FIGURE 1. Expression pattern of hspa5 in developing Xenopus embryos. A–K, spatial expression pattern of hspa5 analyzed by whole mount in situ hybridization.
Embryos are shown in different views (dorsal-lateral and vegetal). A and B, Hspa5 was expressed in the animal half prior to gastrulation stages. C–E,
during gastrulation, hspa5 is strongly expressed in ventral ectoderm, dorsal blastopore lip, and the developing notochord but is absent from the prospective
neural plate. In C and D, the red arrow indicates dorsal blastopore lip; in E, the white arrowhead indicates the notochord. F and G, at neurula stages, hspa5
expression is restricted to the notochord (white arrow) and the region anterior to the neural plate (red arrow) that corresponds to the future cement gland. H
and I, at stage 22, strong expression is found in the cement and hatching glands. H, anterior view; I, lateral view. J and K, at the tail bud stage, hspa5 is expressed
in the cement gland, pronephros (red arrow), ear vesicle, and epidermis. It is notable that the hspa5 signal can also be found in the liver primordium (K, black
arrow). L andM, transverse sections of tail bud stages (J) showing hspa5 expression in pronephros (pn) and notochord (nc) tissue. N and O, transverse sections
of a late tail bud stage embryo (K) show hspa5 expression in the pronephros (pn) and liver (li). P–S, Xbp1 expression in Xenopus embryos at the stages indicated.
T, RT-PCR shows the temporal expression pattern of hspa5. Numbers above the lanes indicate the corresponding embryonic stages. A negative control (RT )
without reverse transcriptase was included. odc expression was used as the internal standard control. U, schematic diagram illustrating the interaction of Xbp1
and Hspa5 in the unfolded protein response. V, real time PCR indicates that hspa5 is up-regulated by overexpression of xbp1. xbp1mRNAat the indicated doses
was injected into Xenopus embryos at the two-cell stage. Animal caps were dissected at stage 9 and cultured for 2 h. The expression of hspa5 was analyzed by
real time PCR, and its expression was normalized to odc. The animal cap assay was performed twice. St, stage.
FIGURE 2. Hspa5MO specifically knocks down Hspa5 in Xenopus embryos. A, the two Hspa5MOs targeted to the ATG sequence are depicted in blue font and
underlined.Thetranslation start site ofhspa5isshownin black with theATGin red.B–E,Hspa5MO1and-MO2reducedendogenousHspa5protein inHspa5morphants.
Xenopus embryos at the two-cell stage were injected with the indicated doses of Hspa5MO1 (B) or Hspa5MO2 (D) and collected at stage 11. Endogenous Hspa5 was
detected by Western blotting using -Hspa5 antibody. Injection of Hspa5MO1 reduced endogenous Hspa5 in a dose-dependent manner in Xenopus embryos. The
knockdown experiments were repeated twice for each MO, and a representative gel is shown in B and D. Western blots shown in B and D were quantified in C and E,
respectively. The signal densities were acquired and analyzed using a GS-800 calibrated imaging densitometer. The relative signals of Hspa5 to tubulin from either
Hspa5MO1- or Hspa5MO2-injected embryos were normalized to those from control embryos. The ratios indicate the decrease of Hspa5 expression. F,Hspa5MO1and
Hspa5MO2 induced the same phenotypes. The injected embryos show a range of phenotypes of short and curved body axis and disruption of eye and other head
structures. The phenotypes of embryos injected with Hspa5MO1 or -MO2 were categorized into mild, moderate, and severe groups. G, quantification of phenotypes
thatwereinducedbyincreasing doses ofHspa5MO1orHspa5MO2.Thenumbersabovethecolumnsindicate thenumberof injectedembryosfromthree experiments,
and the phenotypes were categorized by investigators who were blinded to the treatment. H, silent mutations were introduced into the pCS2-hspa5 sequence to
prevent binding of Hspa5MOs. I, the pCS2-hspa5 mRNA partially rescued the phenotype induced by Hspa5MOs. Quantification of phenotypes induced by the
indicatedHspa5MOsoracombinationofpCS2-hspa5mRNAandHspa5MOisshownin thecolumnchart. Threeindependentexperimentswereperformed,andhspa5
mRNA partially rescued the phenotype induced by Hspa5MO1. The number of injected embryos is listed on the top of the columns.
FIGURE 3. Knockdown of Hspa5 inhibits pronephros formation. A–C, phenotype of Xenopus tadpoles injected with Hspa5MO2 into one ventral-vegetal
blastomere at the eight-cell stage. Either 15 or 30 ng of Hspa5MO2 was co-injected with LacZ mRNA into one of the ventral-vegetal blastomeres of eight-cell
stage embryos. A, anterior-dorsal view of mildly affected embryos at stage 16. B, lateral view of mildly affected embryos at stage 32. LacZ staining indicates the
injected side; the uninjected side was used as a negative control. C, phenotype categorization of embryos injected with 15 or 30 ng Hspa5MO2. Over 90% of
the embryos showed a normal or mild repression of anterior axis development after the injection. The severe phenotype refers to failure of gastrulation. D–F ,
depletion of Hspa5 inhibits expression of pan-pronephros marker genes such as pax2, lhx1, and atp1b1. Eight-cell stage embryos were injected with LacZmRNA
(100 pg) into one of the vegetal-ventral blastomeres as a lineage tracer along with Hspa5MO2 (30 ng/embryo). The injected embryos were collected at stage
32, and the expression of pronephric marker genes as indicated was examined by whole mount in situ hybridization. D, E, and F, the uninjected side. D , E , and
F , expression of pax2, lhx1, and atp1b1 on the MO-injected side of the same embryos, respectively. D , E , and F , transverse sections of embryos in D, E, and F
accordingly. G–H , expression of smp30 (tubule) and nephrin (glomus) at the Hspa5MO2-injected side was inhibited. G and H, the uninjected side. G and H ,
Hspa5MO2-injected side. G and H , transverse sections of embryos shown in G and H, respectively. I–L , expression of pax2, lhx1, smp30, and nephrin in the
tadpoles injected with 30 ng of standard control MO/embryo with 100 pg of LacZ mRNA into one vegetal-ventral blastomere at the eight-cell stage. I–L,
uninjected side. I -–L , standard control MO-injected side (CMO-lacZ).
FIGURE 4. Hspa5 depletion inhibits of pronephric duct and tubule formation.
Single vegetal-ventral blastomeres of eight-cell stage embryos were
injected with LacZ mRNA (100 pg/embryo) and Hspa5MO2 (30 ng/embryo).
Injected embryos were collected at approximately stage 39. Embryos were
stained for LacZ and immunostained with 3G8 and 4A6 antibodies to detect
pronephric ducts and tubules. A, A , B, and B , pronephric tubules in embryos
detected by 3G8 antibody. C, C , D, and D , pronephros ducts in stage 39
embryos detected by 4A6 antibody. A –D , higher magnification of A–D.
White arrow indicates pronephros. pn, pronephros. The white arrow indicates
tubule in A–B and duct in C–D . Uninjected, uninjected side; MO-lacZ,
FIGURE 5. Pronephros induction in animal cap assays. A, schematic diagram of in vitro pronephros induction assay showing animal caps are induced to
differentiate into pronephros tissues using atRA and activin. B and C, Hspa5MO2 (MO2) was injected into both blastomeres at the two-cell stage. Animal caps
were dissected at stage 9 and treated with activin and atRA for 3 h. Subsequently, the animal caps were transferred into normal animal cap culture mediumand
cultured until stage 15 (St15) (B) or stage 32 (St32) (C). Expression analysis was performed by RT-PCR using total RNA extracted from the animal caps. AC, control
animal cap; WE, sibling whole embryos. D and E, knockdown of Hspa5 suppresses lhx1 expression. Either 30 ng of Hspa5MO1 (MO1) (D) or Hspa5MO2 (MO2) (E)
was co-injected with 100 pg of LacZ mRNA into one ventral animal blastomere of four-cell stage embryos. Injected embryos were collected at stage 15. The
injected side was identified by red X-Gal staining, and the expression of lhx1 was examined by whole mount in situ hybridization.
FIGURE 6. Hspa5 is required for transduction ofRAsignaling. A,Hspa5MO2down-regulates lhx1, cdx4, and gbx2 gene expression in animal caps treated with
atRA but not in animal caps treated with activin. Hspa5MO2 (MO2) (30 ng/embryo) was injected into both blastomeres of Xenopus embryos at the two-cell
stage. Animal caps were dissected at stage 9, treated with either atRA or activin separately or a mixture of both for 3 h, and then cultured in normal animal cap
culture medium for another 3 days. Expression of the indicated genes was examined by RT-PCR. Depletion of Hspa5 attenuates expression of lhx1, cdx4, and
gbx2 induced by atRA. Activin treatment up-regulates lhx1, hoxd1, cdx4, gbx2, and chordin expression. Knockdown of Hspa5 has little effect on the indicated
genes except that lhx1 expression is enhanced. Treatment of atRA and activin induces lhx1, hoxd1, and gbx2 expression in control animal caps, whereas
knockdown of Hspa5 causes a reduction of lhx1 but not hoxd1 or gbx2. AC, control animal cap; WE, sibling whole embryos. B, Hspa5MO decreases expression
of atRA-responsive genes. Animal caps were treated with 0.1 or 1 M atRA for 4 h at room temperature. RA-responsive genes lhx1, gbx2, hoxd1, and cdx4 were
induced by atRA treatment in a dose-dependent manner. Depletion of Hspa5 leads to decreased induction of these genes. C, real time PCR shows that HSPA5
shRNA effectively reduces HSPA5mRNAlevels in human HEK293T cells. Expression of HSPA5 in cells transfected with either control shRNA or HSPA5 shRNA was
normalized to the control cells. D–G, endogenous HSPA5 expression is reduced in HSPA5 shRNA-infected (D and E) or HSPA5 siRNA-infected (F and G) HEK293T
cells. The expression of HSPA5 was detected by Western blotting. -Tubulin was used as a loading control (D and F). The quantified HSPA5 signal in either
HSPA5 shRNA-infected cells or control shRNA-infected cells was normalized to the -tubulin signal. The relative expression was normalized to that of control
cells (E and G). The intensity of Western blot signals was quantified by a GS-800 calibrated imaging densitometer. H and I, depletion of HSPA5 by either Hspa5
shRNA (H) or Hspa5 siRNA (I) reduces the luciferase activity of RA luciferase reporter gene in HEK293T cells. atRA treatment enhances the luciferase activity,
whereas HSPA5 shRNA reduces the luciferase activity in a dose-dependent manner. BMS453, an RA signaling inhibitor, also inhibits the luciferase activity of RA
luciferase reporter gene.DMSOtreatment was used as a control. The asterisks indicate the statistically significant difference. con, control; RLU, relative luciferase
units. Error bars represent S.D.
FIGURE 7. Depletion of HSPA5 induces down-regulation of RAR at protein level. A, real time PCR indicates the expression of genes involved in RA
signaling and atRA metabolism in control (blue column) or in HSPA5-depleted HEK293T cells (red column). B–F3, knockdown of HSPA5 reduced RAR protein in
SH-SY5Y as revealed by confocal imaging. Either HSPA5 siRNA or control siRNA was transfected into SH-SY5Y cells. The knockdown efficiency is confirmed by
Western blotting (B). C–F3, confocal imaging illustrates the subcellular expression of endogenous HSPA5 (green; C1–F1) and RAR (red; C2–F2) in SH-SY5Y cells.
The nucleus was stained with DAPI (blue; C–F), and the merged images are shown in C3–F3. A clear reduction of RAR was identified in HSPA5-depleted cells
(D2 and F2) compared with that in control (con) siRNA-transfected cells (C2 and E2). C–D3, lower magnification; E–F3, high magnification. Scale bars represent
the length as indicated.
FIGURE 8. Lhx1 expression partially rescues pronephros inhibition induced by Hspa5MO. A–H , expression of pronephric markers including lhx1, pax2,
nephrin, and smp30 in embryos injected with either Hspa5MO1 (MO1) (30 ng) or a mixture of Hspa5MO1 (30 ng) and lhx1 (100 pg) were examined by whole
mount in situ hybridization. Single ventral-vegetal blastomeres of eight-cell stage embryos were targeted, and LacZ mRNA was used as a lineage tracer. A, C, E,
and G, Hspa5MO-injected side, A , C , E , and G , non-injected (Non-inj) side of the same embryo. B, D, F, and H, Hspa5MO1- and lhx1-injected side. B , D , F , and
H , non-injected side. I, quantification of the rescue experiments of different pronephros markers. The phenotype assessments were performed blinded to the
treatment. The numbers on the tops of the columns indicate the total number of injected embryos.
hspa5 (heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 8, horizontal view, animal up.
hspa5 (heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
hspa5 (heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.
hspa5 (heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35, lateral view, anterior left, dorsal up.