Defining components of early thyroid hormone signalling through temperature-mediated activation of molecular memory in cultured Rana [lithobates] catesbeiana tadpole back skin.

Thyroid hormones (THs) are essential signalling molecules for the postembryonic development of all vertebrates. THs are necessary for the metamorphosis from tadpole to froglet and exogenous TH administration precociously induces metamorphosis. In American bullfrog (Rana [Lithobates] catesbeiana) tadpoles, the TH-induced metamorphosis observed at a warm temperature (24 °C) is arrested at a cold temperature (4 °C) even in the presence of exogenous THs. However, when TH-exposed tadpoles are shifted from cold to warm temperatures (4 â†’ 24 °C), they undergo TH-dependent metamorphosis at an accelerated rate even when the initial TH signal is no longer present. Thus, they possess a "molecular memory" of TH exposure that establishes the TH-induced response program at the cold temperature and prompts accelerated metamorphosis after a shift to a warmer temperature. The components of the molecular memory that allow the uncoupling of initiation from the execution of the metamorphic program are not understood. To investigate this, we used cultured tadpole back skin (C-Skin) in a repeated measures experiment under 24 °C only, 4 °C only, and 4 â†’ 24 °C temperature shifted regimes and reverse transcription quantitative polymerase chain reaction (RT-qPCR) and RNA-sequencing (RNA-seq) analyses. RNA-seq identified 570, 44, and 890 transcripts, respectively, that were significantly changed by TH treatment. These included transcripts encoding transcription factors and proteins involved in mRNA structure and stability. Notably, transcripts associated with molecular memory do not overlap with those identified previously in cultured tail fin (C-fin) except for TH-induced basic leucine zipper-containing protein (thibz) suggesting that thibz may have a central role in molecular memory that works with tissue-specific factors to establish TH-induced gene expression programs.

Uncovering early thyroid hormone signalling events through temperature-mediated activation of molecular memory in the cultured bullfrog tadpole tail fin.

Thyroid hormone (TH) is a critical signalling molecule for all vertebrate organisms, playing a crucial role in postembryonic development. The best-studied mechanism of TH response is through modulating gene expression, however TH's involvement in coordinating the early steps in the TH signal transduction pathway is still poorly understood. The American bullfrog, Rana [Lithobates] catesbeiana, is a useful model to study these early responses as tadpole post-embryonic development in the form of metamorphosis of the tadpole into a frog can be experimentally induced by TH exposure. The rate of TH-induced metamorphosis can be modulated by temperature where sufficiently cold temperatures (5 °C) completely halt precocious metamorphosis. Interestingly, when premetamorphic tadpoles exposed to exogenous THs at 5 °C are shifted to permissive temperatures (24 °C), their metamorphic rate exceeds that of TH-exposed tadpoles at the permissive temperature. This suggests that a molecular memory of TH exposure is retained at 5 °C even after THs are cleared at this low temperature. However, the molecular memory machinery is poorly understood. Herein we use RNA-seq analysis to identify potential components of the molecular memory in cultured tail fin that allows for the recapitulation of the molecular memory phenomenon. Eighty-one gene transcripts were TH-responsive at 5 °C compared to matched controls indicating that the molecular memory is more complex than previously thought. Many of these transcripts encode transcription factors including thyroid hormone-induced B/Zip, thibz, and a novel krüppel-like factor family member, klfX. Actinomycin D and cycloheximide treatment had no effect on their TH induction suggesting that a change in transcription or translation is not required. Rather a change in RNA stability may be a possible mechanism contributing to the molecular memory. The ability to manipulate temperature and TH response in cultured organs provide an exciting opportunity to further elucidate the early TH signalling mechanisms during postembryonic development.

Differential expression of RNA exosome subunits in the amphibian Lithobates catesbeianus during reproductive and non-reproductive periods.

OBJECTIVE: The RNA exosome is an evolutionarily conserved 3'-5' exoribonucleolytic protein complex involved in processing and degradation of different classes of nuclear and cytoplasmic RNAs, and, therefore, important for the posttranscriptional control of gene expression. Despite the extensive in vivo functional studies and the structural data on the RNA exosome, few studies have been performed on the localization and expression of exosome subunits during gametogenesis, process during which gene expression is largely controlled at the posttranscriptional level. RESULTS: We report the identification of exosome subunits in Lithobates catesbeianus and analysis of the differential subcellular localization of RNA exosome core and catalytic subunits in testis cells. In addition, we show seasonal differences in the expression levels of four exosome subunits in different organs. In addition to being part of the RNA exosome complex, its subunits might participate independently of the complex in the control of gene expression during seasonal variation in bullfrog tissues. These results may be relevant for other eukaryotic species.

A novel candidate tumor suppressor, ING1, is involved in the regulation of apoptosis.

We have recently cloned a novel growth inhibitor and candidate tumor suppressor called p33ING1 (I. Garkavtsev et al., Nature Genet., 14: 415-420, 1996). Because some tumor suppressors participate in the regulation of apoptosis, we hypothesized that the ING1 gene may also play a role in this process. Our results show that p33ING1 levels increase upon the induction of apoptosis in P19 teratocarcinoma cells by serum deprivation. Elevated expression of ING1 in P19 and rodent fibroblast cells containing a tetracycline-controlled human c-myc gene enhanced the extent of serum starvation-induced apoptosis. This suggests that the pathway by which ING1 modulates cell death is synergistic with Myc-dependent apoptosis. Conversely, constitutive expression of an antisense construct of INGI conferred protection against apoptosis in these cells. These data support the idea that loss of proper ING1 function may facilitate tumorigenesis, in part, by reducing the cell's sensitivity to apoptosis.

Quiescence versus apoptosis: Myc abundance determines pathway of exit from the cell cycle.

When exposed to diverse growth conditions in vitro, cells can respond by entering states of proliferation, quiescence, differentiation or apoptosis. While the choices among these states can be influenced by proto-oncogene expression, how these disparate outcomes are achieved remains poorly understood. To address these issues, we have generated rodent fibroblast cell lines that harbor a human c-myc gene under the control of a tetracycline-regulated promoter. When Myc-induced cells are deprived of serum growth factors, they rapidly become apoptotic with the onset of apoptosis preceded by a large, transient increase in cdk2 kinase activity that is associated with the induction of cdc25A phosphatase and the later accumulation of p27Kip1 kinase inhibitor. Surprisingly, serum starvation in the absence of myc overexpression, (which leads to quiescence instead of apoptosis) also causes a marked transient elevation in cdk2 kinase activity, an induction of cdc25A and a delayed increase in p27Kip1. Transient elevations in cdk2 kinase activity and cdc25A abundance are required for cell cycle progression, but it is evident that these changes also precede entry to either apoptosis or quiescence in serum-starved cells. These findings suggest that the pathways to both quiescence and apoptosis share regulatory machinery with cell cycle control mechanisms. In addition, the abundance of Myc protein can be critical in the choices among these cellular states.

Detection of environmental endocrine-disruptor effects on gene expression in live Rana catesbeiana tadpoles using a tail fin biopsy technique.

We describe a novel method of rapidly assessing exposure of premetamorphic Rana catesbeiana tadpoles to endocrine-disrupting chemicals. Tail fin biopsy and reverse transcription-polymerase chain reaction analyses of the thyroid hormone (TH)-responsive gene, TH receptor beta (TRbeta), demonstrate for the first time that a known accelerant of TH-induced metamorphosis, acetochlor, can significantly enhance TH-induced TRbeta mRNA levels within 24 h at an environmentally relevant dose. Although we focus on laboratory exposures in this study, this method easily can be adapted for use in field studies.

Isolation and characterization of three estrogen receptor transcripts in Oreochromis mossambicus (Peters).

Exposure of aquatic organisms to 17beta-estradiol (E(2)) induces a variety of estrogen-responsive genes, including vitellogenin (vtg)-the precursor protein of egg yolk in oviparous animals and to date the single most used gene product in screening for estrogenic endocrine disruption. Transcription regulation of vtg by E(2) is dependent on binding of the ligand (E(2)) to a specific nuclear receptor (estrogen receptor, ESR) which in turn binds to an estrogen responsive element (ERE) in the promoter of vtg. Since a local tilapiine, Oreochromis mossambicus (Peters), is targeted as a model for estrogenic endocrine disruption in Southern Africa, a platform of knowledge is necessary for the ontogenic and tissue specific behavior of ESR in this species before vtg levels can be interpreted in relation to such endocrine disruption. Therefore, three ESR cDNA sequences (ESR1, ESR2a and ESR2b) in O. mossambicus were isolated and QPCR protocols were developed to ascertain their quantitative transcript levels in adult brain, gonadal and hepatic tissues. ESR1 transcript levels were highest in female liver tissue compared to males and other tissues, whereas the levels for ESR2a and b were not statistically significantly different between male and female tissues. Quantitative gene levels during development demonstrated a sharp increase in ESR1 during the stage of gonad differentiation (50-60 days post-fertilization) in this species. Finally, an induction experiment in adult male liver tissue confirms the upregulation of ESR1 by E(2).

Characterization of vtg-1 mRNA expression during ontogeny in Oreochromis mossambicus (PETERS).

The yolk-precursor lipoprotein, vitellogenin (VTG) has been widely recognized as a biomarker for the detection of estrogenic activity in water-borne chemical pollutants. We characterized the expression status of this important constituent of reproduction in the Mozambique tilapia (Oreochromis mossambicus), a tilapiine freshwater fish species indigenous to Southern Africa, and investigated its utility in detection of exposure to estrogen using a quantitative real-time polymerase chain reaction (QPCR) assay. We initially isolated a 3kb upstream promoter region of the vtg gene and identified putative binding sites for several regulatory factors including estrogen receptor (ESR). Evidence for the expression of several splice-site vtg mRNA variants was found in a number of tissue types. A quantitative real-time polymerase chain reaction (QPCR) assay was subsequently developed based upon a specific primer pair (OMV6/9) that selectively amplified the liver-enriched transcript. The level of this transcript in liver tissue was high in females and lower, but detectable, in males and was significantly increased in male fish following laboratory exposure to 17beta-estradiol (E(2)). This study further established that juvenile whole body homogenates (WBHs) contained extremely low levels of liver-specific vtg mRNA between 5 and 110 days post-fertilization (dpf) compared to adult male liver. Subsequent exposure of 20 dpf juveniles to E(2) showed a substantial increase in this transcript within hours, and when compared to classic male model under same conditions, the juveniles were remarkably more sensitive. We therefore conclude that the quantification, using QPCR methodology, of vtg mRNA expression in 20 dpf O. mossambicus juveniles has promise for assessing estrogenic EDC activity in aquatic sources.

Temporal expression of two cytochrome P450 aromatase isoforms during development in Oreochromis mossambicus, in association with histological development.

Oreochromis mossambicus is targeted as a sentinel species for assay development to detect disruption of estrogen-dependent pathways in Southern Africa. Aromatase--an enzyme encoded by either of two cytochrome P450 19 (cyp19) genes in O. mossambicus is the only enzyme able to catalyze the aromatization of androgens to estrogens. As part of background studies relating to evaluating the use of early life stages of O. mossambicus as an endocrine disruptor screen, we investigated the tissue specificity and temporal expression of cyp19 transcripts, cyp19a (ovarian cyp19) and cyp19b (brain cyp19) during development and estrogen exposure. The cyp19a transcript was detected only in the ovaries of adults, and expression during development reflected this result as induction of cyp19a coincides with histological development of putative ovaries. Using primers that only identify the ovarian transcript, cyp19a transcript levels demonstrated a typical reflection of puberty - after an initial surge in cyp19a, juveniles expressed very low levels, which increased again at the time histologically discernable vitellogenic ovaries were detected. Moreover, we found evidence of putative alternate transcript of cyp19a whose function is currently unknown. cyp19b transcripts were expressed in brain and muscle tissue of both male and female adults, in addition to ovaries in females. During development, cyp19b transcript levels were increased coincidental with cyp19a at 20 days post fertilization but the expression pattern was distinct from that observed for cyp19a. These studies set the foundation for utilizing this native species as a possible indicator of endocrine disruption and accentuates the importance of understanding "normal" basal levels of transcript levels and the nature of amplification of QPCR targets.

Genistein prevents thyroid hormone-dependent tail regression of Rana catesbeiana tadpoles by targetting protein kinase C and thyroid hormone receptor alpha.

Thyroid hormone (TH)-regulated gene expression is mainly mediated by TH binding to nuclear thyroid hormone receptors (TRs). Despite extensive studies in mammalian cell lines that show that phosphorylation signaling pathways are important in TH action, little is known about their roles on TH signaling in vivo during development. Anuran metamorphosis is a postembryonic process that is absolutely dependent upon TH and tadpole tail resorption can be precociously induced by exogenous administration of 3,5,3'-triiodothyronine (T(3)). We demonstrate that genistein (a major isoflavone in soy products and tyrosine kinase inhibitor) and the PKC inhibitor (H7) prevent T(3)-induced regression of the Rana catesbeiana tadpole tail. T(3)-induced protein kinase C tyrosine phosphorylation and kinase activity are inhibited by genistein while T(3)-induced up-regulation of TRbeta mRNA, but not TRalpha mRNA, is significantly attenuated, most likely through inhibition of T(3)-dependent phosphorylation of the TRalpha protein. This phosphorylation may be modulated through PKC. These data demonstrate that T(3) signaling in the context of normal cells in vivo includes phosphorylation as an important factor in establishing T(3)-dependent tail regression during development.

3,5,3'-Triiodothyronine-induced carbamyl-phosphate synthetase gene expression is stabilized in the liver of Rana catesbeiana tadpoles during heat shock.

One of many changes occurring during spontaneous and 3,5,3'-triiodothyronine (T3)-induced metamorphosis of the Rana catesbeiana tadpole is the permanent transition from an ammonotelic, aquatic larva to a ureotelic, terrestrial adult. T3-induced urea production is preceded by T3-induced elevation in the synthesis and level of liver-specific urea cycle enzymes essential for detoxication of ammonia in a terrestrial environment. This report focuses on establishing the effects heat shock (hs) has on the T3-induced expression of genes encoding three essential urea cycle enzymes. We demonstrate that hs stabilizes the intracellular existing levels of carbamyl-phosphate synthetase I (CPS I), the first enzyme in the urea cycle, while concurrently depressing its new synthesis. To establish the effects of hs on CPS I mRNA levels, we characterized cDNAs encoding an amphibian CPS I and demonstrate that it may represent an evolutionary link between microbial CPS and mammalian CPS I. Using this CPS I cDNA and other R. catesbeiana gene-specific probes, we demonstrate that hs depresses the level of T3-induced thyroid hormone receptor beta mRNAs but does not affect the level of T3-induced CPS I, ornithine transcarbamylase, and arginase mRNAs. These results support the contention that the hs response may involve the selective protection of some pre-existing mRNAs and proteins essential for an organism's survival.

Neuronal Cdc2-like kinase (Nclk) binds and phosphorylates the retinoblastoma protein.

The tumor suppressor retinoblastoma protein (RB) plays a central role in cellular growth regulation, differentiation, and apoptosis. Phosphorylation of RB results in a consequent loss of its ability to inhibit cell cycle progression. However, how RB phosphorylation might be regulated in apoptotic or postmitotic cells, such as neurons, remains unclear. Here we report that neuronal Cdc2-like kinase (Nclk), composed of Cdk5 and a neuronal Cdk5 activator (p25(nck5a)), can bind and phosphorylate RB. Since RB has been shown recently to associate with D-type G1 cyclins and viral oncoproteins through a common peptide sequence motif of LXCXE, Nclk binding may be mediated by a related sequence motif (LXCXXE) found in p25(nck5a). We demonstrate (i) in vitro binding of bacterially expressed p25(nck5a) to a GST-RB fusion protein, (ii) coprecipitation of GST-RB and reconstituted Cdk5.p25(nck5a), and (iii) phosphorylation of GST-RB by bacterially expressed Cdk5.p25(nck5a) kinase and by Cdk5.p25(nck5a) kinase purified from bovine brain. Finally, we show that immunoprecipitation of RB from embryonic mouse brain homogenate results in the coprecipitation of Cdk5 and that Cdk5 kinase activity is maximal during late embryonic development, a period when programmed cell death of developing neurons is greatest. Taken together, these results suggest that Nclk can bind to and phosphorylate RB in vitro and in vivo. We infer that Nclk may play an important role in regulating the activity of RB in the brain, including perhaps in apoptosing neurons.

Expression of novel ING variants is regulated by thyroid hormone in the Xenopus laevis tadpole.

The candidate tumor suppressor gene, ING1, encodes several protein isoforms as a result of alternative splicing that may possess agonistic and antagonistic roles in the control of cell proliferation and apoptosis. Recently a related gene, ING2, was isolated in human whose expression is increased in adenocarcinomas. Little is known about the cellular function and regulation of these ING family members, but the fact that ING proteins contain a plant homeodomain finger suggests that these proteins may modulate transcription factor-mediated pathways. To elucidate how ING may interact in different tissues to modulate function, we used amphibian metamorphosis as a model system in which a single stimulus, thyroid hormone (TH), initiates tissue-specific proliferation, differentiation, and apoptosis. We have isolated the first Xenopus laevis ING2 and demonstrate that transcript levels increase in response to TH treatment. We provide evidence for the existence of splice variants that are differentially expressed in tissues with different TH-induced fates. Western blots using an antibody directed against the highly conserved C-terminal end of ING proteins reveal a tissue-specific pattern of ING isoform expression in adult Xenopus tissues. Analyses of premetamorphic tadpole tissues show a TH-induced accumulation of ING proteins in tail, whereas the levels in the leg are not affected. This TH-induced accumulation is also observed in serum-free tail organ cultures and is prevented by inhibitors of tail apoptosis. Therefore, this work presents the first link between ING expression and a hormonally regulated nuclear transcription factor-mediated apoptotic response opening the possibility that ING family members may be involved in transducing the signal initiated by TH that determines cell fate.