid hormone. So that the kinetics of response to thyroid hormone could be determined, 12-d-old hypothyroid rats were injected with saline (−) or thyroid hormone (+) and killed at various times after injection. Total RNA (10 μg per lane) prepared from cerebellum was used for Northern analysis with ³²P-labeled cDNA probes for hr (top) or Srg1(bottom). (−), Control; (+), TH-treated;hr, hr after injection. Two independent experiments gave the same results. B, hrresponds to thyroid hormone in the absence of protein synthesis. GH1 (rat pituitary) cells were grown in hormone-free media (−) for 2 d. T₃ was added to 10⁻⁷m(TH); cycloheximide (CHX) was added to 10 μg/ml for 16 hr. Total RNA (15 μg per lane) was used for Northern analysis.

Fig. 4.

Developmental expression of hr andSrg1 in brain. Northern analysis of RNA prepared from the brains of embryonic and neonatal rats. Parallel blots (10 μg of total RNA per lane) were hybridized with ³²P-labeled cDNA probes from hr, Srg1,TRβ1, TRα1 (top tobottom). E, Day of gestation;E22/P0, birth; P, days after birth.

Fig. 5.

Tissue distribution of hr andSrg1. A, RNA was prepared from various tissues collected from euthyroid animals on postnatal day 12. Total RNA (15 μg per lane) was used for Northern analysis with³²P-labeled cDNA probes from hr(top) or Srg1 (bottom). Spleen RNA is somewhat degraded; all other RNAs are equivalent, as assessed by ethidium bromide staining. B,hr is regulated by thyroid hormone in brain, but not in skin. Shown is Northern analysis of RNA from cerebellum (CEREB) and skin (SKIN) of hypothyroid and euthyroid neonatal (P14) rats. Eu, Euthyroid; H, hypothyroid.

Fig. 6.

Expression of Srg1 andhr in neonatal cerebellum. Shown is in situ hybridization of coronal sections from cerebellum of P14 rat. Shown are results from Srg1; signal in the cerebellum with an hr probe is comparable (seeB). A, Thionine counterstain (after hybridization); B, hybridization with antisense probe;C, hybridization with sense probe.EGL, External granule cell layer;IGL, internal granule cell layer. Sections (10 μm) of frozen tissue were hybridized with ³⁵S-labeled cRNA probes.B, C, Shown are photomicrographs of an autoradiographic image of the hybridized section exposed directly to x-ray film. D, Expression induced by thyroid hormone injection is indistinguishable from euthyroid expression in neonatal cerebellum. In situ hybridization of coronal sections from cerebella of P14 rats using a cRNA probe from Srg1(top) or hr (bottom).Eu, Euthyroid; H, hypothyroid; H + TH, hypothyroid treated with thyroid hormone for 48 hr. Sections were hybridized simultaneously with the same probe preparation. Shown are photomicrographs of the autoradiographic image of the hybridized section exposed directly to x-ray film.

Fig. 7.

Expression of Srg1 in neonatal rat brain. Shown is in situ hybridization of coronal sections from P14 rat brain. Sections are shown rostral (A) to caudal (D). Hybridization with an antisense ³⁵S-cRNA probe from Srg1 is shown; sense probe gave no detectable signal. Shown are photomicrographs of an autoradiographic image of the hybridized section exposed directly to x-ray film. AAA, Anterior amygdaloid area;ACB, nucleus accumbens; BMA, basal medial amygdala; CA1, CA3, regions of hippocampus;CoAp, cortical nucleus amygdala, posterior part;CP, caudate putamen; DG, dentate gyrus;IsoCtx, isocortex; LA, lateral nucleus;LHA, lateral hypothalamic area; MeA, medial nucleus amygdala; OT, olfactory tubercle;PIR, piriform area; thal, thalamus;VB, ventrobasal complex, thalamus; ZI, zona incerta.