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We have characterized the constitutive and stress-inducible pattern of immunoglobulin-binding protein (BiP) gene expression during Xenopus early development. Whole mount in situ hybridization analysis revealed that BiP mRNA was detected in unfertilized eggs, cleavage and blastula stage embryos. In gastrulae, BiP mRNA was present across the surface of the embryo, while in neurulae BiP mRNA was enriched in the neural plate, neural fold, and around the blastopore. In early and late tailbud embryos, BiP mRNA was found primarily in the dorsal region. Tunicamycin and A23187, the calcium ionophore, enhanced BiP mRNA accumulation first at the neurula stage, while heat shock induced BiP mRNA accumulation first at the gastrula stage. Compared to control, A23187- and heat shock-treated neurulae displayed relatively high levels of BiP mRNA in selected tissues, including the neural plate, neural folds, around the blastopore, and ectoderm. At the early tailbud stage, A23187 and heat shock enhanced BiP mRNA accumulation primarily in the head, somites, tail, and along the spinal cord. A similar situation was found with A23187- and heat shock-treated late tailbud embryos, except that heat-shocked embryos also displayed enhanced BiP mRNA accumulation in the epidermis. These studies demonstrate a preferential accumulation of BiP mRNA in selected tissues during development and in response to stress.
Fig. 1. Relative levels of BiP mRNA during early Xenopus laevis
development. Total RNA was isolated from embryos at the cleavage
stage (C), four different blastula (B) stages, neurula stage (N), and
early (ETB) and late (LTB) tailbud stage. Fifteen μg of RNA was
subjected to Northern hybridization analysis employing the [32P]-
labeled Xenopus BiP cDNA probe. The numbers above each lane
indicate the embryonic stage according to Nieuwkoop and Faber
[1967].
Fig. 2. Spatial pattern of BiP mRNA accumulation during early
Xenopus development. Whole mount in situ hybridization with DIGlabeled
BiP antisense riboprobe was carried out with Xenopus albino
unfertilized eggs (A), cleavage (B, stage 2), gastrula (C, stage 11),
neurula (D, stage 18), early (E, stage 24–25), and late (F, stage 32–33)
tailbud embryos. Panels G and H represent early and late tailbud
stage embryos, respectively, hybridized with DIG-labeled BiP sense
riboprobe. Panels I and J represent neurula stage embryos (stage 18)
hybridized with DIG-labeled L8 sense and antisense riboprobe, respectively.
AN 5 anterior; AP 5 animal pole; VP 5 vegetal pole; CF 5
cleavage furrow; YP 5 yolk plug; BL 5 blastopore lip; NF 5 neural
fold; PO 5 posterior; BP 5 blastopore; NC 5 notochord; SC 5 spinal
cord;A5 anus; S 5 somites; L5 liver diverticulum; PN 5 pronephros;
PD 5 pronephric duct; H 5 heart; OV 5 otic vesicle.
Fig. 3. Effect of tunicamycin and calcium ionophore, A23187, on
BiP mRNA accumulation in Xenopus embryos. (A) Total RNA was
isolated from control (C) and tunicamycin-treated (T; 2 μg/ml for 12 h)
embryos at gastrula (G, stage 11), neurula (N, stage 18), and tailbud
stage (TB; stage 22â23). Fifteen μg of RNA was subjected to Northern
hybridization analysis using a [32P]-labeled Xenopus BiP cDNA. The
RNA blot was then stripped of labeled BiP probe and reprobed with
Xenopus L8 probe (lower panel). (B) Total RNA was isolated from
control (C) and A23187-treated (A; 4 μM for 3 h) embryos at blastula
(B, stage 5), gastrula (G, stage 11), neurula (N, stage 18), and tailbud
stage (TB; stage 22â23). Fifteen μg of RNA was subjected to Northern
hybridization analysis as described above.
Fig. 4. Effect of heat shock on the accumulation of BiP, hsp70 and
L8 mRNA in Xenopus embryos. (A) Total RNA was isolated from
control (C) and heat shocked (HS; 1 h at 33°C) blastula (B, stage 5),
gastrula (G, stage 11), neurula (N, stage 18), and tailbud stage (TB;
stage 22â23) Xenopus embryos. Northern blot analysis was carried out
employing the [32P]-labeled BiP cDNA clone. The same RNA blot was
stripped of labeled BiP probe and reprobed with labeled Xenopus
hsp70 gene (middle panel) and Xenopus L8 cDNA (lower panel). (B)
Effect of cycloheximide on BiP mRNA accumulation in Xenopus
embryos. Total RNA was isolated from control (C) and heat-shocked
(HS; 1 h at 33°C) neurula stage embryos (N, stage 18) which were
incubated in the presence or absence of either 25 or 50 μg/ml
cycloheximide (CX) as outlined in Materials and Methods. Northern
blot analysis was carried out employing a [32P]-labeled BiP cDNA
clone.
Fig. 5. Spatial pattern of BiPmRNAaccumulation in early embryos
after heat shock. Whole mount in situ hybridization with DIG-labeled
BiP antisense riboprobe was carried out with control (A,C,E) and
heat-shocked (B,D,F; 1 h at 33°C) Xenopus albino embryos at neurula
(A,B; stage 18) early (C,D; stage 22–23), and late (E,F; stage 32–33)
tailbud stages. Panels G and H are histological sections taken from
early tailbud embryos shown in panels E and F, respectively. The
hatched lines in panels E and F indicate the orientation of the
sections. Panels I and J represent control and heat-shocked early
tailbud stage embryos (stage 22–23) hybridized with DIG-labeled L8
antisense riboprobe. FB 5 forebrain; SC 5 spinal cord; A 5 anus; S 5
somites; L 5 liver diverticulum; H 5 heart; CN 5 cranial nerve; OV 5
otic vesicle; PN 5 pronephros; T 5 tail; LV 5 lens vesicle; MC 5
mesencephalon; ED 5 epidermis; PH 5 pharynx; EPL 5 eye pigment
layer.
