XB-ART-22324J Biol Chem August 5, 1993; 268 (22): 16270-8.
The changes in gene expression leading to tail resorption that are initiated by thyroid hormone (TH) were studied in Xenopus laevis. Four of the less than 10 genes that are down-regulated during this period have been isolated; their mRNAs decay with identical kinetics. Twenty of the approximately 35 genes that are up-regulated in the first 48 h have been isolated. The up-regulated genes fall into two kinetic patterns. After a lag of several hours, the direct response genes (including thyroid hormone receptor beta) increase their mRNA level steadily for 24-48 h. The delayed genes respond mainly in the second 24 h after TH addition. The importance of these genes for tail resorption is supported by the fact that they are all regulated developmentally during normal metamorphosis in tail and respond to hormone induction when the tail becomes competent to respond to TH. The relatively simple gene expression program leading to tail resorption is contrasted with the complex and multiple periods of gene expression during limb development. The gene expression screen defines the tail resorption program and has isolated the majority of TH-regulated genes.
PubMed ID: 8344914
Article link: J Biol Chem
Species referenced: Xenopus laevis
Genes referenced: crhbp dan4l dio3 dpepe fap fn1 fosl2 hhipl2 itga11 kat8 klf9 mamdc2 mmp11 mmp13l paaf1 tbx2 thdl17 thdl18 thdl20 thibz thra thrb tra
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|FIG. 1. Effect of TH pretreatment on cultured tails. Tails were amputated from stage 54 tadpoles that had been pretreated with 100 nM TB for various times up to 48 h, then cultured in Steinberg’s saline in the presence or absence of 20 pg/ml cycloheximide plus 100 nM anisomycin. Cultured tails from animals pretreated with TH for up to 24 h were identical to controls. Control tails cultured in the presence (A-A) or absence (Mof )th e inhibitors; tails pretreated with TH for 48 h and then cultured in the presence (A-A) or absence (-1 of the inhibitors. Four tails were used. in each assay condition. The standardd eviation of each measurement is indicated at each date point by the vertical bar.|
|FIG. 2. Time course of the TH effect on two down-regulated genes (17 and 19) in tadpole tails assayed by Northern blot. Total RNA was isolated from the tails of stage 54-55 tadpoles which had been treated with 100 nM T S for the indicated times. The quantity of RNA loaded in each lane was standardized using an actin cDNA probe (Mohun et al., 1984).|
|F'IG. 3. Time course of TH induction of isolated up-regulated genes in X. Zaeuis tadpole tails by Northern blot analysis. Total taiRl NA at various time points afteTrS t reatment was analyzed as described in Fig. 2. Multiple exposures to autoradiograms were obtained for quantitation by densitometry. The six representative upregulated genes that are shown are: (0) gene 1, (A) gene 6 (TRB), (0) gene 8, (0) gene 11, (W) gene 14, (A) gene A. TRa mRNA is included (x- - - - -x).|
|FIG. 4. Effect of protein synthesis inhibition on TH-induced up-regulation for three genes. Stage 54 tadpoles were treated with or without 100 nM T3, (TH) for 8h in the presence or absence of protein synthesis inhibitors (CHX) that were added 1 h prior to the addition of TH. Tails were amputated and poly(A)+ RNA was electrophoresed for Northern analysis.|
|FIG. 5. Developmental Northern blot analysis of TH-regulated genes in the tadpole tailE. ach lane contains 5 pg of poly(A)+ RNA from tails at the indicated stages of development. The amount of RNA loaded was standardized by probing with PR28 cDNA (Shi and Brown, 1990). The developmental profile of only one of the down-regulated genes (17) is shown; the other three genes (18- 20) have identical profiles. Genes 1,5,8, and 9 encode multiple mRNAs (Wang and Brown, 1991). Other multiple bands are probably due to partial degradation of mRNAs, especially for the very high molecular mRNAs.|
|FIG. 6. Developmental Northern blot analysis of representative genes in the limb. Each lane contains 15 pg of total RNA extracted from hind limbs at the indicated developmental stages. Developmental expression of genes 1, 2, 3, 6, 13, 16, D, and E is similar to that of gene 12. Patterns of genes 9,11, and A are similar to gene 8. Developmental expression of genes 14 and B is similar to gene C. All of the down-regulated genes are similar to gene 17. Genes 4,7 and 15 have distinct expression patterns in the limb. Gene 5 has no detectable base line of expression in limb.|
|FIG. 7. Northern blot analysis of the developmental expression of TRa and TRb genes in X. laevis hind limbs and tails using poly(A)+ RNA and total RNA. The number of transcripts per cell was estimated by comparing Northern and genomic Southern signals when both types of filters were hybridized in the same hybridization bag (Wang and Brown, 1991). The plasma T3 level in X. laeuis tadpoles during development is illustrated with a dotted line (Leloup and Buscaglia, 1977). limb TRa, (A-A) limb TRB, (Wta)il TRa, (A-A) tail TRB.|
|FIG. 8. Acquisition of competence to TH in the tail during development. Tails were amputated from tadpoles at indicated stages and then cultured in Steinberg's saline in the presence of 100 nM T3. Tail shrinkage waa measured daily using the ratio of the length of TH treated tails to that of control tails after 5 days in culture. Four tails were used for each measurement.|
|Fig. 9. Competence of tail to up-regulate TH response genes. Total RNA was isolated from tails at the indicated stages pretreated with or without1 00 nMT S (TH)fo r2 4 h. The RNA was analyzed by Northern blot.|