Lang L et al. (1999), Spatial pattern of constitutive and heat shock-...

XB-ART-11934

Dev Genet 1999 Jan 01;254:365-74. doi: 10.1002/(SICI)1520-6408(1999)25:4<365::AID-DVG10>3.0.CO;2-2.

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Spatial pattern of constitutive and heat shock-induced expression of the small heat shock protein gene family, Hsp30, in Xenopus laevis tailbud embryos.

Lang L , Miskovic D , Fernando P , Heikkila JJ .

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We employed whole-mount in situ hybridization and immunohistochemistry to study the spatial pattern of hsp30 gene expression in normal and heatshocked embryos during Xenopus laevis development. Our findings revealed that hsp30 mRNA accumulation was present constitutively only in the cement gland of early and midtailbud embryos, while hsp30 protein was detected until at least the early tadpole stage. Heat shock-induced accumulation of hsp30 mRNA and protein was first observed in early and midtailbud embryos with preferential enrichment in the cement gland, somitic region, lens placode, and proctodeum. In contrast, cytoskeletal actin mRNA displayed a more generalized pattern of accumulation which did not change following heat shock. In heat shocked midtailbud embryos the enrichment of hsp30 mRNA in lens placode and somitic region was first detectable after 15 min of a 33 degrees C heatshock. The lowest temperature capable of inducing this pattern was 30 degrees C. Placement of embryos at 22 degrees C following a 1-h 33 degrees C heat shock resulted in decreased hsp30 mRNA in all regions with time, although enhanced hsp30 mRNA accumulation still persisted in the cement gland after 11 h compared to control. In late tailbud embryos the basic midtailbud pattern of hsp30 mRNA accumulation was enhanced with additional localization to the spinal cord as well as enrichment across the embryo surface. These studies demonstrate that hsp30 gene expression can be detected constitutively in the cement gland of tailbud embryos and that heat shock results in a preferential accumulation of hsp30 mRNA and protein in certain tissues.

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Species referenced: Xenopus laevis
Genes referenced: actg1 hsp30c

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	Fig. 1. Relative levels of heat shock-inducible hsp30 mRNA accumulation during early Xenopus laevis development. Total RNA was isolated from control (c) and heat shocked (h; 1 h at 33°C) embryos at the cleavage (C; stage 4), early blastula (B; stage 8), gastrula (G; stage 11), neurula (N; stage 17/18), early (ETB; stage 22/23), mid- (MTB; stage 28) and late (LTB; stage 35) tailbud stage. Three μg of RNA was subjected to Northern hybridization analysis employing DIG-labeled Xenopus hsp30 (A) and cytoskeletal actin (B) antisense riboprobes as outlined in Materials and Methods. The autoradiogram in A was overexposed in order to properly visualize the ETB signal. A slight distortion in the agarose gel resulted in a slightly higher mobility in the MTB lane. Arrows indicate the positions of the different mRNAs. Transcript sizes: hsp30, 1.1 kb; cytoskeleton actin, 1.8 and 2.0 kb.
	Fig. 2. Spatial pattern of hsp30 mRNA accumulation during early Xenopus development. Whole-mount in situ hybridization with DIGlabeled hsp30 antisense riboprobe was carried out with control (A,C,E) and heat shocked (B,D,F; 1 h at 33°C) Xenopus embryos at neurula (A,B; stage 17/18), mid- (C,D; stage 28) and late (E,F; stage 35) tailbud stages. At the late tailbud stage, control embryos (E) display a natural increase in eye pigmentation and melanocyte production. G,H: Control and heat shocked midtailbud stage embryos (stage 28), respectively, hybridized with DIG-labeled hsp30 sense riboprobe. I,J: Control and heat shocked midtailbud stage embryos (stage 28), respectively, hybridized with DIG-labeled actin antisense riboprobe. LP, lens placode; S, somites; P, proctodeum; CG, cement gland; SC, spinal cord; A, anus; ME, melanocytes.
	Fig. 3. Histological analysis of hsp30 mRNA accumulation in sections from Xenopus tailbud embryos. Whole-mount in situ hybridization with DIG-labeled hsp30 antisense riboprobe was performed on control and heat shocked (1 h at 33°C) mid- (stage 28) and late (stage 35) tailbud embryos followed by histological analysis as described in Materials and Methods. A,B: Anterior sections of control and heat shocked midtailbud embryos, respectively, showing the cement gland. C: Cross-section displaying the proctodeum in heat shocked midtailbud embryos. D: An enlarged medial region of a heat shocked midtailbud whole-mount embryo (stage 28) hybridized with DIG-labeled hsp30 antisense riboprobe and demonstrates the abundance of hsp30 mRNAin the somites. E,F: The presence of hsp30 mRNAin the somitic region of heat shocked mid- and late tailbud embryos, respectively. G,H: Cross-sections of the eye of control and heat shocked midtailbud embryos. CG, cement gland; M, mesencephalon; LP, lens placode; OC, optic cup; N, notochord; S, somites; HG, hindgut; P, proctodeum; E, ectoderm.
	Fig. 4. Pattern of hsp30 mRNA accumulation in tailbud embryos during recovery from heat shock. Whole-mount in situ hybridization was carried out with DIG-labeled hsp30 antisense riboprobe. Midtailbud Xenopus embryos (stage 28) were exposed to either 22°C (A) or 33°C for 1 h (B) followed by recovery at 22°C for either 1 h (C), 3 h (D), 7 h (E) or 11 h (F). CG, cement gland; LP, lens placode; P, proctodeum; S, somites.
	Fig. 5. Effect of different temperatures on the spatial patterns of hsp30 mRNA accumulation in Xenopus midtailbud (stage 28) embryos. Wholemount in situ hybridization was carried out with DIG-labeled hsp30 antisense riboprobe. The midtailbud embryos were exposed to 1-h heat treatments at either 22°C (A), 30°C (B), 33°C (C), or 35°C (D). CG, cement gland; LP, lens placode; S, somites; P, proctodeum; SC, spinal cord.
	Fig. 6. Whole-mount immunohistochemical analysis of hsp30 protein accumulation in Xenopus embryos. Late tailbud (stage 35; A,B) and early tadpole (stage 41/42) embryos were incubated at either 22°C (A,C) or 33°C (B,D) for 2 h. The embryos were then processed for immunohistochemical analysis using an affinity-purified polyclonal hsp30 antibody as outlined in Materials and Methods. The outer portion of the tailfin was quite transparent in the late tailbud stage embryos and is not readily visualized in the photograph in C. CG, cement gland; LP, lens placode; SR, somitic region; P, proctodeum.
	hsp30c (heat shock protein 30C) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, lateral view, anterior left, dorsal up.
	hsp30c (heat shock protein 30C) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up. Left embryo: unchallenged, Right embryo heat-shocked
	hsp30c (heat shock protein 30C) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35, lateral view, anterior left, dorsal up. Left embryo: unchallenged, Right embryo heat-shocked