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. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Genesis. 2021 Apr 23;59(5-6):e23420. doi: 10.1002/dvg.23420

Constitutive Expression of Spliced XBP1 Causes Perinatal Lethality in Mice

Qian Xu 1, Hua Zhang 1, Suzhen Wang 1, Chunlin Qin 1, Yongbo Lu 1,*
PMCID: PMC9014887  NIHMSID: NIHMS1796241  PMID: 33891366

Abstract

Upon endoplasmic reticulum (ER) stress, inositol-requiring enzyme 1 (IRE1) is activated and catalyzes a nonconventional splicing of an unspliced X-box binding protein 1 (XBP1U) mRNA to yield a spliced XBP1 (XBP1S) mRNA that encodes a potent XBP1S transcription factor. XBP1S is a key mediator of the IRE1 branch that is essential for alleviating ER stress. We generated a novel mouse strain (referred to as “Xbp1CS/+” mice) that constitutively expressed XBP1S after Cre recombinase-mediated recombination. Further breeding of these mice with Twist2 Cre recombinase (Twist2-Cre) knock-in mice generated Twist2-Cre;Xbp1CS/+ mice. Most Twist2-Cre;Xbp1CS/+ mice died shortly after birth. Reverse-transcription polymerase chain reaction (RT-PCR) showed that constitutive expression of XBP1S occurred in various mouse tissues examined, but not in the brain. Immunohistochemistry confirmed that although the immunostaining signals for total XBP1 (XBP1U and XBP1S) were found in the calvarial bones in both Twist2-Cre;Xbp1CS/+ and control mice, the signals for XBP1S were only detected in the Twist2-Cre;Xbp1CS/+ mice, but not in the control mice. These results suggest that a precise control of XBP1S production is essential for normal mouse development.

Keywords: X-box binding protein 1 (XBP1), mouse, development

INTRODUCTION

Endoplasmic reticulum (ER) maintains a balance between the unfolded proteins that enter the ER and the folding and exporting capacity of the ER, a condition known as “ER homeostasis”. Any physiological or pathological disturbance to this homeostasis may result in an accumulation of misfolded/unfolded proteins within the ER, a condition known as “ER stress”. In response to stress, the ER initiates a collection of signaling pathways, known as “unfolded protein response (UPR),” through which it attempts to restore its homeostasis (Chakrabarti, Chen, & Varner, 2011). The UPR consists of three major branches of signaling pathways that are mediated by three different ER transmembrane proteins (or stress sensors): inositol-requiring enzyme 1 (IRE1), pancreatic ER eukaryotic translation initiation factor (eIF)-2α kinase (PERK), and activating transcription factor 6 (ATF6).

Among the three ER stress sensors, IRE1 is highly conserved across species. It is a Type I transmembrane protein, comprised of an N-terminal ER luminal domain, a transmembrane domain, and a cytoplasmic domain with serine/threonine kinase and endoribonuclease activities (Cox, Shamu, & Walter, 1993; Shamu & Walter, 1996; Tirasophon, Welihinda, & Kaufman, 1998). Under unstressed conditions, ER luminal chaperone immunoglobulin heavy chain-binding protein (BIP), also known as glucose-regulated protein 78 (GRP78), binds to the luminal domain of IRE1, keeping IRE1 in its inactive monomer state (Bertolotti, Zhang, Hendershot, Harding, & Ron, 2000; Kimata et al., 2007; Oikawa, Kimata, Kohno, & Iwawaki, 2009). Upon ER stress, BIP dissociates, resulting in oligomerization, autophosphorylation and activation of the IRE1 endoribonuclease domain (Korennykh, Egea, et al., 2011; Korennykh et al., 2009; Korennykh, Korostelev, et al., 2011; K. P. Lee et al., 2008; Li, Korennykh, Behrman, & Walter, 2010; Oikawa et al., 2009; Shamu & Walter, 1996; Welihinda & Kaufman, 1996).

In mammals, the activated IRE1 endoribonuclease catalyzes a nonconventional splicing of 26 nucleotides from an unspliced X-box binding protein 1 (XBP1U) mRNA to give rise to a spliced XBP1 (XBP1S) mRNA that encodes a potent XBP1S transcription factor (Calfon et al., 2002; Yoshida, Matsui, Yamamoto, Okada, & Mori, 2001). XBP1S enters the nucleus and upregulates the transcription of a variety of genes involved in regulating ER protein folding, ER-associated degradation (ERAD), lipid biosynthesis, lipogenesis, and cell differentiation (Acosta-Alvear et al., 2007; A. H. Lee, Iwakoshi, & Glimcher, 2003; A. H. Lee, Scapa, Cohen, & Glimcher, 2008; Shaffer et al., 2004; Sriburi, Jackowski, Mori, & Brewer, 2004; Tohmonda et al., 2011; Yoshida et al., 2003). In contrast, XBP1U mRNA encodes a protein that is rapidly degraded by proteasome (Calfon et al., 2002; Navon et al., 2010; Tirosh, Iwakoshi, Glimcher, & Ploegh, 2006; Yoshida et al., 2001). Nevertheless, it has been shown that XBP1U protein regulates autophagy, protects endothelial cells from oxidative stress, and maintains the contractile phenotype of vascular smooth muscle cells (VSMCs) (Martin et al., 2014; G. Zhao et al., 2017; Y. Zhao et al., 2013). To determine the roles of XBP1 in vivo, the Xbp1 gene was disrupted in mice by replacing parts of exons 1 and 2 together with the intervening intron with a neomycin resistance gene, thereby, both XBP1U and XBP1S were inactivated; these mice died embryonically due to impaired liver function (Reimold et al., 2000). However, the respective roles of XBP1U and XBP1S in mouse development are largely unknown.

In this study, we generated a novel mouse model (referred to as “Xbp1CS/+”) that carried a modified Xbp1 allele that constitutively expressed XBP1S following Cre-recombinase (Cre)-mediated recombination. We crossed the Xbp1CS/+ mice with Twist2-Cre knock-in mice that express Cre recombinase in Twist2-expressing cells, and obtained Twist2-Cre;Xbp1CS/+ mice to constitutively express XBP1S in the Twist2-expressing cells and in the cells derived from the Twist2-expressing cells. We found that the Twist2-Cre;Xbp1CS/+ mice were smaller, and most of them died perinatally. We further confirmed constitutive expression of XBP1S in a variety of mouse tissues, but not in the brain in the Twist2-Cre;Xbp1CS/+ mice. These findings suggest that an exact control of XBP1S production is essential for normal mouse development.

MATERIALS &METHODS

Animals

All mice were maintained on a C57BL/6 background and were bred and maintained in community housing (≤ 4 mice/cage, 22° C) on a 12 h light/dark cycle with free access to water and standard pelleted food. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Texas A&M University College of Dentistry (Dallas, TX).

Generation of Xbp1sCS/+ and Xbp1sCS/CS mice

We generated the Xbp1CS/+ mouse strain by conventional homologous recombination approach (Figure 1A). Briefly, an Xbp1 targeting vector was constructed in which a neomycin cassette (neo-cassette) flanked by two flippase recognition target (Frt) sites was placed downstream of Xbp1 exon 5. Exons 4 and 5 together with the neo-cassette was further flanked by two loxP sites, and a modified version of Xbp1 exon 4 (E4Δ26) along with exon 5 was positioned downstream of the second loxP site. The exon E4Δ26 is devoid of the intronic sequence of 26 nucleotides. A diptheria toxin gene fragment A (DTA) cassette was cloned downstream to the 3’ homologous arm. The DTA cassette will reduce the frequency of random integration of the targeting construct in the genome in embryonic stem (ES) cells, as random integration, but not homologous recombination, will result in the expression of DTA, which leads to cell death (Melidoni, Dyson, Wormald, & McCafferty, 2013). The targeting vector was linealized and electrophorated into ES cells, which were subsequently selected with G418 antibiotic. G418-resistant clones were first screened by polymerase chain reaction (PCR) (data not shown), and then by Southern-blot analysis of SspI-, BglII- and EcoNI-digested genomic DNA using external 5’ and 3’ probes as well as neo probe, respectively (Figure 1B). With SspI digestion, the wild type allele gave rise to a 16.6 kb DNA fragment and the correctly targeted allele produced a 10.5 kb fragment, and both fragments were detected by the 5’ probe. With BglII digestion, the wild-type allele yielded a 15.0 kb fragment and the correctly targeted allele produced a 9.6 kb fragment, and both fragments were detected by the 3’ probe. The neo probe only detected a 12.5 kb fragment from the correctly targeted allele after EcoNI digestion. Correctly targeted ES clone cells were injected into blastocysts to produce chimeras, which were bred with wild-type C57BL/6 mice to obtain germ-line transmitted Xbp1CS/+ mice. The Xbp1CS/+ mice were intercrossed to generate the Xbp1CS/CS mice.

FIGURE 1. Generation of targeted Xbp1 allele (Xbp1CS) and Δ26 mutant Xbp1 allele (Xbp1Δ26).

FIGURE 1

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(A) A schematic representation of Xbp1 gene targeting strategy. a) wild-type Xbp1 locus. The Xbp1 gene consists of five exons (E1-E5). Green boxes, coding exons (or coding regions of exons); and grey boxes, noncoding regions of exons. The lengths of SspI and BglII genomic restriction fragments are indicated by double-arrow solid lines. The locations of the 5’ and 3’ probes (orange lines) for Southern blot analysis are also shown. b) targeting construct. The neomycin (Neo) cassette (orange box) flanked by two flippase recognition target (Frt) sites (red oval shapes) was inserted downstream of exon 5. Exons 4 and 5 together with the neo-cassette was flanked by two loxP sites (blue triangles), followed by a modified version of Xbp1 exon 4 (E4Δ26) and exon 5. Exon E4Δ26 is devoid of the intronic sequence of 26 nucleotides. A diphtheria toxin gene fragment A (DTA) cassette was placed downstream to the 3’ homologous arm. c) targeted allele (Xbp1CS) generated following conventional homologous recombination. d) Δ26 mutant Xbp1 allele (Xbp1Δ26) generated following Cre-mediated recombination: In the presence of Cre recombinase, exons 4 and 5 along with the neo-cassette were removed from the targeted Xbp1CS allele, and were replaced by downstream exons E4Δ26 and 5. Therefore, there was only one loxP site retained in the intron 3 in the Xbp1Δ26 allele. e) Nucleotide sequences of the wild-type Xbp1 exon 4 (E4) and Δ26 mutant Xbp1 exon 4 (E4Δ26). The bolded nucleotide sequence in E4 is the intronic sequence of 26 nucleotides, which is deleted in E4Δ26. (B) Southern-blotting analysis of the targeted embryonic stem (ES) cell clones. The targeted ES clones were analyzed by Southern blot with 5’ probe, 3’ probe and neo probe. A total of eight correctly targeted ES clones (clones 1-B04, 1-B08, 1-F04, 1-G09, 1-H09, 2-B02, 2-C07, and 2-E04) were identified. WT, wild-type ES cell clone.

PCR genotyping was performed using genomic DNA extracted from mouse tail biopsies with the following primers, forward primer Loxp-F (5’-CTTGCTCTGTAGCTGATAATCCTACCTC-3’) and reverse primer Loxp-R (5’-CAGAGGTGCACATAGTCTGAGTGCTG-3’). The PCR reaction was set at 95 °C for 2 min as an initial denaturation, followed by 35 cycles of 95 °C for 30 sec, 59 °C for 30 sec, 72 °C for 45 sec, and a 7-minute final extension at 72 °C. It produced a product of 495 bp for the wild-type Xbp1 allele and 573 bp for the targeted Xbp1CS/+ allele (Figure 2A).

FIGURE 2. Genotyping strategy.

FIGURE 2

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(A) The Xbp1 allele variants were genotyped by PCR using primers Loxp-F and Loxp-R, which produced a product of 495 bp for the wild-type (Wt) allele and 573 bp for the targeted allele (Xbp1CS). The Twist2-Cre allele was genotyped as previously described (Yu et al., 2003) and generated a product of 370 bp. (B) The Xbp1 allele variants were amplified by PCR using primers Xbp1-F and Xbp1-R1, which produced a product of 1337 bp for the wild-type (Wt) allele, 1415 bp for the targeted allele (Xbp1CS), and 1392 bp for the mutant allele (Xbp1Δ26). The PCR products were digested with or without restriction enzyme ApaLI. The PCR products from the Xbp1+/+ mouse were completely cleaved by ApaL1 into two fragments of 900 bp and 437 bp; the PCR products from the Xbp1CS/CS mouse were completely cleaved into two fragments of 978 bp and 437 bp. The Twist2-Cre;Xbp1CS/CS mouse contained a mixture of the targeted (Xbp1CS) and mutant (Xbp1Δ26) alleles, the PCR products from the Xbp1CS allele were digested into two fragments of 978 bp and 437 bp, whereas those from the Xbp1Δ26 allele were undigested. (C) DNA sequencing. Top: the electropherogram of DNA sequencing result of the undigested PCR products from the Twist2-Cre;Xbp1CS/CS mouse. The downward arrow indicates the deletion of the 26-base-pair intronic sequence. Bottom: the alignment of wild-type (Wt) Xbp1 exon 4 and mutant Xbp1Δ26 exon 4 (Δ26). The 26-base-pair intronic sequence is underlined in the wild-type Xbp1 allele, but it is deleted in the mutant Xbp1Δ26 allele.

Generation of Twist2-Cre;Xbp1sCS/+ and Twist2-Cre;Xbp1sCS/CS mice

The Xbp1sCS/+ mice were mated with Twist2-Cre knock-in mice (Twist2Cre/+; Stock No. 008712, the Jackson Laboratory) to generate Twist2-Cre;Xbp1CS/+ mice. The Twist2-Cre;Xbp1CS/+ mice were crossed with the Xbp1CS/CS mice to generate the Twist2-Cre;Xbp1CS/CS mice. The Twist2-Cre mice carry a Cre recombinase knock-in Twist2 allele, and express Cre recombinase under the control of the entire Twist2 regulatory sequences (Yu et al., 2003). In the presence of Cre recombinase, the floxed exons 4 and 5 together with the neo-cassette would be removed so that the modified exon 4 (E4Δ26) and exon 5 would be brought immediately downstream of Xbp1 exon 3 (Figure 1A). The resulting mutant Xbp1Δ26 allele would express “mutant” spliced XBP1S mRNA, which constitutively express XBP1S protein even in the absence of ER stress (Figure 1A). The Twist2-Cre allele was genotyped (a PCR product of 370 bp), as previously described (Yu et al., 2003) (Figure 2A).

Confirmation of Δ26 mutant Xbp1 allele (Xbp1Δ26) by PCR, enzymatic digestion and DNA sequencing

A combination of PCR, enzymatic digestion and DNA sequencing was conducted to confirm the Δ26 mutant Xbp1 allele (Xbp1Δ26). PCR was performed using the following primers, forward primer Xbp1-F (5’-GAACCAGGAGTTAAGAACACG-3’) and reverse primer Xbp1-R1 (5’-AACATGACAGGGTCCAACTTGTCCA-3’), and Phusion High-Fidelity DNA polymerase (New England Biolabs, Inc., Ipswich, MA), which yielded a product of 1337 bp for the wild-type allele, 1415 bp for the targeted allele (Xbp1CS) and 1392 bp for the mutant allele (Xbp1Δ26). The PCR reaction was set at 98 °C for 30 sec as an initial denaturation, followed by 35 cycles of 98 °C for 5 sec, 60 °C for 15 sec, 72 °C for 50 sec, and a 7-minute final extension at 72 °C. The PCR products were then digested with or without ApaLI restriction enzyme. The 1337 bp PCR products amplified from the Xbp1+/+ mice were completely cleaved by ApaLI into two fragments of 900 bp and 437 bp; the PCR products from the Xbp1CS/CS mice were completely cut into two fragments of 978 bp and 437 bp. The Twist2-Cre;Xbp1CS/CS mice contained a mixture of the targeted and mutant Xbp1 alleles, the PCR products from the targeted Xbp1CS allele were digested into two fragments of 978 bp and 437 bp, whereas those from the mutant Xbp1Δ26 allele were undigested. The 1392 bp undigested PCR products amplified from the mutant Xbp1Δ26 allele were purified from agarose gel using the QIAquick gel extraction kit (Qiagen, Germantown, MD) and were sequenced to verify the deletion of the 26-base-pair intronic sequence in the exon 4 of the mutant Xbp1Δ26 allele (Sequetech, Mountain View, CA).

Photography

The embryonic day 11.5 (E11.5) Xbp1CS/+ and Twist2-Cre;Xbp1CS/+ embryos were photographed using a high-precision stereo Olympus SZX16 microscope (Olympus Corporation, Tokyo, Japan). The newborn mice were photographed using a digital camera.

Reverse-transcription PCR (RT-PCR)

Total RNA was extracted from various tissues, including brain, heart, lung, liver, spleen, pancreas, kidney, skeletal muscle and bone, of newborn Xbp1CS/+ and Twist2-Cre;Xbp1CS/+ mice using TRIzol reagent (Ambion, Carlsbad, CA, Invitrogen). Five hundred nanogram of total RNAs were reverse transcribed into cDNAs using QuantiTect Reverse Transcription Kit (Qiagen, Germantown, MD), according to the manufacturer’s instructions. The XBP1 cDNA was then amplified by PCR using the following primers, forward primer Xbp1-F (5’-GAACCAGGAGTTAAGAACACG-3’) and reverse primer Xbp1-R2 (5’-AGGCAACAGTGTCAGAGTCC-3’), as previously described (Iwawaki, Akai, Kohno, & Miura, 2004). The forward primer is located in exon 3 of the Xbp1 gene, whereas the reverse primer in exon 4, downstream of the intronic sequence of 26 nucleotides; thereby both XBP1U and XBP1S cDNAs were amplified. The plasmid containing spliced XBP1 cDNA (pFLAG.XBP1p.CMV2; Addgene, Watertown, MA) and the plasmid containing unspliced XBP1 cDNA (pFLAG.XBP1u.CMV2; Addgene, Watertown, MA) were a gift from David Ron (Calfon et al., 2002), and were used as the controls. The PCR products were resolved by electrophoresis on a 3% agarose gel. The gels were imaged with Azure C150 gel imaging system (Azure Biosystems, Dublin, CA), and the density of each PCR band was quantified by using ImageJ program (Schneider, Rasband, & Eliceiri, 2012). The ratios of XBP1S to XBP1U mRNAs in each tissue examined in the Xbp1CS/+ and Twist2-Cre;Xbp1CS/+ mice were calculated based on the densities of the PCR bands corresponding to XBP1S and XBP1U mRNAs. Three individual newborn mice were analyzed for each designated tissue and for each genotype. The quantified data shown represented mean ± standard deviation (SD).

Tissue processing and immunohistochemistry (IHC)

Tissue processing and IHC were performed as previously described (Gibson et al., 2013; Liang et al., 2019). Briefly, heads were harvested from newborn Xbp1CS/+ and Twist2-Cre;Xbp1CS/+ mice, and fixed in 4% paraformaldehyde in diethyl pyrocarbonate (DEPC)-treated 0.1M phosphate-buffered saline (PBS) for 24 hours. They were then dehydrated, embedded in paraffin and cut into 5-μm sections. IHC was then conducted to detect total XBP1 (XBP1U and XBP1S) and spliced XBP1S using rabbit anti-XBP1 polyclonal antibody that recognizes both XBP1U and XBP1S (1:200; Abcam, Cambridge, MA) and rabbit anti-XBP1S monoclonal antibody (E9V3E) that is specific to XBP1S (1:50; Cell Signaling Technology, Danvers, Massachusetts), respectively. The anti-XBP1S monoclonal antibody was produced with a synthetic peptide that corresponds to the carboxyl-terminus of XBP1S. The secondary antibody used was biotinylated goat anti-rabbit IgG (H+L) antibody (1:200, Vector Laboratories, Burlingame, CA). The immunostaining signals were visualized using the DAB (3,3’-diaminobenzidine) kit (Vector Laboratories, Burlingame, CA), according to the manufacturer’s instructions. The sections were counterstained with methyl green (Sigma, Saint Louis, MO). Images were taken under an upright BX51 Olympus microscope (Olympus America Inc., Waltham, MA).

Statistical analysis

Statistical analysis was conducted using the GraphPad Prism 9.0 software package (GraphPad Software). A Chi-Square goodness of fit test was performed to determine whether or not the number of survived mice followed the Mendelian inheritance in genotype distribution. Student’s t-test was employed to compare the difference in the ratios of XBP1S/XBP1U mRNAs between the Xbp1CS/+ and Twist2-Cre;Xbp1CS/+ mice. P<0.05 was considered statistically significant.

Results

Generation of the Xbp1CS/+ and Xbp1CS/CS mice

We first generated the Xbp1CS/+ strain by conventional homologous recombination approach. The targeting vector was constructed in which once the floxed DNA sequence, consisting of Xbp1 exon 4 (encoding XBP1U), exon 5 and a neo-cassette, was excised by Cre recombinase, a modified version of exons 4 (E4Δ26, encoding XBP1S) and exon 5 would be brought immediately downstream of exon 3 (Figure 1A). Southern blot analysis of genomic DNA extracted from G418 resistant ES cell clones revealed that eight of the ten targeted clones were correctly targeted ES clones, including 1-B04, 1-B08, 1-F04, 1-G09, 1-H09, 2-B02, 2-C07 and 2-E04) (Figure 1B). The correctly targeted ES clone cells were subsequently injected into blastocysts, and chimera were obtained. The chimera was further crossed with wild-type mice, and established a germ-line transmission of the targeted Xbp1CS/+ allele. The Xbp1CS/+ mice were further intercrossed and generated Xbp1CS/CS mice. Both Xbp1CS/+ and Xbp1CS/CS mice were phenotypically normal. These mice were expected to constitutively express XBP1S following Cre-mediated recombination.

Perinatal lethality of the Twist2-Cre;Xbp1CS/+ and Twist2-Cre;Xbp1CS/CS mice

Next, we introduced Cre recombinase into the Xbp1CS/+ mice by crossing the Xbp1CS/CS mice with the Twist2-Cre knock-in mice. Of 107 mice genotyped, we obtained 83 Xbp1CS/+ mice, but only 24 Twist2-Cre;Xbp1CS/+ mice (Table 1). Chi-Square goodness of fit test demonstrated that there was a significant difference in genotype distribution between the number of mice observed and the number of mice expected from the Mendelian inheritance (Table 1), suggesting that constitutive expression of XBP1 may cause early death of mice. Therefore, we examined the litters harvested from timed matings, and found that the Twist2-Cre;Xbp1CS/+ embryos showed no apparent abnormalities at E11.5, compared to the Xbp1CS/+ control embryos (Figure 3A–B). However, the newborn Twist2-Cre;Xbp1CS/+ mice were smaller, and showed no evidence of sucking milk compared to the Xbp1CS/+ control littermates (Figure 3C–D). Most of the Twist2-Cre;Xbp1CS/+ newborns died shortly after birth (The estimated death rate is approximately 69% for the Twist2-Cre;Xbp1CS/+ pups, assuming 8 pups per litter.). The Twist2-Cre;Xbp1CS/+ mice that survived to adulthood remained smaller compared to the age- and sex-matched Xbp1CS/+ mice, but they were fertile. We crossed them with the Xbp1CS/CS mice in order to generate the Twist2-Cre;Xbp1CS/CS mice. Yet, of 44 mice genotyped, we obtained 23 (52.27%) XBP1CS/+ mice, 13 (29.55%) XBP1CS/CS mice, and eight (18.18%) Twist2-Cre;XBP1CS/+ mice, but did not obtain any Twist2-Cre;Xbp1CS/CS mice who could survive after birth (Table 2). Chi-Square goodness of fit test further confirmed the significant difference in genotype distribution between the number of mice observed and the number of mice expected from the Mendelian inheritance (Table 2). Genotyping of the embryos harvested from the timed pregnant females revealed that the Twist2-Cre;Xbp1CS/CS embryos were similar in overall appearance as the Twist2-Cre;Xbp1CS/+ embryos at E18.5 (data not shown).

Table 1.

Chi-Square goodness of fit test for observed and expected mice in genotype distribution

Genotype Observed n Expected n Observed % Expected %
Xbp1 CS/+ 83 53.5 77.57 50.00
Twist2-Cre;Xbp1 CS/+ 24 53.5 22.43 50.00
Total 107 107 100.00 100.0
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Notes: Data represented 20 litters of survived mice born of the Xbp1CS/CS mice mated with the Twist2-Cre mice. n, the number of mice; X2(1) = 32.533; p < 0.001

FIGURE 3. Gross view of the Twist2-Cre;Xbp1CS/+ mice.

FIGURE 3

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(A-B) Representative embryonic day 11.5 (E11.5) Xbp1CS/+ (A) and Twist2-Cre;Xbp1CS/+ (B) embryos. The Twist2-Cre;Xbp1CS/+ embryos had no apparent abnormalities, compared to the Xbp1CS/+ embryos of the same litter. Scale bars: 1 mm. (C-D) Representative newborn (NB) littermates Xbp1CS/+ (C) and Twist2-Cre;Xbp1CS/+ (D) mice. The Xbp1CS/+ newborn mice showed a clearly visible milk spot on their left sides, indicating milk sucking after birth. However, the Xbp1CS/+ newborn mice appeared to be smaller in size, and had no evidence of sucking milk; and most of them died shortly after birth. Scale bars: 5 mm.

Table 2.

Chi-Square goodness of fit test for observed and expected mice in genotype distribution

Genotype Observed n Expected n Observed % Expected %
Xbp1 CS/+ 23 11 52.27 25.00
Xbp1 CS/CS 13 11 29.55 25.00
Twist2-Cre;Xbp1 CS/+ 8 11 18.18 25.00
Twist2-Cre;Xbp1 CS/CS 0 11 0.00 25.00
Total 44 44 100.00 100.0
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Notes: Data represented eight litters of survived mice born of the Xbp1CS/CS mice mated with the Twist2-Cre;Xbp1CS/+ mice. n, the number of mice; X2(3) = 25.273; p < 0.001.

Δ26 mutant Xbp1 allele (Xbp1Δ26) generated by Cre-mediated recombination

To confirm genomic organization and sequence of the Δ26 mutant Xbp1 allele (Xbp1Δ26) generated by Cre-mediated recombination, we performed PCR to amplify the genomic region spanning part of exon 3, intron 3, exon 4, intron 4 and part of exon 5 of the Xbp1 allele variants using genomic DNA extracted from the Xbp1+/+, Xbp1CS/CS and Twist2-Cre;Xbp1CS/CS mouse embryos (Figure 2B). The length of the PCR products from the wild-type allele was 1337 bp, and was cleaved by ApaL1 into two fragments of 900 bp and 437 bp; the length of the PCR products from the targeted Xbp1CS allele was 1415 bp, and was digested into two fragments of 978 bp and 437 bp; and whereas that of the mutant Xbp1Δ26 allele was 1392 bp, and could not be digested by ApaL1 enzyme (Figure 2B). The undigested Xbp1Δ26 allele PCR products from the Twist2-Cre;Xbp1CS/CS embryos were purified and sequenced. As expected, DNA sequencing confirmed that the mutant Xbp1Δ26 allele had the same genomic organization as the wild-type Xbp1 allele (Suppl Figure 1). Moreover, it lacked the intronic sequence of 26 nucleotides in the modified exon 4 (E4Δ26) (Figure 2C and Suppl Figure 1), and contained only one loxP site in the middle of intron 3, compared to the wild-type allele (Suppl Figure 1). Therefore, the mutant Xbp1Δ26 allele would constitutively express spliced XBP1S mRNA and protein, regardless of the presence/absence of ER stress, in the Twist2-Cre;Xbp1CS/+ and Twist2-Cre;Xbp1CS/CS embryos/mice.

Constitutive expression of XBP1S in various tissues of the Twist2-Cre;Xbp1CS/+ mice

RT-PCR and IHC were performed to determine the levels of spliced/unspliced XBP1 mRNA and protein, respectively, in the various tissues of the Twist2-Cre;Xbp1CS/+ mice. RT-PCR showed that the level of XBP1S mRNA was dramatically increased in the most tissues examined, including heart, lung, liver, spleen, pancreas, kidney, skeletal muscle and bone, but not in the brain, in the Twist2-Cre;Xbp1CS/+ mice, compared to those in the Xbp1CS/+ control mice (Figure 4). Moreover, IHC analyses showed that the immunostaining signals for total XBP1 (XBP1U and XBP1S) were detected in the calvarial bone (Figure 5A and B) and brain (Figure 5E and F) in both Xbp1CS/+ and Twist2-Cre;Xbp1CS/+ mice, even though the signals became slightly weaker in the Twist2-Cre;Xbp1CS/+ mice. In contrast, when IHC analyses were done with a monoclonal antibody that specifically recognizes the carboxyl-terminus of XBP1S, the XBP1S immunostaining signals were only detected in the calvarial bone in the Twist2-Cre;Xbp1CS/+ mice (Figure 5D), but not in the Xbp1CS/+ mice (Figure 5C); and the XBP1S signals were not detected in the brains in either Xbp1CS/+ (Figure 5G) or Twist2-Cre;Xbp1CS/+ (Figure 5H) mice. The tissue distribution of the XBP1S mRNA and protein is consistent with the Twist2-Cre expression profile, which is active in the mesoderm-derived tissues, but not in the neural tissues including the brain (Yu et al., 2003). Taken together, these results suggest that the mutant Xbp1Δ26 allele, as expected, constitutively expressed the XBP1S mRNA and protein in the Twist2-Cre;Xbp1CS/+ mice.

FIGURE 4. Increased expression of XBP1S mRNA in newborn Twist2-Cre;Xbp1CS/+ mice.

FIGURE 4

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RT-PCR was performed using total RNAs isolated from various tissues of newborn Xbp1CS/+ (A) and Twist2-Cre;Xbp1CS/+ (B) mice. The tissues were analyzed including brain, heart, lung, liver, spleen, pancreas, kidney, skeletal muscle and bone. PCR products corresponding to XBP1U mRNA and XBP1S mRNA are indicated. The ratios of XBP1S to XBP1U mRNAs in different tissues in the Xbp1CS/+ and Twist2-Cre;Xbp1CS/+ mice were calculated based on the densities of the PCR bands determined by ImageJ (C). The data represent mean ± standard deviation (SD) (n=3). The Twist2-Cre;Xbp1CS/+ newborn mice displayed increased expression of XBP1S mRNA in various tissues, compared to the Xbp1CS/+ newborn mice. M, DNA molecular weight markers; pXBP1U, unspliced XBP1 plasmid control; pXBP1S, spliced XBP1 plasmid control; 1:5, the ratio of pXBP1S plasmid to pXBP1U plasmid; and 5:1, the ratio of pXBP1S plasmid to pXBP1U plasmid.

FIGURE 5. Immunohistochemical analyses of total XBP1 and spliced XBP1S.

FIGURE 5

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Shown are the representative immunohistochemical staining results (signal in brown) of total XBP1 (XBP1U and XBP1S) in the calvarial bone (A-D) and brain (E-H) of the newborn Xbp1CS/+ (A, C, E and G) and Twist2-Cre;Xbp1CS/+ (B, D, F and H) mice. Note that the immunostaining signals for total XBP1 was found in the calvarial bones and brains in both Xbp1CS/+ and Twist2-Cre;Xbp1CS/+ mice, but the signals became slightly weaker in the Twist2-Cre;Xbp1CS/+ mice. Also note the nuclear localization of the immunostaining signals for XBP1S in the calvarial bone of the Twist2-Cre;Xbp1CS/+ mice, but not in Xbp1CS/+ mice, and the lack of the XBP1S signals in the brains in both Xbp1CS/+ and Twist2-Cre;Xbp1CS/+ mice. Scale bars: 20 μm in A-H.

DISCUSSION

XBP1S is a key mediator of the IRE1 branch of the UPR, which has been implicated in a wide range of physiological and pathological processes. In this study, we generated a novel mouse model, Twist2-Cre;Xbp1CS/+, that constitutively expressed XBP1S in the Twist2-expressing cells as well as in the cells derived from the Twist2-expressing cells and found that most Twist2-Cre;Xbp1CS/+ mice died shortly after birth.

We demonstrated that the Twist2-Cre;Xbp1CS/+ mice constitutively expressed XBP1S in the mesoderm-derived tissues, including heart, lung, liver, spleen, pancreas, kidney, skeletal muscle and bone, due to the presence of the mutant Xbp1Δ26 allele. In addition to the Xbp1Δ26 allele, the Twist2-Cre;Xbp1CS/+ mice had a wild-type Xbp1 allele, so they continued to express XBP1U mRNA and protein. Previous studies showed that even though the Xbp1-null mice died embryonically, the Xbp1 heterozygous mice appeared to be phenotypically normal (A. H. Lee, Chu, Iwakoshi, & Glimcher, 2005; Reimold et al., 2000). Moreover, Ishikawa et al reported that while the medaka fish was normal if one allele of the Xbp1 locus had the Δ26 mutation and constitutively expressed spliced XBP1S, the medaka fish failed to survive till 2 months after hatching if both alleles of the Xbp1 locus carried the Δ26 mutation (Ishikawa et al., 2017). Taken together, these studies support that excessive expression of XBP1S caused the perinatal lethality of the Twist2-Cre;Xbp1CS/+ mice.

The level and activity of XBP1S is tightly regulated at the translational and posttranslational levels within a cell. First of all, XBP1S is only produced when the unspliced XBP1 mRNA is converted into spliced mRNA by IRE1α endoribonuclease that is activated upon ER stress, whereas XBP1U is constitutively translated from the unspliced XBP1 mRNA (Calfon et al., 2002; Yoshida et al., 2001). Secondly, XBP1U undergoes ubiquitin-dependent and -independent proteasomal degradation after synthesis (Calfon et al., 2002; Navon et al., 2010; Tirosh et al., 2006; Yoshida et al., 2001; Yoshida, Oku, Suzuki, & Mori, 2006), and it also binds to and targets XBP1S for proteasomal degradation during the recovery phase of ER stress, thereby reducing the level of XBP1S once ER stress is relieved (Yoshida et al., 2006). Thirdly, XBP1S itself is subject to ubiquitin-mediated proteasomal degradation (J. Lee et al., 2018; Sun et al., 2020; Y. Wang et al., 2019). Lastly, other posttranslational modifications, such as phosphorylation, acetylation/deacetylation and sumoylation/desumoylation, can affect XBP1S protein stability and activity as well (Jiang et al., 2012; J. Lee et al., 2011; Liu et al., 2016; F. F. Wang et al., 2016; F. M. Wang, Chen, & Ouyang, 2011). These different mechanisms ensure that XBP1S is only produced and functional when it is needed during ER stress.

In this study, we have demonstrated that the Twist2-Cre;Xbp1CS/+ mice still expressed a moderate level of XBP1U due to the presence of the wild-type Xbp1 allele. It is also very likely that these mice may have normal posttranslational mechanisms, through which the protein stability and activity of XBP1S is regulated. Nevertheless, the Twist2-Cre;Xbp1CS/+ mice showed a higher level of XBP1S, compared to the control mice. These findings indicate that the amount of constitutively expressed XBP1S may overwhelm the mechanisms through which XBP1 is degraded. In other words, our current findings suggest that the conversion of unspliced XBP1 mRNA into spliced mRNA by activated IRE1 endoribonuclease may be the pivotal step to control the level and activity of XBP1S in a cell.

In summary, we have shown that constitutive expression of XBP1S resulted in perinatal lethality in mice. As Xbp1 is widely expressed in many cell types, future studies are warranted to delineate the detrimental effects of continuous presence of excessive XBP1S in each cell type by generating cell-type-specific expression of XBP1S using cell-type-specific Cre-recombinase.

Supplementary Material

Supplemental Figure 1 Sequence alignment of wild-type Xbp1 allele (Wt) and mutant Xbp1Δ26 allele (Δ26)

ACKNOWLEDGEMENTS

The authors thank Biocytogen for generating the Xbp1CS/+ mouse strain. Q.X. and H.Z. contributed to design, data acquisition, analysis and interpretation, drafted and critically revised the manuscript. S.W. contributed to data acquisition, analysis, drafted and critically revised the manuscript; C.Q. contributed to conception, interpretation, drafted and critically revised the manuscript; Y.L. contributed to conception, design, interpretation, drafted and critically revised the manuscript. All authors approved the final version of the submitted manuscript and agreed to be accountable for all aspects of the work. The authors declare that they have no conflict of interest with respect to the content of this article. This work was supported by grant DE027345 from National Institute of Dental and Craniofacial Research.

Grant support:

grant DE027345 from National Institute of Dental and Craniofacial Research (NIDCR)

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Associated Data

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Supplementary Materials

Supplemental Figure 1 Sequence alignment of wild-type Xbp1 allele (Wt) and mutant Xbp1Δ26 allele (Δ26)
NIHMS1796241-supplement-Supplemental_Figure_1.docx (15.6KB, docx)

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