Douglas Forrest

Abnormal heart rate and body temperature in mice lacking thyroid hormone receptor alpha 1

Thyroid hormone, acting through several nuclear hormone receptors, plays important roles in thermogenesis, lipogenesis and maturation of the neonatal brain. The receptor specificity for mediating these effects is largely unknown, and to determine this we developed mice lacking the thyroid hormone receptor TRα1. The mice have an average heart rate 20% lower than that of control animals, both under normal conditions and after thyroid hormone stimulation. Electrocardiograms show that the mice also have prolonged QRS‐ and QTend‐durations. The mice have a body temperature 0.5°C lower than normal and exhibit a mild hypothyroidism, whereas their overall behavior and reproduction are normal. The results identify specific and important roles for TRα1 in regulation of tightly controlled physiological functions, such as cardiac pacemaking, ventricular repolarisation and control of body temperature.

TARGETED DISRUPTION OF NMDA RECEPTOR 1 GENE ABOLISHES NMDA RESPONSE AND RESULTS IN NEONATAL DEATH

In vitro studies have suggested that the NMDA receptor consists of an essential subunit, NR1, and various modulatory NR2 subunits. To test this hypothesis directly in vivo, we generated mice carrying a disrupted NR1 allele. NMDA-inducible increases in intracellular calcium and membrane currents were abolished in neurons from homozygous null mutants (NR1-/-). Thus, NR1 has a unique role, which cannot be substituted by any other subunit, in determining the activity of the endogenous NMDA receptor. A concomitant reduction in levels of NR2B but not NR2A occurred in NR1-/- mice, demonstrating that there is an interdependence of subunit expression. NR1-/- mice died 8-15 hr after birth, indicating a vital neonatal function for the NMDA receptor. Although the NMDA receptor has been implicated in several aspects of neurodevelopment, overall neuroanatomy of NR1-/- mice appeared normal. Pathological evidence suggested that respiratory failure was the ultimate cause of death.

Recessive resistance to thyroid hormone in mice lacking thyroid hormone receptor beta: evidence for tissue-specific modulation of receptor function.

The diverse functions of thyroid hormone (T3) are presumed to be mediated by two genes encoding the related receptors, TRalpha and TRbeta. However, the in vivo functions of TRalpha and TRbeta are undefined. Here, we report that targeted inactivation of the mouse TRbeta gene results in goitre and elevated levels of thyroid hormone. Also, thyroid‐stimulating hormone (TSH), which is released by pituitary thyrotropes and which is normally suppressed by increased levels of thyroid hormone, was present at elevated levels in homozygous mutant (Thrb−/−) mice. These findings suggest a unique role for TRbeta that cannot be substituted by TRalpha in the T3‐dependent feedback regulation of TSH transcription. Thrb−/− mice provide a recessive model for the human syndrome of resistance to thyroid hormone (RTH) that exhibits a similar endocrine disorder but which is typically caused by dominant TRbeta mutants that are transcriptional inhibitors. It is unknown whether TRalpha, TRbeta or other receptors are targets for inhibition in dominant RTH; however, the analysis of Thrb−/− mice suggests that antagonism of TRbeta‐mediated pathways underlies the disorder of the pituitary‐thyroid axis. Interestingly, in the brain, the absence of TRbeta may not mimic the defects often associated with dominant RTH, since no overt behavioural or neuroanatomical abnormalities were detected in Thrb−/− mice. These data define in vivo functions for TRbeta and indicate that specificity in T3 signalling is conferred by distinct receptor genes.

Neurodevelopmental control by thyroid hormone receptors.

Recent studies have provided insights into the neurodevelopmental functions of thyroid hormone signaling. The nuclear thyroid hormone receptors (TRs) are ligand-activated transcription factors and a variety of TR isotypes, generated by two genes, mediate distinct processes. In addition, deiodinase enzymes that regulate levels of the main active form of thyroid hormone, T3, are likely to cooperate closely with TRs in specifying a localized and timely response to thyroid hormones in target tissues. Some of the most sensitive processes controlled by these pathways are in the auditory and visual sensory systems.

Novel insight from transgenic mice into thyroid hormone resistance and the regulation of thyrotropin

Patients with resistance to thyroid hormone (RTH) exhibit elevated thyroid hormone levels and inappropriate thyrotropin (thyroid-stimulating hormone, or TSH) production. The molecular basis of this disorder resides in the dominant inhibition of endogenous thyroid hormone receptors (TRs) by a mutant receptor. To determine the relative contributions of pituitary versus hypothalamic resistance to the dysregulated production of thyroid hormone in these patients, we developed a transgenic mouse model with pituitary-specific expression of a mutant TR (Delta337T). The equivalent mutation in humans is associated with severe generalized RTH. Transgenic mice developed profound pituitary resistance to thyroid hormone, as demonstrated by markedly elevated baseline and non-triodothyronine (T3)-suppressible serum TSH and pituitary TSH-beta mRNA. Serum thyroxine (T4) levels were only marginally elevated in transgenic mice and thyrotropin-releasing hormone (TRH) gene expression in the paraventricular hypothalamus was downregulated. After TRH administration, T4 concentrations increased markedly in transgenic, but not in wild-type mice. Transgenic mice rendered hypothyroid exhibited a TSH response that was only 30% of the response observed in wild-type animals. These findings indicate that pituitary expression of this mutant TR impairs both T3-mediated suppression and T3-independent activation of TSH production in vivo. The discordance between basal TSH and T4 levels and the reversal with TRH administration demonstrates that resistance at the level of both the thyrotroph and the hypothalamic TRH neurons are required to elevate thyroid hormone levels in patients with RTH.

Retinoid-related orphan nuclear receptor RORβ is an early-acting factor in rod photoreceptor development

Rods and cones are morphologically and developmentally distinct photoreceptor types with different functions in vision. Cones mediate daylight and color vision and in most mammals express M and S opsin photopigments for sensitivity to medium-long and short light wavelengths, respectively. Rods mediate dim light vision and express rhodopsin photopigment. The transcription factor networks that direct differentiation of each photoreceptor type are incompletely defined. Here, we report that Rorb−/− mice lacking retinoid-related orphan nuclear receptor β lose rods but overproduce primitive S cones that lack outer segments. The phenotype reflects pronounced plasticity between rod and cone lineages and resembles that described for Nrl−/− mice lacking neural retina leucine zipper factor. Rorb−/− mice lack Nrl expression and reexpression of Nrl in Rorb−/− mice converts cones to rod-like cells. Thus, Rorb directs rod development and does so at least in part by inducing the Nrl-mediated pathway of rod differentiation.

The Thyroid Hormone Receptor β Gene: Structure and Functions in the Brain and Sensory Systems

Thyroid hormone profoundly influences the development of the vertebrate nervous system. The thyroid hormone receptor beta gene (Thrb) is a key mediator of many of these actions. The Thrb gene is complex, spanning up to 400 kb in mammals, and differentially expresses distinct receptor subtypes through independent tissue-specific promoters and alternative splicing. These receptors serve a range of functions in the brain as well as particularly sensitive functions in the auditory and visual sensory systems. The Thrb gene illustrates how versatility in neurodevelopmental control can be achieved at the receptor level.

The two thyroid hormone receptor genes have opposite effects on estrogen-stimulated sex behaviors.

The two genes coding for thyroid hormone receptors (TR) α 1 and β have opposite effects on female sex behaviors. Deletion of TRα 1 reduced them, whereas deletion of TRβ actually increased them. These results could not be attributed to altered levels of hormones in the blood, general alterations in estrogen responsiveness or altered general activity. Instead, they indicate a previously unknown molecular mechanism upon which the two TR genes exert opposite influences.

Beta receptor isoforms are not essential for thyroid hormone-dependent acceleration of PCP-2 and myelin basic protein gene expression in the developing brains of neonatal mice

In rat pups, thyroid hormone dependent brain development coincides with the appearance of the thyroid hormone receptor (TR)beta1 isoform. This finding led to the suggestion that TRbeta1 plays an essential role in brain development. The recent availability of a mouse TRbeta knockout strain allowed us to test this possibility by determining whether TRbeta is essential for the normal developmental pattern of expression of two thyroid hormone regulated brain genes, myelin basic protein (MBP), and Purkinje cell protein 2 (Pcp-2). Northern analysis of total mRNA from the brains of wild-type mice established that, as in the rat pup, the initial rate of rise of the MBP and Pcp-2 mRNA is slowed in the hypothyroid state. Supporting the effectiveness of TRbeta gene deletion was the finding that the thiiodothyronine (T3) nuclear binding capacity in the livers and brains of knockout animals was consistent with the fractional contribution of TRbeta1 to total binding capacity in the wild-type tissues. Further, no TRbeta1 could be detected by isoform-specific immunoprecipitation of nuclear receptor extracts. However, deletion of the functional TRbeta in the TRbeta knockout mice did not affect the normal ontogeny of expression of the Pcp-2 and MBP genes in the postnatal pup. We conclude that TRbeta is not essential for the normal developmental expression of these T3 dependent brain genes.

Uroporphyrinogen III Synthase AN ALTERNATIVE PROMOTER CONTROLS ERYTHROID-SPECIFIC EXPRESSION IN THE MURINE GENE

Uroporphyrinogen III synthase (URO-synthase, EC4.2.1.75) is the fourth enzyme of the heme biosynthetic pathway and is the defective enzyme in congenital erythropoietic porphyria. To investigate the erythroid-specific expression of murine URO-synthase, the cDNA and ∼24-kilobase genomic sequences were isolated and characterized. Three alternative transcripts were identified containing different 5′-untranslated regions (5′-UTRs), but identical coding exons 2B through 10. Transcripts with 5′-UTR exon 1A alone or fused to exon 1B were ubiquitously expressed (housekeeping), whereas transcripts with 5′-UTR exon 2A were only present in erythroid cells (erythroid-specific). Analysis of the TATA-less housekeeping promoter upstream of exon 1A revealed binding sites for ubiquitously expressed transcription factors Sp1, NF1, AP1, Oct1, and NRF2. The TATA-less erythroid-specific promoter upstream of exon 2A had nine putative GATA1 erythroid enhancer binding sites. Luciferase promoter/reporter constructs transfected into NIH 3T3 and mouse erythroleukemia cells indicated that the housekeeping promoter was active in both cell lines, while the erythroid promoter was active only in erythroid cells. Site-specific mutagenesis of the first GATA1 binding site markedly reduced luciferase activity in K562 cells (<5% of wild type). Thus, housekeeping and erythroid-specific transcripts are expressed from alternative promoters of a single mouse URO-synthase gene.