A characterization of BiP gene expression in Xenopus laevis embryos and A6 kidney epithelial cells

Abstract

We have characterized a full length cDNA clone encoding a Xenopus laevis immunoglobulin binding protein, BiP. The BiP cDNA sequence includes an open reading frame of 1,965 bp encoding a 655 amino acid protein with an N-terminal hydrophobic leader sequence and a C-terminal KDEL tetrapeptide which has been found in other lumenal proteins of the endoplasmic reticulum. The 3' untranslated region contains a polyadenylation and an adenylation control element (ACE) as well as a putative mRNA instability sequence. Although the Xenopus BiP amino acid sequence displayed high identity with BiP from other vertebrates including chicken (91.3%), rat (90.7%), and human (89.9%), the identity with Zenopus hsp70 (57%) and hsc70.1 (55.2%) was much lower. Northern hybridization analysis demonstrated that BiP mRNA was present constitutively in the Xenopus A6 kidney epithelial cell line and that BiP mRNA levels could be enhanced by treatment of the cells with galactose-free media, 2-deoxyglucose, 2-deoxygalactose, glucosamine, tunicamycin, heat shock, dithiothreitol, and the calcium ionophore, A23187. Although BiP mRNA was detected in all of the adult tissues examined, the relative level of BiP mRNA differed dramatically between organs. Relatively high levels of BiP mRNA were detected in liver with moderate levels in testis, ovary and heart and reduced levels in eye and muscle tissue. Constitutively expressed BiP mRNA was detected in all stages of Xenopus early development from unfertilized egg to 4-day-old tadpole. Relative BiP mRNA levels rose slightly at the early neurula stage and then increased dramatically in early and late tailbud stage embryos. Whole mount in situ hybridization employing a DIG-labeled BiP antisense RNA probe revealed that BiP mRNA was present constitutively in the animal pole of the unfertilized egg, cleavage and blastula stage embryos. At the gastrula stage, BiP mRNA was present throughout the embryo with slightly less accumulation in the yolk plug region. At the neurula stage, BiP mRNA levels were enriched in the neural plate and around the blastopore as well as along the neural folds. In the early and late tailbud stage embryos BiP mRNA was distributed primarily along the dorsal region of the embryo in the somitic region, spinal cord, cranial nerves, otic vesicle and in the forebrain. BiP mRNA was also present in the heart, gills, liver diverticulum, pronephros, pronephric duct and around the anus. BiP mRNA was first tunicamycin and A23187 inducible at the neurula stage. A23187 treatment of neurula stage embryos increased BiP and mRNA levels throughout the embryo with enhanced accumulation in the neural plate, along the neural folds, around the blastopore and in the ectoderm. At the tailbud stage, ionophore treatment increased accumulation of BiP mRNA primarily in the head region as well as along the spinal cord, in the somites, tail, pronephros, pronephric duct, heart and liver. In heat shock studies, the incubation of embryos for 1 h at 33*C resulted in an enhanced accumulation of BiP mRNA immediately after MBT at the gastrula stage. In gastrulae, heat shock enhanced the accumulation of BiP mRNA throughout the embryo. In neurule embryos BiP mRNA levels were enhanced by heat shock in the neural plate, along the neural folds, around the blastopore and in portions of the epidermis. At the early tailbud stage heat shock increased the accumulation of BiP mRNA along the spinal cord as wella sin the somites, the forebrain, the tail and around the anus. At the late tailbud stage heat-induced accumulation of BiP mRNA increased dramatically in a global fashion throughout the embryo.

Since the BiP gene is a member of hsp70 family we were interested in comparing its pattern of expression in embryos with the expression of cytosolic hsc70 and hsp70. In situ hybridization experiments employing DIG-labeled hsc70 antisense RNA probe demonstrated that hsc70 mRNA was constitutively present during development in a global fashion and did not appear to increase upon heat shock. In situ hybridization analysis of Xenopus embryos with DIG-labeled hsp70 antisense RNA probe confirmed that hsp70 mRNA was not constitutively present but was heat-shock inducible in post-midblastula stages of Xenopus development including late blastula, gastrula, neurula and tailbud stages. Give n the strong induction of hsp70 gene expression at 33*C we examined the effect of a range of temperatures from 22*C to 35*C on hsp70 mRNA accumulation in early and late tailbud stage embryos. This study revealed a preferential induction of hsp70 gene expression in selected tissues of tailbud embryos at lower heat shock temperatures. Placement of early tailbud embryos a 30*C resulted in the accumulation of hsp70 mRNA in the olfactory placode, lens vesicle, optic cup, cranial nerves, otic vesicle, spinal cord and in the somatic region along the spinal cord. Hsp70 mRNA accumulation in late tailbud embryos was first induced at 26*C in the somitic region of the growing tail. At 30*C hsp70 mRNA was more pronounced in the somitic region and was also present in the fore brain, cranial nerves, otic vesicle, heart and liver. Hsp70 mRNA accumulation in the late tailbud stage embryos exposed to 33 and 35*C was strongly induced and displayed a global pattern of distribution.