Sec13 Safeguards the Integrity of the Endoplasmic Reticulum and Organogenesis of the Digestive System in Zebraﬁsh

Developmental Biology 367 (2012) 197–207

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Developmental Biology

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Sec13 safeguards the integrity of the endoplasmic reticulum and organogenesis of the digestive system in zebraﬁsh

Xubo Niu a,1, Chuan Gao b,1, Li Jan Lo a, Yue Luo a, Chunmei Meng c, Jian Hong c, Wanjin Hong d,e, Jinrong Peng a,n a Key Laboratory for Molecular Animal Nutrition, Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou, PR China b Department of Biological Sciences, National University of Singapore, Singapore c Institute of Biotechnology, Zhejiang University, Hangzhou, PR China d School of Pharmaceutical Sciences, Xiamen University, Xiamen, PR China e Institute of Molecular and Cell Biology, Agency for Science and Technology Research (A*STAR), Singapore article info abstract

Article history: The Sec13-Sec31 heterotetramer serves as the outer coat in the COPII complex, which mediates protein Received 25 October 2011 trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus. Although it has been studied in Received in revised form depth in yeast and cultured cells, the role of COPII in organogenesis in a multicellular organism has not. 12 April 2012 We report here that a zebrafish sec13sq198 mutant, which exhibits a phenotype of hypoplastic digestive Accepted 4 May 2012 organs, has a mutation in the sec13 gene. The mutant gene encodes a carboxyl-terminus-truncated Available online 15 May 2012 Sec13 that loses its affinity to Sec31a, which leads to disintegration of the ER structure in various Keywords: differentiated cells in sec13sq198, including chondrocytes, intestinal epithelial cells and hepatocytes. Sec13 Disruption of the ER structure activates an unfolded protein response that eventually causes the cells to COPII complex undergo cell-cycle arrest and cell apoptosis, which arrest the growth of developing digestive organs in Digestive organs the mutant. Our data provide the first direct genetic evidence that COPII function is essential for the Liver development Zebrafish organogenesis of the digestive system. & 2012 Elsevier Inc. All rights reserved.

Introduction factors (Gata4/5/6), FoxA factors (FoxA1/2/3), and other factors, such as Def, Npo and Elys, are essential for the normal organo- The vertebrate digestive system is formed by the alimentary genesis of the entire digestive system (Chen et al., 2005; de Jong- tract and digestive glands that originate from the endoderm germ Curtain et al., 2009; Mayer and Fishman, 2003). The signaling layer (Wells and Melton, 1999). In zebrafish, endoderm cells are molecules Bmp2a/b, Fgf, and Wnt2bb are apparently crucial distributed in a salt–pepper pattern by the end of gastrulation factors for the development of the liver and pancreas (Huang (Warga and Nusslein-Volhard, 1999). The salt–pepper patterned et al., 2008; Ober et al., 2006; Shin et al., 2007). endoderm cells migrate medially and form a solid rod along the In eukaryotes, protein trafficking in the secretory pathway is midline at 24 h post-fertilization (hpf), from which the alimen- essential for normal cellular activities, and is mediated by tary tract and digestive glands are derived. The liver and pan- vesicular carriers (or vesicles), which are composed by the creatic buds are clearly identifiable at the fore-part of the rod at delicately regulated assembly of coat protein complexes. The 50 hpf (Field et al., 2003; Ng et al., 2005; Ober et al., 2003; COPII complex is a specialized coat protein complex that is Wallace et al., 2005; Wallace and Pack, 2003). Morphologically, responsible for transporting newly synthesized secretory and the liver and pancreas in adult fish are comparable to those in membrane proteins from the endoplasmic reticulum (ER) to the other vertebrates. However, the zebrafish alimentary tract does Golgi apparatus in eukaryotic cells (Antonny and Schekman, not develop a distinct stomach but only forms an expanded 2001). There are five core COPII components: the small GTPase foregut bulb (Wallace et al., 2005). The nodal factors Cyclop and Sar1, the Sec13/Sec31 heterotetramer and the Sec23/Sec24 het- Squint, and the Sox-type factors, Casanova and Sox17, are essen- erodimer (Barlowe et al., 1994; Huh et al., 2003). Biogenesis of tial for the specification of the whole endoderm. In contrast, Gata the COPII complex begins with the activation of Sar1 from a GDP- bound to a GTP-bound form by its guanine-nucleotide-exchange factor Sec12 at the ER exit site (ERES) (Nakano et al., 1988;

n Nakano and Muramatsu, 1989). The GTP-bound Sar1 then serves Corresponding author. Fax: þ86 571 88982233. E-mail address: [email protected] (J. Peng). as an anchor to recruit the Sec23/Sec24 complex through its 1 These authors contributed equally to this work. carboxyl-terminal region interaction with Sec23 (Bi et al., 2002).

The Sar1/Sec23/Sec24 complex enriches cargo proteins and helps Huang et al., 2008). For WISH using fabp10a, fabp2 and trypsin to deform the membrane into a tubule-like pre-budding complex. three probes together, fabp10a and fabp2 were labeled with DIG The Sec13/Sec31 complex is then recruited through a physical and trypsin was labeled with fluorescein. interaction between Sec23 and Sec31, which results in the transformation of the pre-budding complexes into COPII transport Detection of alkaline phosphatase activity and Alcian blue staining vesicles (Antonny, 2006; Kuehn et al., 1998). Although COPII components are found in all vertebrates and Embryos were fixed in 4% paraformaldehyde/phosphate buf- plants, their functions have mainly been investigated in yeast and fered solution (PBS) and washed in PBS before being assayed for mammalian cells and rarely in whole organisms. One report alkaline phosphatase activity using nitro blue tetrazolium/5- showed that the loss-of-function mutation F382L in human bromo-4-chloro-30-indolyphosphate staining buffer or for Alcian SEC23A leads to human cranio-lenticulo-sutural dysplasia. Mutant blue staining as described previously (Chen et al., 2005). cells carrying the F382L mutation exhibit mislocalization of Sec31 and gross dilatation of the ER lumen (Boyadjiev et al., 2006). It BrdU incorporation assay, phosphorylated histone 3 (pH3) was also found that the zebrafish crusher mutant carries a immunostaining and TUNEL assay mutation in the sec23a gene and exhibits a phenotype that is comparable with cranio-lenticulo-sutural dysplasia. Chondro- For BrdU incorporation assay, embryos at 3.5 dpf were injected cytes in crusher accumulate proteins in bloated ER, resulting in with 1 nanolitre (nl) of 10 mM BrdU solution. Four hours after a severe reduction in extracellular matrix (ECM) deposits in the injection embryos were harvested for immunostaining of BrdU cartilage (Lang et al., 2006). Recently, Townley et al. (2008) positive cells using an anti-BrdU antibody (AbD seroTec, reported briefly that zebrafish Sec13 morphants also exhibited a OBT0030) in dilution 1:200 as described. TUNEL assay was cranial facial defect but they did not characterize the Sec13 performed using the In Situ Cell Death Detection Kit, TMR red morphants in detail. Disruption of the COPII complex not only (Roche) and pH3 immunostaining using the monoclonal antibody blocks protein trafficking but also activates the unfolded protein against pH3 (Santa Cruz, SC-8656-R) in dilution 1:200 as pre- response (UPR). In metazoa, the UPR is mediated by three distinct viously described (Chen et al., 2005). membrane-bound stress sensors—IRE1a, ATF6, and PERK (Hetz and Glimcher, 2009; Wiseman et al., 2010). The UPR is a cellular Rescue and morpholino mimicking of sec13sq198 phenotype response to cope with ER stress (Higashio and Kohno, 2002; Saito et al., 2011), such as that caused under pharmacological or WT sec13 and mutant sec13sq198 coding regions were cloned pathological condition (Cinaroglu et al., 2011; Pyati et al., 2011; into a pCS2þ vector. mRNAs were synthesized by mMessage Thakur et al., 2011). However, a prolonged UPR usually triggers mMachine SP6 Kit (Ambion). A 0.5-ng aliquot of in-vitro tran- cell apoptosis by activating pro-apoptotic factors such as the scribed WT or mutant sec13sq198 mRNA was injected into one cell- CCAAT enhancer-binding homologous protein (CHOP) (Pino et al., stage embryos. Injected embryos were subjected to WISH using a 2009;Yamaguchi and Wang, 2004). digoxigenin-labeled fabp10a probe at 3.5 dpf. We report here the identification and characterization of a Morpholinos were ordered from Gene Tools (Philomath, USA). zebrafish mutant, sec13sq198. sec13sq198 is hypoplastic in major The sec13 sp2 morpholino (50- TATTTGGCTGTGAATACCTGCGAGC- digestive organs, including the liver, exocrine pancreas and 30) was designed to target the junction of exon 7 and intron 7 of intestine. The mutation is caused by a thymdine (T) to adenine sec13 and 1 nL (0.2 nmol/mL) was injected into one cell-stage (A) substitution that creates a new splicing acceptor site in intron embryos. The sec31a morpholino (Sec31a-ATG MO) (50-CGGTT- 7 of the sec13 gene, which encodes the zebrafish Sec13 protein. AATTTCTTTCAGCTTCATCC-30) was designed to target the sec31a This mutation leads to disruption of the ER structure and translation start site, and 1 nL (0.75 nmol/mL) was injected into secretory pathway in mutant cells. We show that the defective one cell-stage embryos. A human b-globin antisense morpholino secretory pathway in this mutant activates the UPR pathway and (50-CCTCTTACCTCAGTTACAATTT-30) was used in parallel as the up-regulates pro-apoptotic factors such as CHOP, which is asso- standard control (st-MO). ciated with cell-cycle arrest and cell apoptosis, causing under- development of the digestive organs and branchial cartilage. RNA and protein analysis

RNA sample preparation and northern blot hybridization were Materials and methods performed as described previously (Cheng et al., 2006), as were total protein extraction and western blotting analysis (Chen et al., Fish lines 2009). The rabbit anti-Sec31a antibody was generated by the laboratory of Prof. Wanjin Hong. The mouse anti-Sec13 antiserum The WT AB strain of zebrafish (Danio rerio) was used in this was generated by immunizing mice with full-length zebrafish study. The sq198 small-liver mutant was identified from screen- Sec13 over-expressed in bacteria. Rabbit anti-Bip (Sigma, G9043; ing an ethylnitrosourea-induced mutated AB population (Huang 1:1000 dilution), rabbit anti-Chop (Sigma, G6916; 1:1000 dilu- et al., 2008). Heterozygous sq198 were mated with the poly- tion), rabbit anti-eIF2a (Cell Signaling Technology, 9720 S; 1:2000 morphic ecotype WIK to generate a mapping population. Zebra- dilution), and rabbit anti-P-eIF2a (Cell Signaling Technology, 9721 fish were raised and maintained in a standard Zebrafish Unit S; 1:2000 dilution) antibodies were used in western blotting (produced by Aisheng Zebrafish Facility Manufacturer Company, analysis. Beijing, China). Quantitative real-time polymerase chain reaction (qRT-PCR) Whole-mount in-situ hybridization (WISH) Embryos were collected at 3, 4, and 5 dpf and genotyped. Total WISH was performed as described previously (Mayer et al., RNA was extracted and purified. cDNA synthesis was performed 2003). RNA probes prox1, fabp10a, trypsin, insulin, fabp2, foxa1, using a reverse transcriptase kit (Invitrogen). Gene specific foxa3, gata6 and hhex were labeled with digoxigenin (DIG, Roche primers were designed through DNAStar software. Aliquots of Diagnostics) and used as previously described (Chen et al., 2005; 0.5 mL of each prepared cDNA and corresponding primers were X. Niu et al. / Developmental Biology 367 (2012) 197–207 199 added to SsoFast EvaGreen Super mix (Biorad). The CFX96 Real- whole-mount RNA in-situ hybridization (WISH) with various Time System (Biorad) was used to obtain the threshold cycle (Ct) early endoderm markers. Because the sq198 mutant was initially value. The relative expression of each gene was determined characterized by its small-liver phenotype at 3 dpf (Supplemental after being normalized to elongation factor 1a (elf1a) (10680.1 RKPM). Fig. 1A), we first compared the liver specification and budding in Primer sequences were: atf6 (NM_001110519) forward: CTgg- sq198 mutants and WT embryos by examining the expression AggCgCTggTgAAAAgTg, reverse: CCggACgggAgATgggAACA; bip patterns of two early hepatic markers, prox1 and hex, at 30 hpf, a (NM_213058) foward: ggTCggCggCTCCACTCgTAT, reverse: Ag- stage at which hepatoblasts are specified and a discrete liver bud CCggTTCTggTCgTTggTAATg; chop (NM_001082825) forward: starts to form. No discernible differences were revealed by these ggAgCCCgAgTTTTTggATgTTTT, reverse: AgTgCgCCgCCTCgTTTT- two markers within the progenies from crosses of sq198 hetero- CTT; grp94 (NM_198210) forward: TCCCgCACCgATgACgA, reverse: zygotes (Supplemental Fig. 1C and D). We then investigated the gTAgAAgCCCACACCgAACTgAC; ire1a (NM_001020530) forward: phenotypes of the liver, pancreas and intestine using pan-endo- ATggCgTggggAgTgTgC, reverse:gTATTCTgTgCggCCAAggTAAA; perk derm markers (foxa1, gata6, and shh), a liver marker (prox1), and a (NM_001077149) forward: CCgCggggCAACAgAgT, reverse: ggTggC- pancreatic marker (pdx1)insq198 mutants and WT embryos at AgCgATACAgAAgAAgAT. 2 dpf. No obvious differences between the homozygous mutants and WT embryos were revealed at this stage (Supplemental Sec13 and Sec31a interaction assay Fig. 1E–I). These results suggest that the sq198 mutation does not impair specification and early morphogenesis of the digestive Coding sequences of WT and mutant sec13 were cloned into organs. PDHA and PDMyc, respectively. HA-tagged WT and mutant Sec13 After 2 dpf, zebrafish digestive organs undergo rapid cell were expressed in HEK-293 T cells. The co-immunoprecipitation proliferation and differentiation, which is reflected by significant study was performed using the ProFoundTM HA system (Pierce) organ expansion and expression of genes essential for digestive following the manufacturer’s instructions. Myc-tagged WT and functions. We examined the expression of the hepatocyte marker, mutant Sec13 were expressed in HeLa cells grown on coverslips in fatty acid-binding protein 10a (fabp10a), the exocrine pancreas a six-well dish for the immunostaining assay. marker, trypsin, the islet marker, insulin, and the intestine marker, fabp2 using WISH in sq198 mutants and siblings at 3 and 4 dpf. Immunofluorescence microscopy Results revealed that expansion of both the liver and exocrine pancreas was arrested in homozygous sq198 mutant embryos Zebrafish embryos were fixed, embedded and sectioned as (Fig. 1A and B). At 3 and 4 dpf, the mutant intestine was less described (Huang et al., 2008). Primary antibodies against PDI expanded at the intestinal bulb region while the gut-looping (Sigma) and Collagen II (Lifespan) were used at a 1:100 dilution. process appeared to be relatively normal (Fig. 1C). Interestingly, Polyclonal antibody against Laminin (Sigma, L9393) was used at the mutant islet grew normally (Fig. 1D; Supplemental Fig. 2). In 1:50 dilution. Secondary antibodies conjugated with Alexa Fluor addition to defective digestive organs, the sq198 mutant exhibits 488 and 647 were used at a 1:400 dilution. 40,6-Diamidino-2- malformed skeleton cartilage as revealed by Alcian blue staining phenylindole (DAPI) was used to label nuclei. Images were taken in 4 and 5 dpf embryos (Fig. 1E). Staining 3-dpf sq198s mutant with a Leica TCS SP5 confocal microscope. embryos for internal alkaline phosphatase activity showed that blood vessels were normal (Fig. 1F), whereas the head region of Transmission electron microscopy (TEM) the pronephric ducts was thinner (Fig. 1G), suggesting that the sq198 mutation differentially affects the development of meso- 5 dpf zebrafish embryos were fixed in 2% glutaradehyde and derm-derived tissues. Altogether, these data demonstrated that 1% paraformaldehyde in PBS for 2 h followed by post-fixation in the expansion, but not the cell differentiation, of the liver, 1% osmium tetroxide for 1 h at room temperature. Fixed embryos exocrine pancreas, and intestine and the development of the were gradually dehydrated with ethanol and embedded indivi- skeletal cartilage are impaired by the sq198 mutation. dually into resin blocks; 50-nm cross-sections were obtained with an Ultracut E microtome (Leica) and collected on copper grids Hypoplasia of the liver, exocrine pancreas and intestine in sq198 is a (Structure Probe), followed by staining with 5% uranyl acetate and result of cell-cycle arrest and abnormal cell apoptosis 2% lead citrate. Images were acquired using a Philips CM-10 TEM or a Hitachi H-7650 TEM. To learn more about the nature of hypoplatic digestive organs in sq198, we studied the fate of cells in the liver, pancreas and intestine by analyzing the cell cycle and cell apoptosis status in Results WT and mutant embryos. We first performed immunofluorescence staining using an anti-phosphorylated histone 3 (pH3) The sq198 mutation causes growth arrest but does not affect antibody, a cell-cycle marker of G2 to M transition (Supplemental functional differentiation of digestive organs Fig. 3A and B). By examining transverse cryosections from three embryos in each case (Supplemental Fig. 3C, (Supplemental The sq198 small-liver mutant was identified during screening Table 1), we found that the ratio of pH3-positive cells (16 positive an ethylnitrosourea-induced mutated population (Supplemental cells out of 997 cells counted at 3 dpf; 11 out of 1666 counted at Fig. 1A) (Huang et al., 2008). Under a dissection microscope, the 4 dpf) in the mutant intestine was 2.6- and 2.8-fold lower, sq198 mutant embryo is visibly indistinguishable from a wild- respectively, than that in WT intestine at the same stage (54 type (WT) embryo before 2 day post-fertilization (dpf). However, positive cells out of 1299 cells counted at 3 dpf; 16 out of 997 at 5 dpf, the sq198 mutant can be readily identified by the lack of counted at 4 dpf). The ratio of pH3-positive cells in the mutant an inflated swim-bladder, fully expanded intestinal bulb and pancreas (1 positive cell out of 724 cells counted) was signifi- mouth opening, and also by a bigger yolk (Supplemental Fig. 1B). cantly lower (4.7-fold) at 4 dpf compared with that in wildtype The liver, pancreas and intestine all originate from the endo- (13 out of 1963 cells counted) but not at 3 dpf (Supplemental derm. In zebrafish, the lineage specification and budding of these Fig. 3C). Interestingly, we observed no significant reduction in the organs are accomplished by 2 dpf. To determine at which stage ratio of pH3-positive cells between WT and mutant liver (Supple- the sq198 mutation starts to affect development, we performed mental Fig. 3C). We also performed BrdU labeling experiment and 200 X. 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of 5-dpf sq198 and WT embryos at the subcellualr level. Trans- mission electron microscopy (TEM) results revealed that micro- villi in the intestinal epithelium were reduced in number, shorter in length and less organized in sq198 (Supplemental Fig. 4A and B). More importantly, the mutant epithelial cells were no longer tightly linked to each other like those in the WT, but had many gaps in between, apparently due to the lack of junction proteins between epithelial cells (Fig. 3A and B; Supplemental Fig. 4C and D, white arrows). Co-immunostaining of the basal layer component Laminin and the ER resident protein disulfide isomerase (PDI) revealed that Laminin forms a nice lining (yellow arrows) that separates the intestine and the liver at 5 dpf and is not accumulated in the ER (Fig. 3C, E, G). In contrast, although Laminin secretion did happen in the mutant (likely duo to the function of the maternal Sec13) the regular pattern of Laminin is disrupted and Laminin is clearly accumulated in the ER (white arrowheads) in the mutant (Fig. 3D, F, H) at 5 dpf. Interestingly, it appeared that Laminin is also expressed in the mutant hepatocytes (Fig. 3H). TEM results also revealed that the chondrocytes were deformed and disorganized (Fig. 4A, B) while the ECM was significantly less dense in sq198 (Fig. 4 A0 and B0). It was also evident that the mutant chondrocytes accumulated dilated vacuole-like structures in the cytoplasm (Fig. 4B, green asterisks). These results suggest that the mutant cells suffered severe defects in exporting secretory and membrane proteins from the ER (Bonfanti et al., 1998). Normal chondrocytes are known to secrete matrix protein collagen II (Col2a). Double immunostaining using antibodies against PDI (Fig. 4C and F) and Col2a (Fig. 4E and H) showed that almost all of the Col2a was secreted in the ECM in Fig. 1. The sq198 mutation confers hypoplastic digestive organs and malformed 5-dpf WT chondrocytes (Fig. 4D). In contrast, the Col2a was skeletal cartilage. (A–D) WISH using markers specifically for the liver (fabp10a) (A), exocrine pancreas (trypsin) (B), intestine (fabp2) (C), and endocrine pancreas retained in the discrete ER vacuoles in sq198 (Fig. 4G). (insulin) (D) on 3 dpf wildtype (WT) and mutant (mu) embryos. (E) Alcian blue The above results strongly suggest that the ER structure might staining showing the branchial arches 1–7 in WT and sq198 (mu) at 4 dpf. (F,G) be affected in sq198. Next, we examined the ER structures in Histochemical staining of alkaline phosphatase (AP) did not reveal obvious sq198 and WT embryos using TEM. Typical rough ER structures difference between 3 dpf wildtype (WT) and mutant (mu) blood vessels were easily identified in 5-dpf WT chondrocytes, hepatocytes and (F) whilst the head region of the pronephric ducts appears thinner in the mutant (G). intestinal epithelial cells (Fig. 5A, C and E, red arrow). In contrast. The normal ER morphology in 5-dpf mutant chondrocytes and hepatocytes was disrupted and appeared as large number of found that the ratio of BrdU positive cells were markedly reduced discrete vacuoles containing electron-dense material (Fig. 5B in the mutant liver (14.6% versus 22.8% in WT), intestine (13.3% and D, red asterisks), which probably represents undelivered versu 36.1% in WT) and pancreas (11.7% versus 32.3% in WT) at protein cargoes accumulated in the dilated ER. The ER in the 3.5 dpf while the ratio in the somites did not show drastic mutant intestinal epithelial cells did not forming discrete difference between the mutant and WT (7.6% versus 9.4%) vacuoles, but was also severely distended and dilated (Fig. 5F). (Fig. 2A–D; (Supplemental Table 2). The terminal deoxynucleoti- Interestingly, the distended ER lumen in the intestinal epithelium dyl transferase dUTP nick end labeling (TUNEL) assay revealed no did not contain electron-dense materials, which may reflect the or only a few apoptotic cells in the WT digestive organs (Fig. 2E, different nature or amount of the products secreted by the (Supplemental Table 3)(Cole and Ross, 2001). In contrast, intestinal epithelial cells (Fig. 5F). apoptotic activity was drastically high in mutant liver and intestine (Fig. 2F; (Supplemental Table 3): 2.4% (10 positive cells out of A T to A substitution in sq198 creates a new splicing acceptor site in 412 cells counted) and 5.3% (35 out of 660 cells counted) in the intron 7 of sec13 liver and 2.3% (28 out of 1208 cells counted) and 2.9% (45 out of 1555 counted) in the intestine at 3 and 4 dpf, respectively The initial positional cloning of the sq198 mutant gene deter- (Fig. 2G). Abnormal apoptotic activity was observed in the mined by bulked-segregant analysis (Shimoda et al., 1999) pancreas at 4 dpf (7/433) but not at 3 dpf (Fig. 2G). Hence, we mapped the mutation on chromosome 22 at a position between concluded that cell-cycle arrest and subsequent abnormal cell two existing simple sequence length polymorphism markers, apoptosis lead to hypoplastic digestive organs in sq198. z230 and z10321. Successive use of a series of markers on 1332 individual mutants placed the sq198 mutation between two sq198 mutation disrupts the integrity of ER structure and impairs closely linked markers, 257E and 257H, spanning a 54.3-kb protein secretion genomic DNA fragment (Fig. 6A). Sequencing analysis of sec13, one of the four genes encompassed by this genomic region, We have shown that sq198 suffers from hypoplasia of digestive identified a T to A substitution in intron 7 of sec13 that creates organs (Fig. 1A) and exhibits a deformed skeletal cartilage a new mRNA splicing acceptor site eight nucleotides away from phenotype (Fig. 1E) which is more severe than that observed in exon 8 (Fig. 6B and C, panel for genomic DNA sequence). This the crusher mutant (Lang et al., 2006). These observations results in the addition of eight nucleotides of intronic origin into prompted us to compare the intestinal epithelium and cartilage the final transcripts in sq198 (Fig. 6C, panel for cDNA sequence). 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Fig. 2. The sq198 mutation causes cell cycle arrest and cell apoptosis in the digestive organs. (A–D) BrdU incorporation assay showed that, at 3.5 dpf, the number and ratio of BrdU positive cells were obviously reduced in the digestive organs of the sec13sq198 mutant (mu) compared with the wild-type (WT). (A) Transverse section of the wildtype control through the liver (lv), intestine (in) and pancreas (pa). (B) Transverse section of a mutant embryo through the liver (lv) and intestine (in). (C) Transverse section of a mutant through the intestine (in) and pancreas (pa). (D) Histogram showing the ratios of BrdU positive cells versus total cells in a speciﬁc organ. Data were obtained from sections derived from three wild-type and three sec13sq198 mutant embryos, respectively. S: somite, L: liver, I: intestine, P: pancreas; **: Po0.01. (E–G) TUNEL assay showing cell apoptosis (cells in red) in wild-type (WT) (E) and mutant (mu) (F) cryosections. Data shown in (G) were obtained by counting apoptotic cells against total cells in a speciﬁc organ (e.g., intestine) in sections from three wild-type and three mutant embryos, respectively. *: Po0.05; **: Po0.01.DAPI was to stain the nuclei. G, gut; L, liver; P, pancreas; S, somite.

This sq198 mutant transcript appears to dominate the native sec13 exhibits dynamic expression patterns in zebrafish transcript because no WT splicing product was found in the cDNA cloned from nearly 100 5-dpf mutant embryos (data not shown). We investigated the temporal and spatial expression patterns To prove that this T to A mutation in sec13 is indeed of sec13 during early embryogenesis by northern blot analysis and responsible for the sq198 mutant phenotypes, in-vitro transcribed WISH hybridization. Northern blot analysis showed that sec13 WT or mutant sec13 mRNA was injected into one cell-stage mRNA is deposited maternally and its expression spans the early embryos derived from heterozygote crosses. The injected embryonic stages from 1 dpf to 5 dpf (Fig. 6F). WISH analysis embryos were analyzed by WISH using a fabp10a probe at 3.5 showed that sec13 is distributed ubiquitously in the unfertilized dpf (Supplemental Fig. 5A). In total, the small-liver in 144/194 egg. After fertilization, sec13 is evenly distributed into daughter mutant embryos was successfully restored to normal by injection cells after the first two rounds of cell division. At 2 and 3 dpf, in of WT sec13 mRNA. In addition, injection of sp2-MO, which addition to its continuous expression in the head and trunk, sec13 specifically targets the splice junction of exon7/intron7 of sec13 is also enriched in the developing digestive organs, including the (Fig. 6D; Supplemental Fig. 5B), into WT zebrafish embryos liver, intestine and exocrine pancreas (Fig. 6G). At 4 and 5 dpf, resulted in a small-liver phenotype resembling that in sq198 strong sec13 mRNA expression is apparent in the intestine while a (Fig. 6E). These data demonstrated that the T to A substitution weak signal is observed in the liver, exocrine pancreas and in the sec13 gene in sq198 confers the hypoplastic phenotype to skeletal cartilage (Fig. 6G). the digestive organs. This mutation was therefore designated We then studied the temporal protein expression pattern of as sec13sq198. Sec13 and that of its known interaction partner, Sec31a, using 202 X. Niu et al. / Developmental Biology 367 (2012) 197–207

Fig. 3. The sq198 mutation causes loosening of the cell-cell junction in the digestive organs. (A,B) TEM analysis of intestinal epithelium in 5 dpf wildtype and mutant embryos. The epithelial cells are tightly adhered to each other in the wildtype (WT) (A, white arrows) but are separated by clearly visible gaps in the mutant (mu) (B, white arrows). (C–H) Double immunofluorescence staining using the antibodies against the basement component Laminin (red) and the ER marker PDI (green). In the wild type control at 5 dpf, Laminin forms the normal lining along the basement layer (C) that nicely separates the liver and intestine (yellow arrows) (G). No apparent accumulation of Laminin in the ER (E,G). In contrast, the mutant did not form an apparent basement layer to separate the liver and intestine (white arrowheads) (D,H) and the mutant cells accumulated Laminin in the ER (white arrowheads) (D,F,H). western blot analysis. Consistent with its mRNA expression immunostaining with anti-Sec13 and anti-Sec31a antibodies in dynamics, Sec13 protein is deposited maternally and was 5 dpf WT cross-sections. The results showed that Sec13 and Sec31a detected in all stages investigated (Fig. 6H). However, expression appear to be expressed in a variety of tissues and cells differentially of its interacting partner, Sec31a, was barely detectable before in zebrafish (Supplemental Fig. 5C). For instance, while Sec13 is 2 dpf, although it exhibited strong and consistent expression from expressed abundantly in the neural tube, Sec31a is only enriched 3 to 5 dpf (Fig. 6H). These findings imply that the COPII complex peripherally to the neural tube (Supplemental Fig. 5C). At the functions minimally during the earlier stages of development and cellular level, the co-localization patterns of Sec13-Sec31a in becomes very active from 3 dpf onwards by regulating the protein hepatocytes (Fig. 7A), pancreatic cells (Fig. 7B), intestinal epithelial levels of Sec31a and therefore functional Sec13-Sec31a complex. cells (Fig. 7C), and chondrocytes (Fig. 7D) are strikingly similar to those observed in yeast and human cells (Tang et al., 2000). Sec13 and Sec31a are co-localized in a variety of cell types in zebrafish sec13sq198 encodes a carboxyl-terminal-truncated protein that is unable to interact physically with Sec31a The most essential function of Sec13 in the COPII complex is to form the outer coat together with Sec31. We investigated the The sec13sq198 mutant mRNA is predicted to encode a polypep- in-vivo cellular localization of Sec13 and Sec31a in zebrafish by tide with 85 amino acids truncated from the carboxyl-terminus of X. Niu et al. / Developmental Biology 367 (2012) 197–207 203

Fig. 5. The sq198 mutation disrupts the ER structure in chondrocytes, hepatocytes 0 0 Fig. 4. The sq198 mutation impairs the protein secretory pathway. (A,A ,B,B ) TEM and intestinal epithelium. (A–F) TEM analysis of the ER structure in chondrocytes analysis. Compared to wildtype (WT), the mutant (mu) exhibited disorganized (A,B), hepatocytes (C,D), and intestinal epithelium (E,F) in wildtype (WT) and 0 0 chondrocytes (compare A and B) and less-accumulated ECM (compare A and B ). mutant (mu) embryos at 5 dpf. Rough ER in WT cells are indicated with red arrows 0 0 A and B represent high resolution pictures of boxed regions in A and B, (A,C,E), disrupted ER structures in the mutant cells are highlighted with red respectively. e: ECM; green asterisk: unknown vesicles in a mutant chondrocyte. asterisks (B,D,F). (C–H) Double immunostaning of the ER resident marker PDI and the secretory protein Collagen II (Col2a) in chondrocytes in 5 dpf wildtype (WT) (C–E) and mutant (mu) (F–H) embryos. (C,F) Immunostaining of PDI (green). (E,H) Immu- co-localized with endogenous SEC31a which marked the ERES nostaining of Col2a secretion (red). (D,G) Superimpose of PDI and Col2a staining accordingly. (Fig. 8C). A similar co-localization pattern was also observed in HeLa cells that expressed Myc-tagged zebrafish Sec13 (Fig. 8D), suggesting that zebrafish Sec13 is a functional ortholog of human WT Sec13 and the addition of 34 new amino acids due to frame- SEC13. In contrast, Myc-SEC13sq198 appeared to be uniformly shift in the new open reading frame (Fig. 8A; Supplemental distributed in the cytoplasm, and showed no evidence of co- Fig. 6A). Human and zebrafish Sec13 and Sec31a share high localization with endogenous SEC31a in the transfected cells sequence homology (Supplemental Fig. 6B; Supplemental Fig. 7). (Fig. 8E). This result suggests that loss of interaction with Sec31a To determine whether the truncated carboxyl-terminus of Sec13 is resulted in defective recruitment of the mutant Sec13 to the ERES. important for its interaction with Sec31a, we created a human mutant Sec13 equivalent to the zebrafish sec13sq198 by inserting Knockdown of Sec31a results in hypoplastic digestive organs the excess eight nucleotides in the zebrafish sec13sq198 mRNA into the comparable site of human SEC13. Both human SEC13 and To demonstrate the necessity of the Sec13-Sec31a complex SEC13sq198 were tagged with HA epitope and expressed in the during organogenesis of the digestive system, we designed a HEK-293T cells. An anti-HA monoclonal antibody was used to morpholino (31a-ATGMO) that specifically targets the translation immunoprecipitate HA-SEC13 or HA-SEC13sq198 from the protein start region of sec31a. Immunoblots performed with rabbit anti- extracts from HEK-293T cells. A rabbit anti-SEC31a antibody was Sec31a antibody revealed that the level of Sec31a in 4-dpf sec31a- used to detect the co-immunoprecipitated endogenous SEC31a. ATGMO morphants was approximately three fold lower than that The result showed that endogenous SEC31a was efficiently co- in the WT, while Sec13 remained unchanged (Fig. 9A). WISH immunoprecipitated with HA-SEC13 but not with the mutant HA- examination of the sec31a-ATGMO morphants using fabp10a, SEC13sq198 (Fig. 8B). This result defined the mechanistic basis for trypsin and fabp2 probes showed that all morphant embryos the loss of function of the mutant because it was no longer able to displayed a phenotype of hypoplastic liver (Fig. 9B), exocrine interact with Sec31a. pancreas (Fig. 9C) and intestinal tube (Fig. 9D). In fact, majority of Myc-tagged human SEC13 (Myc-SEC13) and SEC13sq198 (Myc- Sec31a-ATGMO morphant embryos exhibited largely diminished SEC13sq198) were expressed in HeLa cells. Immunofluorescence digestive organs (Fig. 9B–D). To find out whether this is due to the staining was then carried out to investigate the subcellular failure of the endoderm development at all we examined the relationship between endogenous SEC31a and over-expressed Sec31a-ATGMO morphants at 24 and 36 hpf with early pan- Myc-SEC13 and Myc-SEC13sq198. The results showed that Myc- endodermal markers foxa3 and gata6, respectively. The result SEC13 was enriched in the perinuclear region and was essentially showed that the endoderm development was clearly evident in 204 X. Niu et al. / Developmental Biology 367 (2012) 197–207

Fig. 6. The sec13sq198 mutation creates a novel splice receptor site in intron7 of sq198 sec13. (A) Diagram summarizing map-based cloning of the sec13 mutant gene. Fig. 7. Sec13 and Sec31a co-localize in hepatocytes, exocrine pancreatic cells, Genetic markers used in mapping are shown on top with their corresponding intestinal epithelium and chondrocytes. (A–D) In 5 dpf embryos Sec31a and Sec13 number of recombinants shown below in bracket. The mutant gene (mu) was co-localize in hepatocytes (A), exocrine pancreatic cells (but not in endocrine located between markers 257H and 257E spanning a 54.3 kb genomic DNA pancreatic cells, white arrow) (B), apical side of the intestinal epithelium (C), and fragment contained in two overlapping BACs (CH211-165I22 and CH211- chondrocytes (white arrow) (D). (A1,B1,C1,D1) Immunostaining of Sec31a (red). 257F16). (B,C) Analysis of sec13 genomic DNA sequence in sq198 identiﬁed a T (A3,B3,C3,D3) Immunostaining of Sec13 (green). (A2,B2,C2,D2) Superimpose of to A substitution (indicated by red arrows in B) that creates a novel splice receptor Sec31a and Sec13 staining accordingly. DAPI was used to stain the nucleus. site (underlined in red in C, panel for Genomic DNA sequence) in intron 7 of sec13. Native splice receptor site for Intron7/Exon8: underlined in blue. Intron sequence: black letters; exon sequence: red letters. An eight nucleotides insertion (black letters) was identiﬁed in the sec13 cDNA derived from sec13sq198 (C, panel for cDNA sequence). (D,E) Injection of the sec13 splicing blocking morpholino sp2 that compromised Sec31 function would lead to deformation the altered sec13 transcripts with 66 nucleotides deletion (D) that led to the small ER structure in yeast (Wuestehube et al., 1996). Co-immunostain- liver phenotype as conferred by the sec13m198 mutation when examined with the ing of PDI and Col2a revealed that, as observed for the sec13sq198 liver marker fabp10a at 3 dpf (E). (F) Northern analysis of sec13 transcripts in mutant, the ER structure of chondrocytes in the Sec31a-ATGMO unfertilized eggs (Mt) and 1dpf to 5 dpf embryos using a sec13 probe. 18S RNA was morphants was distended and formed discrete vacuoles (Supple- used as loading control. (G) WISH analysis of sec13 expression patterns in unfertilized eggs (Mt), zygotic eggs at 1-cell, 2-cell and 4-cell stages, and in mental Fig. 12). These results demonstrate that the functional embryos at 3 dpf and 4 dpf. bc: branchial arch; in: intestine; lv: liver; pa: pancreas. Sec13-Sec31a complex is essential for the development of the (H) Western analysis of Sec13 and Sec31a protein expression, respectively, in digestive organs. unfertilized eggs (Mt) and 1 dpf to 5 dpf embryos. Tubulin was used as loading control. sec13sq198 mutation activates the UPR pathway

UPR is a highly regulated process that plays an essential role in the Sec31a-ATGMO morphants (Supplemental Fig. 8). We also cellular protein homeostasis (Lin et al., 2008; Ron and Walter, cross-sectioned the embryos after WISH using fabp10a, fabp2 and 2007). Previous reports have shown that ER stress caused by trypsin three probes for checking the liver, intestine and exocrine disruption of components of the COPII complex can activate pancreas simultaneously. The result showed that morphant UPR (Chang et al., 2004; Higashio et al., 2002; Saito et al., 2009). embryos exhibiting greatly diminished digestive organs were Because sec13sq198 mutation causes disintegration of the ER actually not abolished of endoderm-derived cells (Supplemental structure and accumulation of secretory cargoes, which are Fig. 9A–C). Alcian blue staining showed that the Sec31a-ATGMO expected to generate intracellular ER stress, we examined the morphant embryos also suffered from malformation of the expression of six UPR-related genes (bip, chop, grp94, atf6, ire1a cartilage skeleton (Suplemental Fig. 10). Interestingly, WISH using and perk) in mutant embryos at 3, 4, and 5 dpf. All six genes fabp10a and insulin two probes together showed that, in contrast were signiﬁcantly up-regulated in the mutant, and chop showed to the drastic reduction of the liver size, both the Sec31a-ATGMO the highest magnitude of up-regulation at all three stages morphant and sec13sq198 mutant developed a normal islet as that examined (Fig. 10A). Using chop as the probe, WISH showed that observed in the WT (Supplemental Fig. 11A–D). It was reported its expression was mainly elevated in the digestive organs X. Niu et al. / Developmental Biology 367 (2012) 197–207 205

Fig. 8. Sec13sq198 mutant protein is a C-terminal truncated Sec13 which is unable to interact with Sec31a. (A) Alignment of the C-terminal sequences of Sec13 and Sec13sq198. Sec13sq198 mutant protein lacks the C-terminus of Sec13 starting from D236 (blue arrow) and contains 34 new amino acids after Q235 (blue arrow). (B) Co- immunoprecipitation (Co-IP) analysis. HEK-293T cells were transfected with HA- SEC13 or HA-SEC13sq198 plasmids. An anti-HA antibody was used to immunopre- Fig. 9. Knockdown of Sec31a resulted in hypoplastic digestive organs. (A) Western cipitate (IP) the tagged proteins. The input (1st and 2nd panels) and the IP blotting analysis of Sec31a and Sec13 in sec31a-ATG MO (31a-ATG MO) and products (3rd and 4th panels) were blotted using an anti-SEC31A antibody (2nd standard control MO (st MO) injected embryos at 4 dpf, respectively. b-Actin was and 4th panels) and an anti-Sec13 mosue serum (1st and 3rd panels). (C-E) In used as the loading control. (B-D) WISH using the fabp10a (B), trypsin (C) and fabp2 human Hela cells, Myc-SEC13 (C) or zebraﬁsh Myc-Sec13 (D) co-localizes with (D) probes to examine the development of the liver, exocrine pancreas and endogenous SEC31a whilst SEC13sq198 is evenly distributed in the cytoplasm (E). intestine in wildtype (WT), standard control MO (st MO), and 31a-ATG MO (C1,D1,E1) Immunoﬂuorescence staining of endogenous SEC31a (red). (C3,D3,E3) embryos at 4 dpf. Number of embryos displaying the showed phenotype of the Immunostaining of Myc-tagged hSEC13 (C3) or zSec13 (D3) or hSEC13sq198 (E3) total embryos examined was provided at the bottom right side of each represen- (green). (C2,D2,E2) Superimposed. tative embryo.

(Fig. 10B). We then examined the protein expression of Bip, Chop and intron7 mimicked the sec13sq198 mutant phenotype. In addi- and phosphorylated eIF2a (p-eIF2a) in the mutant. The results tion, the fact that the endogenous Sec31 still formed a nice showed that although the protein level of Bip was not obviously crescent around the nuclei in the presence of Sec13sq198 in the altered, the levels of Chop and p-eIF2a were markedly up- cultured HeLa cells suggests that the Sec13sq198 mutant protein regulated in the mutant (Fig. 10C). appears not to inhibit the recruitment of Sec31 to the ER exit site. Based on these data we conclude that the mutant protein unlikely contributed to the mutant phenotype. Discussion Immunostaining in 5-dpf WT embryos revealed that Sec13 and Sec31a are expressed and co-localized in chondrocytes, hepato- We investigated the zebrafish sec13sq198 mutant, which exhi- cytes and intestinal epithelial cells. TEM of these cell types in bits digestive organ growth arrest. Positional cloning revealed sec13sq198 revealed a dilated ER structure and/or accumulation of that sec13sq198 carries a T to A substitution in intron 7 of the sec13 dilated vacuoles. Further studies showed that the ECM protein gene, which leads to the addition of eight nucleotides of intronic Col2a and Laminin were not correctly transported to the outer origin to the mature mRNA and results in a carboxyl-terminal- surface of mutant chondrocytes and intestinal epithelial cells, truncated Sec13 protein. The main function of Sec13 is to form the respectively, but were retained in the dilated ER lumen. This outer layer of the COPII complex with Sec31. This mutation demonstrated that the COPII export function is deficient in these devastates the integrity of normal COPII formation because mutants. Prolonged cargo retention in the ER is expected to Sec13sq198 loses its affinity to Sec31a. All evidence presented induce cellular ER stress that in turn activates the UPR. We found demonstrate that sec13sq198 is a loss-of-function mutation: 1) that six hallmark genes for the UPR were all up-regulated in the sec13sq198 is a recessive mutant; 2) WT sec13 could rescue the 5-dpf mutants. More importantly, the pro-apoptotic factor Chop mutant phenotype and 3) knockdown of sec13 expression using was most notably up-regulated in the digestive organs, suggest- sec13 specific morpholino targeting the junction between exon7 ing that cells in these organs likely underwent apoptosis. 206 X. Niu et al. / Developmental Biology 367 (2012) 197–207

Fig. 10. Activation of the UPR pathway in the sec13sq198 mutant. (A) Quantitative RT-PCR analysis of UPR-related genes bip, chop, grp94, atf6, ire1a and perk in WT and the mutant, respectively. The relative expression level of the genes was shown in fold change as normalized to the housekeeping gene, elf1a. (B) WISH analysis of chop expression in WT and mutant (mu) embryos at 5 dpf. (C) Western blot analysis of Bip, Chop, phosphorylated eIF2a (p-eIF2a) and total eIF2a (eIF2a) in WT amd mutant (mu). b-Actin was used as the loading control.

The BrdU incorporation assay, pH3 staining and TUNEL assay organs suggests that Sec31a is the one to form the COPII complex revealed that the progression of the cell cycle was arrested in with Sec13 during early embryogenesis. When cell proliferation mutant cells which finally resulted in abnormal apoptotic activ- and organ growth rely increasingly on COPII from 3 dpf onwards ities in the mutant digestive organs. These results provide direct (as shown by the increased level of Sec31a), the function of Sec13 evidence to explain the sec13sq198 phenotypes. First, cell-cycle is similarly more important and maternal Sec13 is no longer arrest and increased cell apoptosis lead to reduced cell number, as sufficient, which leads to a defective COPII function that is not reflected by decreased expansion of the digestive organs and able to meet developmental needs in the sec13sq198 mutant. cartilage. Second, compromised transport of membrane and Our study provides the first genetic evidence that sec13 and secretive proteins disrupts the normal patterns of organs, such the COPII complex safeguard the normal establishment of the as the intestinal epithelium and cartilage, because insufficient digestive organs. In addition to its major function in the COPII material is available to build up the ECM and tissue boundary. It is complex, Sec13 also plays a role in the nuclear pore complex worth to point out that trafficking of small molecules does not (Loiodice et al., 2004). Recent studies have shown that the nuclear depend upon the recruitment of Sec13/Sec31 dimer to the ER exit pore complex plays an important role in regulating the expression sites. The organogenesis of digestive organs requires secreted of certain developmental induced genes by specifically binding to signaling molecules such as Fgfs and Bmps. Whether the secretion their regulatory sites in the genome (Capelson et al., 2010). of these small molecules is affected in the mutant is an interesting Therefore, whether this function of Sec13 is related to the question to be addressed in the future. observed organ-specific subcellular defects in the mutant fish In addition, Sec13 and Sec31a displayed different spatial and will be of great interest in our future study. Meanwhile, it would temporal expression patterns during the early stages of develop- be interesting to use sec13sq198 to study the role of the nuclear ment, which raises an interesting possibility that regulated pore complex structure in organogenesis in the future (de Jong- expression of Sec31a or Sec13 may be employed to regulate the Curtain et al., 2009). Although the currently known function of capacity of secretory pathway, depending on physiological needs. Sec31 is solely attributed to form the COPII complex we cannot Sec13 is maternal protein. The sharply increased expression of exclude the possibility that Sec31 might function independently Sec31a around 3 dpf compared with the low or undetectable in the organogenesis of the digestive organs. For example, we levels of expression before this time-point coincides with found that the endocrine pancreas region appeared to be devoid increased cell proliferation and organ size, implying that the level of Sec13, suggesting that Sec31a might play a special role in of Sec31a is regulated to accommodate the needs of organ organogenesis of the endocrine pancreas or the secretion of growth. This is also in agreement with our analysis of the Sec13 endocrine hormones. Apparently, it will need extensive future mutant, in which early development and cell specification were work to unravel this myth. essentially not affected, and is consistent with the hypothesis that minimal activity of COPII (low level of Sec31a and maternal Sec13) is sufficient for early development. Alternatively, Sec31b, a paralog of Sec31a (Stankewich et al., 2006) (Supplemental Fig. Acknowledgments S13), may cooperate with maternal Sec13 to ensure COPII function during embryogenesis before 2 dpf. This argument is sup- We thank Low Swee Ling for assisting in the genetic mapping ported by the observation that sec31b transcripts are detected of the sec13sq198 mutation, Ong Yan Shan for technical help in abundantly during the early stages of embryogenesis (Supple- handling human cell lines and Dr Jun Chen and Dr Honghui Huang mental Fig. S14) although future work is needed to demonstrate for their suggestions and technical support. This work was the presence of Sec31b protein at these stages. However, the fact supported financially by a grant from the National Natural that knockdown of Sec31a disrupted the ER structure and greatly Science Foundation of China (30825025) and a ‘‘973 Program’’ affected the development of digestive organs, with majority of grant (2012CB944500) to JRP and from the Agency for Science, Sec31a morphants showing loss of almost all of their digestive Technology and Research in Singapore to WJH. X. Niu et al. / Developmental Biology 367 (2012) 197–207 207

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