mcb accepts, published online ahead of print on 28 july

1

Absence of a red blood cell phenotype in mice with hematopoietic deficiency of SEC23B 1

Running title: Hematopoietic SEC23B deficiency 2

Rami Khoriaty1*, Matthew P. Vasievich2*, Morgan Jones3, Lesley Everett2, Jennifer 3

Chase3, Jiayi Tao4, David Siemieniak3, Bin Zhang4, Ivan Maillard1,3, David 4

Ginsburg1,2,3,5,6# 5

1Department of Internal Medicine, University of Michigan, Ann Arbor, MI; 2Department of 6

Human Genetics, University of Michigan Medical School, Ann Arbor, MI; 3Life Sciences 7

Institute, University of Michigan, Ann Arbor, MI; 4Genomic Medicine Institute, Cleveland 8

Clinic Lerner Research Institute, Cleveland, OH; 5Department of Pediatrics and 9

Communicable Diseases, University of Michigan, Ann Arbor, MI; 6Howard Hughes 10

Medical Institute, University of Michigan, Ann Arbor, MI 11

Matt Vasievich’s current address: Department of Dermatology, Cleveland Clinic, 12

Cleveland, OH. 13

Jiayi Tao’s current address: Case Western Reserve University, Cleveland, OH 14

# Corresponding author: 15

Email: [email protected] 16

Phone: 734-647-4808 17

Fax: 734-936-2888 18

* Authorship note: Rami Khoriaty and Matt P. Vasievich contributed equally to the work. 19

MCB Accepts, published online ahead of print on 28 July 2014Mol. Cell. Biol. doi:10.1128/MCB.00287-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Abstract word count: 196 20

Article length for abstract, introduction, results, discussion, and figure legends: 28,079 21

characters (excluding spaces). 22

Word count for the Materials and Methods section: 1,659. 23

Combined word count for the Introduction, Results, and Discussion sections: 2,810. 24

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Abstract 25

Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease 26

of ineffective erythropoiesis characterized by increased bi/multi-nucleated erythroid 27

precursors in the bone marrow. CDAII results from mutations in SEC23B. The SEC23 28

protein is a core component of the coat protein complex II-coated vesicles, which 29

transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. 30

Though the genetic defect underlying CDAII has been identified, the pathophysiology of 31

this disease remains unknown. We previously reported that SEC23B-deficient mice die 32

perinatally, exhibiting massive pancreatic degeneration, with this early mortality limiting 33

evaluation of the adult hematopoietic compartment. We now report that mice with 34

SEC23B-deficiency restricted to the hematopoietic compartment survive normally and 35

do not exhibit anemia or other CDAII characteristics. We also demonstrate that 36

SEC23B-deficient hematopoietic stem cells (HSC) do not exhibit a disadvantage at 37

reconstituting hematopoiesis when compared directly to wild type HSC in a competitive 38

repopulation assay. Secondary bone marrow transplants demonstrated continued 39

equivalence of SEC23B-deficient and WT HSC in their hematopoietic reconstitution 40

potential. The surprising discordance in phenotypes between SEC23B-deficient mice 41

and humans may reflect an evolutionary shift in SEC23 paralog function and/or 42

expression, or a change in a specific COPII cargo critical for erythropoiesis. 43

4

Introduction 44

Congenital dyserythropoietic anemia type II (CDAII), also known as Hereditary 45

Erythroblastic Multinuclearity with a Positive Acidified-Serum lysis test (HEMPAS), is an 46

autosomal recessive disease characterized clinically by mild to moderate anemia 47

resulting from ineffective erythropoiesis (median hemoglobin 9.1-9.8 g/dL), the presence 48

of bi-/multi-nucleated erythroblasts in the bone marrow (BM), jaundice from indirect 49

hyperbilirubinemia, and splenomegaly (1, 2). CDAII is distinguished from other 50

congenital anemias by a characteristic double membrane appearance on red blood cell 51

(RBC) electron microscopy resulting from residual endoplasmic reticulum (ER) (3), a 52

faster migrating and narrower band on SDS-PAGE for the RBC membrane protein band 53

3, and lysis of RBCs in some, but not all, acidified normal sera (Ham’s test) (1, 2). 54

CDAII results from homozygous or compound heterozygous mutations in SEC23B (4), 55

one of the two mammalian paralogs of SEC23. SEC23 is a core component of the coat 56

protein complex II (COPII)-coated vesicles that transport cargo proteins from the ER to 57

the Golgi apparatus (2, 5). More than 60 different SEC23B mutations have been 58

identified in CDAII patients (4, 6-14), affecting every domain of the protein (7). No 59

patient with two nonsense SEC23B mutations has been reported, suggesting that 60

complete loss of SEC23B may be lethal. Homozygosity for specific missense mutations 61

in SEC23A, the paralog of SEC23B, results in cranio-lenticulo-sutural-dysplasia (15), 62

thought to be due to defective collagen secretion. 63

Despite the identification of the genetic defect, the molecular mechanism by which 64

deficiency of SEC23B results in the CDAII phenotype remains unknown (2). Nearly all 65

5

proteins destined for secretion from the cell or export to the cell membrane or lysosome 66

(~1/3 of the mammalian proteome) are dependent on COPII vesicles for transport from 67

the ER to Golgi. Thus, it is surprising that deficiency of a key, ubiquitously expressed 68

component of the COPII coat, SEC23B, results in a phenotype apparently restricted to 69

the red blood cell. Reports of curative allogeneic bone marrow transplantation for CDAII 70

suggest that the mechanism responsible for the erythroid defect is intrinsic to the 71

hematopoietic compartment (9, 16, 17). 72

We previously reported that mice homozygous for a Sec23b gene-trap allele 73

(Sec23bgt/gt) die within 24 hours of birth, exhibiting degeneration of the pancreas and 74

other professional secretory tissues (18). The perinatal lethality precluded assessment 75

of adult hematopoietic function in these mice. We now report that chimeric mice with 76

SEC23B-deficiency restricted to the hematopoietic compartment can support the normal 77

production of adult RBCs, with no apparent abnormality in hematopoiesis. Competitive 78

hematopoietic stem cell (HSC) transplantation assays also fail to demonstrate a 79

disadvantage of Sec23bgt/gt HSC at reconstituting hematopoiesis compared to WT HSC. 80

81

6

Materials and Methods 82

Generation of SEC23B-deficient mice 83

Two Sec23b mutant mouse lines, one with a gene-trap (gt) cassette insertion into 84

Sec23b intron 19 (Sec23bgt), and the second with a conditional gt insertion in Sec23b 85

intron 4 (Sec23bcgt), were generated as previously described (Fig. 1A and B) (18). All 86

Sec23bgt mice used in this study were generated from heterozygous mice backcrossed 87

to C57BL/6J mice for > 10 generations. The Sec23bcgt allele was derived from 88

C57BL/6J ES cells (18), and maintained on a pure background by backcrosses 89

exclusively to C57BL/6J. Sec23bcgt/+ mice were crossed to a mouse ubiquitously 90

expressing FLPe under the control of the human β-actin promoter (β-actin FLP) 91

(Jackson laboratory stock # 005703) to excise the gt cassette and generate the Sec23b 92

floxed allele (Sec23bfl), with exons 5 and 6 flanked by loxP sites (Fig. 1B). Mice with 93

complete deficiency of SEC23B (Sec23b-) were generated by crossing the Sec23bfl 94

allele to a mouse expressing Cre recombinase driven by an EIIA promoter (EIIA Cre) 95

(Jackson laboratory stock # 003724). Deletion of Sec23b exons 5 and 6 results in a 96

frameshift and downstream stop codon in exon 7. Mice were housed at the University of 97

Michigan and all procedures were in accordance with the regulations of the Animal Care 98

and Use Committee. 99

PCR genotyping 100

Genotyping for the Sec23bgt allele was performed as previously described (18). The 101

Sec23bcgt allele was genotyped by a three-primer polymerase chain reaction (PCR) 102

assay using a forward primer (F1) located in Sec23b intron 4 upstream of the insertion 103

7

site and two reverse primers, one (cgtB1) located in the gene-trap insertion cassette 104

between the two FRT sites and the second (cgtR1) located in intron 4 downstream of 105

the FRT sites. This PCR reaction results in a 475 base pair (bp) product from the wild 106

type (WT) allele (F1:cgtR1) and a 344 bp product from the Sec23bcgt allele (F1:cgtB1), 107

which are resolved on 2% (weight/volume) agarose gel electrophoresis (Fig. 1C). 108

Genotyping for the Sec23bfl and Sec23b- alleles was performed with a three-primer 109

competitive PCR assay consisting of the forward primer F1, a second forward primer 110

(F2) located in intron 6 between the two loxP sites, and a common reverse primer (R1) 111

located in intron 6 downstream of the insertion site. This reaction produces a 235 bp 112

product from the WT allele (F2:R1), a 269 bp product from the Sec23bfl allele (F2:R1), 113

and a 336 base pair product from the Sec23b- allele (F1:R1), which are resolved by 3% 114

agarose gel electrophoresis (Fig. 1D). Locations of the genotyping primers are indicated 115

in figures 1A and 1B. Primer sequences are shown in Table 1. 116

Fetal liver cell (FLC) transplants 117

Timed matings were performed by intercrossing Sec23b+/gt mice or Sec23b+/- mice. The 118

following morning, designated as E0.5, matings were separated. Pregnant female mice 119

were euthanized at E17.5 post-coitus. Recovered fetuses were separated and placed 120

individually in Petri dishes on ice under sterile conditions. A tail biopsy was obtained 121

from each fetus for genotyping. Fetal livers were individually disrupted and dispersed 122

cells were suspended in RPMI 1640 (Gibco) supplemented with 2% fetal bovine serum 123

(FBS) at 4°C. FLC were washed twice in RPMI 1640 + 2% FBS, and then suspended in 124

65% RPMI 1640, 25% FBS, and 10% DMSO, frozen at -80°C overnight, and stored in 125

vapor phase liquid nitrogen at -186°C. 126

8

Six to twelve week old C57BL/6J recipient mice were lethally irradiated with two doses 127

of 550 rads separated by 3 hours in a Cs Gammacell 40 Exactor irradiator (MDS 128

Nordion). Three hours following completion of irradiation, frozen WT and SEC23B-129

deficient FLC (Sec23bgt/gt or Sec23b+/- FLC) were thawed in a 37°C water bath. 106 cells 130

were suspended in 300 μl RPMI 1640 with 2% FBS and injected into the retro-orbital 131

venous sinus of recipient mice. For each transplant experiment, control mice were 132

injected with media only. No control mice survived beyond 12 days. Transplanted mice 133

were provided with acidified water (pH = 2.35) for 3 weeks post-transplantation. 134

Competitive FLC transplants 135

C57BL/6J mice expressing high levels of green fluorescent protein (GFP) in all tissues 136

including hematopoietic cells (19) (under the control of the human ubiquitin C promoter 137

(UBC-GFP mice)) were obtained from the Jackson Laboratory (stock # 004353). FLC 138

from crosses between male mice homozygous for the UBC-GFP transgene (UBC-139

GFPtg/tg) and female C57BL/6J mice were harvested at day 17.5 post-coitus and stored 140

as described above. UBC-GFPtg/+ FLC were mixed in a 1:1 ratio with either Sec23bgt/gt 141

FLC (experimental arm) or WT FLC (control arm) and transplanted into lethally 142

irradiated C57BL/6J recipients as described above. 143

Bone marrow cells were isolated from the hind limbs of each chimeric mouse. The 144

number of GFP(-) cells per 2 hind limbs was calculated for each hematopoietic lineage 145

by multiplying the ratio of GFP(-)/GFP(+) cells in each lineage by the total number of 146

cells per lineage. The number of GFP(-) cells per two hind limbs should be proportional 147

9

to the contribution of GFP(-) cells to each lineage, corresponding to Sec23bgt/gt cells in 148

the experimental arm and WT cells in the control arm. 149

GFP(-) mature myeloid cells (Mac1+ Gr1+) were FACS sorted from bone marrows 150

harvested from chimeric recipient mice. Myeloid cells were genotyped for Sec23b. 151

For secondary transplants, whole BM cells were harvested from these chimeric recipient 152

mice twenty weeks after the competitive FLC transplant, and 2 x 106 cells were 153

transplanted into lethally irradiated secondary C57BL/6J recipients. 154

Complete blood counts (CBC) and BM analysis 155

Twenty microliters of blood were drawn from the retro-orbital venous sinuses of mice 156

anesthetized with isoflurane. Blood was diluted 1:10 in 5% Bovine Serum Albumin 157

(BSA) in Phosphate Buffered Saline (PBS) pH 7.4. CBC were performed on the 158

Advia120 whole blood analyzer (Siemens) according to the manufacturer’s instructions. 159

Following pentobarbital-induced anesthesia, BM was flushed from femurs and tibias of 160

each mouse using either Hank’s balanced salt solution (Gibco) or RPMI 1640, 161

supplemented with 5% FBS. BM cells were used for flow cytometry (below) and a 162

subset (~ 160,000 cells) was collected by centrifugation in a cytospin (Thermo scientific 163

cytospin 4 cytocentrifuge), stained with the HEMA 3 kit (Fisher), and examined under 164

light microscopy. BM cytospins were evaluated by an investigator blinded to mouse 165

genotype. 166

Flow cytometry 167

10

Peripheral blood or BM single cell suspensions were incubated with various antibodies. 168

The following antibodies were obtained from BioLegend, eBiosciences, or BD 169

Biosciences: anti-Ter119, Gr1 (RB6-8C5), Mac1 (M1/70), CD3 (145-2C11), CD16/CD32 170

(2.4G2), CD45R/B220 (RA3-6B2), CD150 (TC15-12F12.2), Sca1 (D7), CD117 (2B8), 171

CD48 (BCM1), CD19 (6D5), TCRβ (H57-597), CD8 (53-6.7), CD11c (N418), CD4 172

(RM4-4), NK1.1 (PK136), and TCRγ/δ (GL3). The following antibody cocktail was used 173

to exclude lineage positive (Lin+) cells: anti-Ter119, CD11b, CD11c, Gr1, C220, CD19, 174

CD3, TCRβ, TCRγ/δ, CD8, and NK1.1. Stained cells were analyzed by flow cytometry 175

using flow cytometers FACSCanto II, FACSAria II, or FACSAria III (Becton Dickinson 176

Biosciences). Dead cells were excluded with DAPI where appropriate (Sigma). Files 177

were analyzed with FlowJo (Tree Star). 178

Cell sorting 179

Ter119+ erythroid precursors were sorted from reconstituted bone marrows of recipient 180

mice using FACSAria II. Ter119+ erythroid precursors were also purified from E17.5 181

FLC. Mononuclear cells prepared from E17.5 livers were incubated with APC-182

conjugated anti-Ter119+ antibody (Biolegend) for 30 minutes on ice, washed twice with 183

PBS containing 4% FBS, and then treated with anti-APC conjugated magnetic beads 184

(Miltenyi biotech) for 15 minutes. Cells were then washed once and suspended in PBS 185

+ 4% FBS. Ter119+ positive cells were collected using LS MACS separation columns 186

(Miltenyi biotech) mounted on a magnet stand according to manufacturer’s instructions. 187

RBC ghost preparation 188

11

Seventy microliters of peripheral blood were centrifuged at 2300 g. The pellet was 189

washed twice with PBS (pH 7.4) and then lysed by suspension in ghost lysis buffer (5 190

mM Na2PO4, 1.3 mM EDTA, pH = 7.6) containing protease inhibitor (1 protease 191

inhibitor tablet (Roche, stock # 11873580001) per 50 ml ghost lysis buffer). Lysates 192

were centrifuged at 16,000 g and the supernatants containing the RBC membrane 193

fraction were collected and washed 4-6 times in ghost lysis buffer. RBC ghosts were 194

stored at -80°C in lysis buffer. 195

Electron microscopy 196

Cells were fixed in 2.5% glutaraldehyde in 0.1M Sorensen’s buffer (pH 7.4) overnight at 197

4°C. After 2 rinses with 10-20 milliliters of Sorensen’s buffer, cells were fixed with 1% 198

osmium tetroxide in 0.1M Sorensen’s buffer, rinsed in double distilled water, and then 199

en bloc stained with aqueous 3% uranyl acetate for 1 hour. Cells were dehydrated in 200

ascending concentrations of ethanol, rinsed twice in 100% ethanol, and embedded in 201

epoxy resin. Samples were ultra-thin sectioned at 70 nm in thickness and stained with 202

uranyl acetate and lead citrate. Sections were examined on a Philips CM100 electron 203

microscope at 60kV. Images were recorded digitally using a Hamamatsu ORCA-HR 204

digital camera system operated with AMT software (Advanced Microscopy Techniques 205

Corp., Danvers, MA). 206

Western blot 207

Proteins were separated by SDS gel electrophoresis using 4-20% tris-glycine gels 208

(Invitrogen) and tris-glycine running buffer or using 4-12% Bis-Tris gels (Invitrogen) and 209

MOPS running buffer (Invirogen). Proteins were then transferred onto nitrocellulose 210

12

membranes (BioRad). For X-ray development, membranes were blocked in 5% 211

milk/TBST (weight/volume), probed with primary antibody, washed 3 times in TBST, 212

probed with peroxidase coupled secondary antibodies (Thermo Scientific), washed 3 213

times in TBST, and developed using Western Lightning Plus-ECL (Perkin-Elmer). 214

Quantitative western blots were performed using Odyssey (LICOR Biosciences) 215

according to the manufacturer’s instructions. Secondary antibodies utilized were IRDye 216

680RD or IRDye 800 CW. Band intensities were quantified using the Odyssey software. 217

SEC23A band intensity was normalized to beta-actin or RalA. 218

Antibodies 219

Anti-SEC23B and anti-SEC23A antibodies were generated in rabbit against peptides 220

LTKSAMPVQQARPAQPQEQP and DNAKYVKKGTKHFEA respectively. Anti-Band3 221

and anti-GAPDH antibodies were obtained from Millipore. Anti-actin antibody was 222

obtained from Santa Cruz. 223

qRT-PCR 224

RNA was isolated with Trizol. Reverse transcription was performed using the 225

Superscript First-Strand Synthesis System for RT-PCR (Invitrogen) with random 226

primers. Real-time PCR amplification was performed in triplicates with Power SYBR 227

Green PCR Master Mix (Applied Biosystems) using the Applied Biosystems 7900HT 228

Fast real-time PCR System. Relative gene expression was calculated using the 2-ΔΔCT 229

method. Beta-actin or GAPDH were used as internal controls. Two samples of each 230

genotype were analyzed, each in triplicate. qPCR primer sequences are listed in table 231

1. 232

13

Results 233

Transplantation of SEC23B-deficient HSC does not result in a CDAII phenotype 234

SEC23B deficient mice die perinatally, exhibiting degeneration of their professional 235

secretory tissues but no evidence of anemia at birth (18). To assess the impact of 236

SEC23B-deficiency on adult mouse hematopoietic function, equal numbers of FLC 237

collected from either Sec23bgt/gt or WT E17.5 embryos were transplanted into lethally 238

irradiated C57BL/6J recipient mice. Livers harvested from Sec23bgt/gt and WT E17.5 239

embryos exhibited no significant differences in total cell counts or numbers of long-term 240

HSCs (ckit+ Sca1+ CD48- CD150+ Lin-) (20) measured by flow cytometry (Fig. 2A, B, 241

and C). Hemoglobin (Fig. 3A) and hematocrit (Fig. 3B) levels measured at weeks 6, 8, 242

12, and 25 post-transplantation were all the within the normal range and 243

indistinguishable between mice transplanted with Sec23bgt/gt FLC and recipients of WT 244

FLC, as were spleen weights (Fig. 3C) and bone marrow myeloid to erythroid ratios 245

(Fig. 3D). There was also no increase in the number of bi/multi-nucleated RBC 246

precursors observed in the recipients of Sec23bgt/gt BM (Fig. 3E). 247

Characteristic RBC abnormalities in humans with CDAII include a “double membrane” 248

appearance on transmission electron microscopy, and narrower band size together with 249

a shift in the mobility of membrane protein band 3 on sodium dodecylsulfate-250

polyacrylamide gel electrophoresis. RBC from mice transplanted with Sec23bgt/gt FLCs 251

did not exhibit either of these abnormalities (Fig. 4A-C). 252

SEC23B deficient FLC and WT FLC are equivalent in reconstituting erythropoiesis 253

14

To assess for a more subtle hematopoietic defect, SEC23B deficient FLC were tested 254

for their ability to reconstitute hematopoiesis as compared to WT FLC in a competitive 255

repopulation assay. In this experiment, Sec23bgt/gt FLC were mixed with UBC-GFPtg/+ 256

Sec23b+/+ FLC in a 1:1 ratio and co-transplanted into lethally irradiated C57BL/6J 257

recipient mice. Following engraftment, hematopoietic cells from recipient mice were 258

characterized by GFP expression to distinguish cells of Sec23bgt/gt or WT FLC origin. 259

Control mice were co-transplanted with a 1:1 ratio of WT FLCs cells with or without the 260

UBC-GFPtg/+ transgene. 261

Over the course of 18 weeks of follow-up, WT FLC exhibited no competitive advantage 262

at reconstituting erythropoiesis compared to SEC23B-deficient FLC (Fig. 5A). Similarly, 263

no defects were observed in the ability of Sec23bgt/gt FLC to differentiate into neutrophils 264

(Fig. 6 A) or lymphocytes (Fig. 6B and C). 265

Eighteen weeks following transplantation, reconstituted bone marrows and thymi were 266

harvested from transplant recipients and the relative contribution of Sec23bgt/gt and WT 267

cells to each hematopoietic compartment was evaluated. Erythroid cells were stratified 268

by forward scatter and CD71 expression to identify primitive progenitors (larger cells 269

expressing higher levels of CD71), mature cells (smaller cells expressing low levels of 270

CD71), and erythroid cells in intermediate stages of development (average or small size 271

cells expressing high levels of CD71) (21). Sec23bgt/gt and WT cells contributed similarly 272

to all populations of erythroid cells examined (Fig. 5B). 273

The contribution of GFP(-) Sec23bgt/gt cells to the populations of long-term HSC (Fig. 274

6D) and myeloid cells (Fig. 6E) in the BM, and to all subgroups of T-lymphocytes (Fig. 275

15

6F) in the thymus was equivalent to that of GFP(+) WT cells. There was a trend for 276

some subsets of T-lymphocytes to be under-represented (Sec23bgt/gt CD8+ TCRβ 277

immature single positive cells, CD4+ CD8+ double positive cells, and CD4+ T-278

lymphocytes); however, this did not reach statistical significance after correction for 279

multiple observations. In contrast, BM Sec23bgt/gt B-lymphocytes (Fig. 6G) were under-280

represented relative to their WT counterparts (p = 0.005). 281

To exclude the possibility that the reconstituted GFP(-) hematopoietic cells in recipient 282

mice were derived from host reconstitution rather than Sec23bgt/gt FLC, GFP(-) mature 283

myeloid cells (Mac1+ Gr1+) were FACS sorted from bone marrows of mice co-284

transplanted with GFP(-) Sec23bgt/gt FLC and GFP(+) WT FLC. The genotype of the 285

isolated myeloid cells was confirmed to be Sec23bgt/gt (Fig. 7). 286

SEC23B deficient HSC and WT HSC are equivalent in their hematopoietic 287

reconstitution potential 288

To further test for the hematopoietic reconstitution potential of Sec23bgt/gt HSC, bone 289

marrows chimeric for Sec23bgt/gt and WT HSC were harvested from the competitive FLC 290

transplant recipients (described above) and transplanted into secondary WT recipients. 291

Over the course of 18 weeks of follow-up, the contribution of Sec23bgt/gt and WT HSC to 292

the reconstituted erythroid, myeloid, B-cell and T-cell compartments was equivalent 293

(Fig. 8A, and Fig. 9A-C). Bone marrows from a subset of secondary transplant 294

recipients were analyzed at 26 weeks post-transplantation. Persistence of GFP(-) 295

Sec23bgt/gt erythroid cells in all stages of erythroid differentiation (Fig. 8B) and 296

Sec23bgt/gt long-term HSC (Fig. 8C) was observed. 297

16

A second Sec23b null allele confirms the absence of a RBC phenotype in 298

SEC23B-deficient mice 299

Sec23bgt/gt murine embryonal fibroblasts express a SEC23B/βGEO fusion protein 300

resulting from the gene-trap insertion into intron 19 of Sec23b. Though SEC23B/βGEO 301

co-immunoprecipitates with SEC24 (binding partner for SEC23) (18), a similar 302

pancreatic phenotype was observed for Sec23bgt/gt mice and a second targeted allele. 303

However, to rule out any residual function of the Sec23bgt allele masking a 304

hematopoietic phenotype, another set of transplant experiments was performed with 305

FLCs derived from a second Sec23b mutant allele, Sec23b- (excision of exons 5 and 6 306

(Fig. 1)), which should result in a protein truncated after amino acid 130 (the full length 307

SEC23B protein contains 767 amino acids). 308

FLC were harvested from E16.5 Sec23b-/- embryos and transplanted into lethally 309

irradiated C57BL/6J recipients ubiquitously expressing GFP (UBC-GFPtg/+). 310

Reconstituted hematopoietic cells in recipient mice were GFP(-), confirming donor stem 311

cell engraftment. At 2 months and 5 months post-transplantation, transplant recipients 312

of Sec23b-/- FLC exhibited normal RBC counts (Fig. 10A), hemoglobin (Fig. 10B), and 313

hematocrit (Fig. 10C) levels, indistinguishable from recipients of control FLCs. RBC 314

ghosts prepared from reconstituted Sec23b-/- peripheral blood demonstrated no 315

evidence of an alteration in band 3 glycosylation compared to control ghosts by western 316

blot (Fig. 10D). Additionally, Ter119+ erythroid precursors isolated from Sec23b-/- FLC 317

did not exhibit the “double membrane” appearance on transmission electron microscopy 318

(Fig. 10 E) that is characteristic of human CDAII. 319

17

BM SEC23B deficient erythroid cells are normally distributed among stages of 320

erythroid development 321

Ter119+ erythroid cells were determined by flow cytometry to comprise 37% (standard 322

deviation (SD) = 17%) and 42% (SD = 18%) of the total number of live BM cells in mice 323

transplanted with Sec23b-/- and WT FLC, respectively (Fig. 10D). Mice transplanted with 324

Sec23b-/- FLC did not exhibit an increase in the percentage of BM bi-/multi-nucleated 325

RBC precursors (Fig. 10E). 326

To test for stage-specific defects in erythroid maturation resulting from murine SEC23B-327

deficiency, BM Ter119+ erythroid cells from mice transplanted with either Sec23b-/- or 328

WT FLC were stratified by Ter119 expression, CD71 expression, and forward scatter 329

into 5 distinct populations of erythroid development, designated stages I to V (22). 330

Stage I is the earliest stage and consists predominantly of proerythroblasts. Erythroid 331

cells progress through stages I, II, III, and IV in chronological order, and ultimately reach 332

the final stage, stage V, which encompasses primarily mature RBCs. 333

The distribution of BM erythroid cells among the 5 stages of erythroid development was 334

comparable in mice transplanted with Sec23b-/- FLC and control mice transplanted with 335

WT FLC (Fig. 10F). 336

SEC23A protein level is increased in SEC23B deficient erythroid precursors 337

Anti-SEC23A and anti-SEC23B anti-peptide antibodies were generated and tested for 338

specificity to their respective paralogs (Fig. 11A and B). SEC23B protein was 339

undetectable by western blot in Sec23b-/- FLC and in sorted Ter119+ Sec23b-/- erythroid 340

precursors (Fig. 11C and D). Sec23b mRNA isolated from Sec23b-/- erythroid 341

18

precursors was markedly reduced, likely due to nonsense mediated decay resulting 342

from the exon 5-6 deletion and resulting frameshift (23) (Fig. 11E). To assess any 343

potential change in SEC23A protein levels in response to loss of SEC23B, quantitative 344

western blot analysis was performed, revealing an ~50% increase in the steady state 345

levels of SEC23A protein in SEC23B-deficient compared to WT erythroid cells (Fig. 346

11F). However, no change In the mRNA expression of the four Sec24 paralogs, the two 347

Sar1 paralogs or TRAPPC3 were observed between SEC23B-deficient and WT 348

erythroid cells as measured by qPCR (Fig. 11G). 349

19

Discussion 350

Homozygous or compound heterozygous SEC23B mutations in humans result in CDAII, 351

with the clinical phenotype restricted to a characteristic set of RBC abnormalities, and 352

no reported non-hematologic clinical manifestations. In contrast, SEC23B-deficient mice 353

die perinatally, exhibiting degeneration of multiple professional secretory tissues, with 354

apparently normal RBCs. However, the failure of these mice to survive beyond the 355

immediate perinatal period precluded detailed RBC analysis and characterization of 356

adult hematopoiesis. To address this issue, we performed FLC transplantation 357

experiments to generate chimeric mice with SEC23B-deficiency restricted to the 358

hematopoietic compartment. Surprisingly, no RBC abnormalities characteristic of 359

human CDAII could be detected in these animals. In addition to the absence of anemia, 360

SEC23B-deficient RBCs lacked the duplicated membrane and band 3 glycosylation 361

defects that are characteristic of CDAII in humans. Erythroid hyperplasia and 362

multinucleated RBC precursors were also absent from the BM. Competitive transplants 363

and secondary transplant experiments also failed to uncover even a subtle defect in 364

erythropoiesis or reconstitution of myeloid cells and T-lymphocytes. SEC23B deficient B 365

lineage cells appeared under-represented in the BMs of mice transplanted with equal 366

numbers of Sec23bgt/gt and WT FLC. However, this finding was not associated with 367

a reduction in the number of SEC23B null B-lymphocytes in the peripheral blood of 368

these chimeric animals, thus its significance remains unclear. Furthermore, secondary 369

recipients of BM harvested from these mice did not exhibit a decreased contribution of 370

the peripheral blood Sec23bgt/gt B-lymphocytes compared to their WT counterparts. We 371

confirmed the absence of SEC23B protein in Sec23b-/- erythroid precursors, with qPCR 372

20

analysis of mRNA isolated from Sec23b-/- erythroid precursors demonstrating a marked 373

reduction in Sec23b mRNA, likely due to nonsense mediated decay (23). 374

SEC23B is ubiquitously expressed in various tissues (18, 24, 25) and is an integral 375

component of COPII vesicles, which facilitate the transport of ~ 8000 proteins from the 376

ER to the Golgi apparatus (26). Despite this broad and fundamental function, SEC23B-377

deficiency in humans results in a phenotype restricted to the RBC compartment. 378

Though deficiencies of the inner COPII coat components, SEC23, SEC24, and SAR1, 379

are all lethal in yeast, the corresponding COPII proteins for which deficiencies have 380

been reported in mice or humans show a wide range of phenotypes. SEC23A deficiency 381

in humans (discussed below) results in cranio-lenticulo-sutural-dysplasia (15), whereas 382

SEC24A, SEC24B and SEC24D deficiency in mice result in low plasma cholesterol 383

(27), chraniorachischisis (28), and early embryonic lethality (29), respectively. SAR1B 384

mutations in humans result in a disease of lipid malabsorption and chylomicron 385

accumulation in the enterocytes (30). It is interesting to note that other genetic 386

deficiencies affecting a large portion of the proteome also selectively disrupt the 387

erythroid compartment, including mutations in genes encoding several ribosomal 388

proteins resulting in Diamond-Blackfan syndrome. These observations suggest that the 389

demanding process of RBC production may be particularly sensitive to perturbations of 390

the basic cell machinery. 391

The mouse is a well established model for the study of human hematopoiesis (31), with 392

numerous gene targeted mice closely recapitulating the eythropoietic phenotypes of the 393

corresponding human diseases (32-36). The lack of conservation in SEC23B deficient 394

21

phenotypes between humans and mice is particularly surprising, given the previous 395

report of a CDAII-like phenotype in SEC23B-deficient zebrafish embryos (4). 396

The mammalian genome encodes 2 SEC23 paralogues, SEC23A and SEC23B. In 397

humans, SEC23A mutations result in cranio-lenticulo-sutural-dysplasia (15), an 398

autosomal recessive disease thought to result from abnormal collagen secretion. This 399

disease is characterized by skeletal abnormalities, late closure of fontanelles, 400

dysmorphic features, and sutural cataracts. Though SEC23A-deficient mice have not 401

been reported, Sec23a deficient zebrafish exhibit abnormal cartilage development 402

reminiscent of the human phenotype (15, 37). The SEC23A and SEC23B proteins 403

exhibit a high degree of sequence similarity (~ 85% identical at the amino acid level), 404

suggesting that the 2 SEC23 paralogs may overlap extensively in function and that the 405

disparate phenotypes of SEC23B deficiency in humans and mice could be due to a shift 406

in tissue-specific expression patterns during mammalian evolution. Consistent with this 407

hypothesis, recently reported analyses of SEC23A/B in cultured erythroid progenitors 408

(38) and transcriptomes for human and murine erythroid cells at several stages of 409

terminal maturation (39, 40), demonstrate different patterns of SEC23A/SEC23B 410

expression in humans and mice. This is evident particularly in the latest stage of 411

erythroid maturation, with SEC23B the predominant paralog in humans and SEC23A in 412

mice. However, an additional unique function for SEC23B in the human erythroid 413

compartment that is not required in mice, cannot be excluded. 414

Of note, western blot analysis demonstrates an increase of steady state SEC23A 415

protein levels in SEC23B-deficient erythroid progenitors, suggesting a balance between 416

the SEC23A and B cytoplasmic pools, potentially mediated via SEC23/24 heterodimer 417

22

formation. This is similar to the increase in SEC24B observed in hepatocytes of 418

SEC24A deficient mice (27). However, there was no apparent change in the mRNA 419

expression of other core components of the COPII vesicles. 420

Reports of curative bone marrow transplantation for CDAII (9, 16) indicate that the 421

pathologic defect in this disease is confined to a transplanted cell. However, the 422

mechanism by which human SEC23B-deficiency results in the unique erythropoietic 423

phenotype of CDAII remains unknown. The role of SEC23 in ER-to-Golgi transport 424

suggests that CDAII results from the impaired secretion of one or more key cargo 425

proteins that depend on SEC23B for export from the ER. Mutation of scl4a1 (the gene 426

encoding band 3) in zebrafish results in increased binucleated erythroblasts, suggesting 427

that band 3 could be the critical cargo, with CDAII resulting from a selective block in its 428

transport to the membrane (41). However, humans with band 3 mutations exhibit 429

hereditary spherocytosis and other RBC shape disorders, but not CDAII (42, 43). The 430

observation that RBCs from CDAII patients are lysed in some but not all acidified 431

normal sera (Ham’s test) (1, 2), may provide a clue as to the identity of the critical 432

SEC23B dependent secretory cargo(s). 433

SEC23B interacts directly with SEC31, a component of the outer layer of the COPII 434

coat. SEC23B also interacts with Bet3 (44), a component of the tethering complex 435

TRAPPI, and with p150Glued (45), a component of the dynactin complex. Whether 436

these direct SEC23B interactions contribute to the pathophysiology of CDAII is 437

unknown. 438

23

In conclusion, we have shown that mice with hematopoietic deficiency of SEC23B 439

support a normal erythroid compartment. Future studies aimed at understanding the 440

functional overlap between SEC23A and SEC23B, as well as the specific protein cargos 441

dependent on SEC23A/B for exit from the ER, should provide further insight into the 442

pathophysiology of CDAII. 443

24

Acknowledgments 444

This work was supported by National Institute of Health Grants R01 HL039693 and 445

P01-HL057346 (DG), R01 AI091627 (IM), and R01 HL094505 (BZ). David Ginsburg is a 446

Howard Hughes Medical Institute investigator. 447

The authors would like to thank Sasha Meshinchi and Jeff Harrison from the Microscopy 448

and Image-Analysis Laboratory at the University of Michigan for help with electron 449

microscopy. The authors would like to acknowledge Elizabeth Hughes, Keith Childs, 450

and Thomas Saunders for preparation of gene targeted mice and the Transgenic 451

Animal Model Core of the University of Michigan’s Biomedical Research Core Facilities. 452

Core support was provided by the University of Michigan Cancer Center (P30 453

CA046592). 454

455

Authorship and conflict of interest: 456

Contribution: RK, MV, and DG conceived the study and designed experiments. RK, MV 457

and MJ performed most of the experiments. BZ, LE, JT, and DS contributed to the 458

execution of the experiments. RK and DG wrote the paper. All authors contributed to the 459

integration and discussion of the results. 460

Conflict of interest disclosure: The authors declare no competing conflicts of interest. 461

25

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601

28

Figure Legends: 602

Figure 1. Sec23b mutant alleles. (A) Schematic of the first Sec23b gene-trap allele 603

demonstrating a gene-trap insertion into intron 19.(18) SA, splice acceptor cassette; β-604

Geo, β-galactosidase-neo fusion; pA, poly-adenylation sequence. (B) The Sec23b 605

conditional gene-trap allele (Sec23bcgt) contains a gene trap insertion in intron 4 flanked 606

by 2 FRT sites. Mice carrying this allele were crossed to β-actin FLP transgenic mice. 607

Mice heterozygous for the resulting Sec23b floxed allele (Sec23b+/fl) were crossed to 608

EIIACre transgenic mice to excise exons 5 and 6, and generate the Sec23b null allele 609

(Sec23b-). Gray boxes represent exons with exon number indicated in the box. F1, F2, 610

R1, cgtB1, and cgtR1 represent Sec23b genotyping primers. En2 SA, splice acceptor of 611

mouse En2 exon 2; IRES, encephalomyocarditis virus internal ribosomal entry site; 612

lacZ, E.coli β-galactosidase gene; pA, SV40 plyadenylation signal; βact:neo, human β-613

actin promoter-driven neomycin cassette. (C) A three-primer PCR assay (F1, cgtB1, 614

cgtR1) distinguishes the Sec23bcgt allele (F1:cgtB1, 344 bp) and the WT allele 615

(F1:cgtR1, 475 bp). (D) A three-primer PCR assay (F1, F2, R1) distinguishes the 616

alleles: Sec23b+ (F2:R1, 235 bp), Sec23bfl (F2:R1, 269 bp), and Sec23b- (F1:R1, 336 617

bp). Location of the primers are indicated in Figures 1A and 1B. The gene structures 618

depicted in figures 1A and 1B are not according to scale. 619

Figure 2. FACS analysis of E17.5 Sec23bgt/gt and WT FLC. (A) Livers were harvested 620

from 3 Sec23bgt/gt and 3 WT E17.5 embryos. Flow cytometry on FLC single cell 621

suspensions demonstrated equivalent total number of recovered cells from Sec23bgt/gt 622

and WT fetal livers. Each liver is represented by 1 point, and horizontal lines indicate 623

mean value for each group. (B) The number of long-term hematopoietic stem cells (ckit+ 624

29

Sca1+ CD48- CD150+ Lin-) was equivalent for Sec23bgt/gt and WT fetal livers. (C) FACS 625

distributions for cell sorts used to calculate long-term hematopoietic stem cells in B. 626

Figure 3. Transplant recipients of Sec23bgt/gt FLC do not exhibit CDAII. Mice 627

transplanted with Sec23bgt/gt FLC demonstrated equivalent (A) hemoglobin and (B) 628

hematocrit levels as compared to mice transplanted with control WT FLC over the 629

course of 25 weeks of post-transplantation follow-up (p > 0.05 for all time points). N = 5-630

7 mice per group. Error bars represent standard deviation. (C) Spleens harvested from 631

transplant recipients of Sec23bgt/gt and WT FLC were equivalent in weights. Recipients 632

of Sec23bgt/gt FLC exhibited equivalent (D) myeloid : erythroid ratios and (E) number of 633

bi/multi-nucleated RBC precursors (evaluated independently by two investigators) 634

compared to control mice transplanted with WT FLC. Each dot represents results from 635

one mouse. Horizontal lines indicate means and error bars indicate standard deviation. 636

Figure 4. RBC from mice transplanted with Sec23bgt/gt FLC do not exhibit a band 3 637

glycosylation defect or a double RBC membrane. (A) RBC ghosts were isolated 638

from Sec23bgt/gt and WT RBC and fractionated on sodium dodecylsulfate-639

polyacrylamide gel electrophoresis. Coomassie blue stain revealed no difference in the 640

appearance of the RBC membrane protein band 3 in Sec23bgt/gt RBC ghosts. Each lane 641

represents a sample from a different individual mouse. (B) Similarly, band 3 protein 642

appeared indistinguishable on western blotting bteween Sec23bgt/gt and WT RBC 643

ghosts. (C) Sec23bgt/gt RBC lack the “double membrane” appearance on transmission 644

electron microscopy characteristic of human CDAII. RBC were evaluated at three 645

different magnifications, illustrated in the right lower corner of each figure. Arrows 646

indicate RBC membrane. 647

30

Figure 5. Sec23bgt/gt FLC do not exhibit a competitive disadvantage at 648

reconstituting erythropoiesis compared to WT FLC. C57BL/6J mice were co-649

transplanted with a 1:1 mix of GFP(-) Sec23bgt/gt FLC and UBC-GFPTg+ Sec23b+/+ FLC 650

in a competitive transplant assay (experimental arm). Following engraftment, the 651

percent of GFP(-) cells in the peripheral blood/BM of recipient mice indicate the percent 652

of cells derived from Sec23bgt/gt FLC. Control mice were co-transplanted with a 1:1 mix 653

of WT GFP(-) and GFPTg+ Sec23b+/+ FLC. (A) By peripheral blood FACS, Ter119+ RBC 654

were determined to be derived from both Sec23bgt/gt and WT FLC. Sec23bgt/gt RBC 655

persisted at a stable level throughout the 18-weeks follow-up period, suggesting no 656

competitive advantage to WT FLC compared to Sec23bgt/gt FLC at reconstituting 657

erythropoiesis. (B) In the BM, the contribution of GFP(-) Sec23bgt/gt cells to the 658

populations of Ter119+ erythroid precursors was equivalent to the contribution of the 659

GFP(-) WT cells in the control arm. Erythroid cells were further stratified by forward 660

scatter and CD71 expression to identify more primitive progenitors as larger cells 661

expressing higher levels of CD71, more mature smaller cells with lower expression of 662

CD71, and intermediate cells. Sec23bgt/gt cells contributed to all subsets of erythroid 663

cells. 664

Figure 6. Analysis of peripheral blood and BM hematopoietic compartments of 665

mice co-transplanted with a 1:1 mix of GFP(-) Sec23bgt/gt FLC and UBC-GFPTg+ 666

Sec23b+/+ FLC (experimental arm) and control mice co-transplanted with a 1:1 mix 667

of GFP(-) and GFPTg+ Sec23b+/+ FLC. By peripheral blood FACS, (A) Mac1+ Gr1+ 668

neutrophils, (B) B220+ B-lymphocytes, and (C) CD3+ T-lymphocytes were found to be 669

derived from both Sec23bgt/gt and WT FLC. The Sec23bgt/gt peripheral blood cells 670

31

persisted at a stable level throughout the 18-week follow-up period, suggesting no 671

competitive advantage to WT FLC compared to Sec23bgt/gt FLC at reconstituting 672

hematopoiesis. In the BM, the contribution of GFP(-) Sec23bgt/gt FLC to (D) the long-673

term hematopoietic stem cells (ckit+ Sca1+ CD48- CD150- Lin-), and to (E) myeloid 674

cells (Mac1+ GR1+) in the experimental arm was equivalent to the contribution of the 675

GFP(-) WT cells in the control arm. (F) Similarly, the contribution of GFP(-) Sec23bgt/gt 676

T-lymphocytes in the thymus was equivalent to that of GFP(-) WT cells in the control 677

arm. *There was a trend for some subsets of GFP(-) Sec23bgt/gt T-lymphocytes to be 678

under-represented; however this did not reach statistical significance after correction for 679

multiple observations using the Holm-Sidak method or the Bonferroni method. ISP, 680

immature single positive cells; DP, CD4+ CD8+ double positive T-lymphocytes; NS, not 681

significant. (G) In contrast, GFP(-) Sec23bgt/gt CD19+ CD220+ BM B-lymphocytes were 682

under-represented. Each point represents one mouse. Lines represent mean values for 683

each group and error bars indicate standard deviation. 684

Figure 7. Reconstituted GFP(-) hematopoietic cells in mice co-transplanted with a 685

1:1 mix of GFP(-) Sec23bgt/gt FLC and UBC-GFPTg+ Sec23b+/+ FLC were derived 686

from Sec23bgt/gt FLC and not from host reconstitution. GFP(-) myeloid cells isolated 687

from mice transplanted with a 1:1 ratio of UBC-GFPtg/+ FLC and Sec23bgt/gt FLC and 688

from control mice transplanted with 1:1 ratio of UBC-GFPtg/+ FLC and WT FLC were 689

genotyped for Sec23bgt/gt. Six mice from each group were examined. Each lane 690

represents the genotype of myeloid cells isolated from a single mouse. Genotype of a 691

Sec23b+/gt control DNA is shown. The lower and upper bands correspond to the 692

expected PCR products for the Sec23b WT and gt alleles, respectively. 693

32

Figure 8. Secondary bone marrow transplantation experiments demonstrate 694

continued equivalence of Sec23bgt/gt and WT FLC at reconstituting erythropoiesis. 695

BMs harvested from mice chimeric for GFP(-) Sec23bgt/gt and GFP(+) WT hematopoietic 696

cells were transplanted into lethally irradiated secondary recipients (experimental arm). 697

Control mice were transplanted with BMs harvested from mice chimeric for GFP(-) and 698

GFP(+) WT hematopoietic cells. (A) By peripheral blood FACS, the contribution of 699

Sec23bgt/gt GFP(-) cells to the population of Ter119+ RBC in the experimental arm was 700

equivalent to the contribution of WT GFP(-) cells in control mice over the course of 18 701

weeks of follow-up. Peripheral blood analysis was performed on N = 13-15 mice per 702

group. Error bars represent standard deviation. Bone marrow cells were isolated from 703

both hind limbs of each secondary transplant recipient mouse. The contribution of 704

Sec23bgt/gt GFP(-) (B) Ter119+ erythroid cells at various stages of differentiation and (C) 705

hematopoietic stem cells (ckit+ Sca1+ CD48- CD150- Lin-) in the experimental mice 706

was equivalent to the contribution of WT GFP(-) cells in the control arm. Each point 707

represents one mouse. Lines represent mean values for each group. P values were 708

calculated by Student’s t-test. 709

Figure 9. Secondary bone marrow transplantation experiments demonstrate 710

continued equivalence of Sec23bgt/gt and WT FLC at reconstituting 711

hematopoiesis. BMs harvested from mice chimeric for GFP(-) Sec23bgt/gt and GFP(+) 712

WT hematopoietic cells were transplanted into lethally irradiated secondary recipients 713

(experimental arm). Control mice were transplanted with BMs harvested from mice 714

chimeric for GFP(-) and GFP(+) WT hematopoietic cells. Peripheral blood was obtained 715

from recipient mice. By FACS, the contribution of Sec23bgt/gt GFP(-) cells to the 716

33

population of (A) Mac1+ Gr1+ neutrophils, (B) B220+ B-lymphocytes, and (C) CD3+ T-717

lymphocytes in the experimental arm was equivalent to the contribution of WT GFP(-) 718

cells in control mice over the course of 18 weeks of follow-up. n = 13-15 mice per group. 719

Error bars represent standard deviation. 720

Figure 10. Mice transplanted with Sec23b-/- FLC do not exhibit an erythroid 721

phenotype. Lethally irradiated UBC-GFPtg/+ mice were transplanted with GFP(-) FLC 722

harvested from either Sec23b-/- or WT E.16.5 embryos. Reconstituted hematopoietic 723

cells in recipient mice were GFP(-), confirming donor engraftment. Recipients of 724

Sec23b-/- FLC had indistinguishable (A) RBC counts, (B) hemoglobin, and (C) 725

hematocrit levels as compared to control recipients of WT FLC. (D) Sec23b-/- RBC 726

ghosts did not exhibit a band 3 glycosylation defect by western blot, (E) nor did Ter119+ 727

erythroid precursors demonstrate a “double membrane” by transmission electron 728

microscopy. (F) By FACS analysis, Ter119+ erythroid cells comprised 36.67% (± 16.69 729

SD) and 41.56% (± 17.68 SD) of total live BM cells harvested from mice transplanted 730

with Sec23b-/- and WT FLC, respectively. (G) Mice transplanted with Sec23b-/- FLC did 731

not exhibit an increase in the percent of bi/multi-nucleated RBC precursors. (H) BM 732

erythroid compartments were stratified by forward scatter and CD71 expression into 5 733

stages of erythroid development (stages I through V in chronological order) (22). The 734

distribution of erythroid cells among stages I through V was comparable in mice 735

transplanted with Sec23b-/- FLC and mice transplanted with WT FLC. Error bars indicate 736

standard deviation. Means and standard deviation are indicated by horizontal lines. 737

Figure 11. SEC23A protein level is increased in Sec23b-/- erythroid precursors. 738

Lysates from COS cells transfected with GFP-tagged SEC23A or SEC23B were 739

34

examined by immunoblotting with antipeptide antibodies raised against (A) SEC23A or 740

(B) SEC23B, demonstrating a high degree of specificity of these antibodies for their 741

respective paralogs. Western blotting of whole cell lysates from (C) WT control and 742

Sec23b-/- FLC and (D) sorted control and Sec23b-/- Ter119+ erythroid precursors 743

demonstrated no detectable SEC23B protein in Sec23b-/- cells. (E) qPCR analysis 744

showed a marked reduction of Sec23b mRNA in Sec23b-/- compared to WT Ter119+ 745

cells. (F) SEC23A protein level normalized to β-actin or to RalA was increased in 746

Ter119+ erythroid precursors compared to WT controls, as determined by quantitative 747

western blot analysis (p = 0.029 and 0.030 for normalization to β-actin and RalA 748

respectively). (G) mRNA levels measured by qPCR for the four Sec24 paralogs, the two 749

Sar1 paralogs, and TRAPPC3 were all indistinguishable between Sec23b-/- and WT 750

Ter119+ erythroid cells. 751

752

Primer sequences

Primer 5’ à 3’ Sequence

Sec23b F1 ATAGACCAGGCTGGCCTCAGTC

Sec23b cgt B1 CCACAACGGGTTCTTCTGTT

Sec23b cgt R1 CGAGCACAGAGAGACCCAAT

Sec23b R CAAGTGAGTGCCTCCTCACA

Sec23b F2 AACAGCCCAGGTGACTAGGA

Sec23b qPCR F CCCTACGTCTTTCAGATTGTCA Sec23b qPCR R CGGGCAAAATGGTGTCTATAA Sec24a qPCR F GAGCAGAGATGGAGCGTTCCT Sec24a qPCR R TTCTTCCAACCCAAAGCATCA Sec24b qPCR F GACCCGAGAAGGCGCTTT Sec24b qPCR R TTTGCCAACCCAAATGTAGAAA Sec24c qPCR F TTATGCGGGTTCGGACAAG Sec24c qPCR R CTCATATAGAAAGCGCCAAAGAAAT Sec24d qPCR F CTCATATAGAAAGCGCCAAAGAAAT Sec24d qPCR R TCATGTACACAGGCAGCACCTT Sar1a qPCR F TCGGTGGGCATGAGCAA Sar1a qPCR R GCCATTAATCGCTGGGAGATAA Sar1b qPCR F GGGTGGGCACGTGCAA Sar1b qPCR R TGCCATTGATAGCAGGAAGGT TRAPPC3 qPCR F GCACGGAGAGCAAGAAAATGA TRAPPC3 qPCR R GGTGACAAGCGCTCCATAGG beta-‐actin qPCR F GATCTGGCACCACACCTTCT beta-‐actin qPCR R GGGGTGTTGAAGGTCTCAAA GAPDH qPCR F TGTGTCCGTCGTGGATCTGA

GAPDH qPCR R ACCACCTTCTTGATGTCATCATACTT

Table 1. Genotyping and qPCR primer sequences.

S 23bgt

4 5 6 7 8 9 18 19 20

β

Sec23b+(wild type)

F1 cgtR1 F2 R1A

4 5 6 7 8 9 18 19 20Sec23bgt(gene-trap)

SA β-geo pA

FRT FRT loxPloxP loxP

F1 cgtR1cgtB1BSec23bcgt(conditional gene-trap)

4 5 6 7 8 9 20

FLPF1 F2 R1

pAlacZIRESEn2 SA pAβact:neo

Cre

4 7 8 9

FRT loxPSec23bfl

(floxed)5 6

loxP

20

Sec23b-(null)

4 7 8 9

FRT loxP

20

F1 R1

C DC D

Sec2

3b+/

+

Sec23b

+/cgt

500 bp

Sec23b

+/fl

Sec23b

+/-

Sec2

3b+/

+

(-) band: 336 bp

300 bp

400 bp

500 bp(+) band: 475 bp

(-) band: 344 bp 200 bp

300 bp( ) band: 336 bp

(fl) band: 269 bp(+) band: 235 bp

4 106

6×106

8×106

pe

r to

tal l

ive

r

p = 0.55

SC

pe

r to

tal l

ive

r

1500

2000

2500

p = 0.45

A B

23b+/+

23bgt/g

t0

2×106

4×106

Ce

ll n

um

be

r p

Nu

mb

er

of L

T-H

S

c23b

+/+

23bgt/g

t0

500

1000

Sec2

Sec23 Sec

2

Sec23

19.7 6.4

Sec23b+/+

C

ckit

Lin

ckit

Sca1 CD150CD

48 18.1

Sec23b /

15.9 6.03

16

15.9

t t 48

Sec23bgt/gt

16

ckit

Lin

ckit

Sca1 CD150

CD4

10

15

bin

(g/d

l)

40

50

60

crit

(%)

A B

0 10 20 300

5

Weeks post-transplant

Hem

oglo

b

Sec23b+/+

sec23bgt/gt

0 10 20 3020

30

40


Hem

atoc

Sec23b+/+

Sec23bgt/gt

C D E

100

150

mas

s (m

g)

p = 0.16

4

6

8

10

Eryt

hroi

d ra

tio

p = 0.19 10

15

clea

ted

eryt

hrob

last

s

Observer 1p = 0.12

Observer 2p = 0.31

C D E

Sec23

b+/+

Sec23

bgt/g

t0

50

Sple

en

Sec23

b+/+

Sec23

bgt/g

t0

2

4

Mye

loid

: E

Sec23

b+/+

Sec23

bgt/g

t

Sec23

b+/+

Sec23

bgt/g

t0

5

% B

M b

i/mul

ti-nu

c

WT Sec23bgt/gt

A B

148

250 kDa

98

50

64

Band 3 Band 3Sec23b+/+ Sec23bgt/gt

36 β-actin

T

C

7900x 64000x 130000x

WT

Sec23b

gt/gt

7900x 64000x 130000x

60

80

100

ood

GFP

(-) R

BC Sec23b+/+ and UBC-GFPtg/+ FLC

Sec23bgt/gt and UBC-GFPtg/+ FLC

A

5 10 15 200

20

40


% P

erip

hera

l blo

B

6×106

8×106

1×107

P (-)

ery

thro

id c

ells

nd

lim

bs

Sec23b+/+ and UBC-GFPtg/+ FLCSec23bgt/gt and UBC-GFPtg/+ FLC

p=0.09

p=0.73

p=0.99p=0.87

B

CD71 high FSC high

CD71 high FSC m

id

CD71 high FSC lo

w

CD71 lo

w FSC low

0

2×106

4×106

Erythroid cells in different stages of maturation

Num

ber o

f BM

GFP

per 2

hin


60

80

100

GF

P (-

) n

eu

tro

ph

ils


60

80

100

P (-

) B-ly

mphocyte

s


A B

5 10 15 200

20

40

Weeks post-transplant% P

eri

ph

era

l blo

od

G

5 10 15 200

20

40

60


% P

eri

phera

l blo

od G

FP

C D

0

20

40

60

80

100

he

ral b

loo

d G

FP

(-)

T-ly

mp

ho

cy

tes


0

1000

2000

3000

4000

5000

Nu

mb

er

of B

M G

FP

(-) L

T-H

SC

p

er

2 h

ind

lim

bs

p=0.97

5 10 15 200


% P

eri

ph

Sec23b+/+

and

UBC-GFP

tg/+

FLC

Sec23bgt/gt a

nd

UBC-GFP

tg/+

FLC

N

1.5×107

) my

elo

id

mb

s

p=0.08

72×1073×107

ho

cy

tes


p=0.18

p=0.04 (NS)*

E F

Sec23b+/+

and

UBC-GFP

tg/+

FLC

Sec23bgt/gt a

nd

UBC-GFP

tg/+

FLC

0.0

5.0×106

1.0×107

Nu

mb

er

of B

M G

FP

(-)

cel

ls p

er

2 h

ind

lim

ISP c

ells

DP cel

ls

CD4+ c

ells

CD8+ c

ells

0

500000

1000000

1500000

2000000

1×107

T-lymphocytes subsets

Nu

mb

er

of G

FP

(-)

T-ly

mp

hp

er

thy

mu

s

p=0.06

p 0.04 (NS)

p=0.05 (NS)*

U

4 106

6×106

8×106

1×107

FP

(-) B

-lym

ph

oc

yte

s

hin

d li

mb

s

p=0.005

G

Sec23b+/+

and

UBC-GFP

tg/+

FLC

sec23bgt/gt a

nd

UBC-GFP

tg/+

FLC

0

2×106

4×106

Nu

mbe

r o

f BM

Gp

er

2

WT Sec23bgt/gt (+/gt)WT Sec23bgt/gt (+/gt)

60

80

100o

od

GF

P (-

) RB

C


A

5 10 150

20

40


% P

eri

ph

era

l blo

B

2 0 106

4.0×106

6.0×106

8.0×106

1.0×107

1.2×107

f BM

GFP

(-) e

ryth

roid

cells

per

2 h

ind li

mbs

Sec23b+/+ and UBC-GFPtg/+ FLCSec23bgt/gt and UBC-GFPtg/+ FLCp=0.29

p=0.20

p=0.53

B

Ter 1

19+ C

D71 h

igh

Ter 1

19+ C

D71 m

id

Ter 1

119+

CD71

low

0.0

2.0×106


Num

ber

of

C

500

1000

1500

2000

2500

mb

er

of G

FP

(-) L

T-H

SC

p

er

2 h

ind

lim

bs

p=0.19

C

Sec23b+/+

and

UBC-GFP

tg/+

FLC

Sec23bgt/gt a

nd

UBC-GFP

tg/+

FLC

0Nu

m

80

100

(-) n

eu

tro

ph

ils


A

5 10 150

20

40

60

Weeks post-transplant% P

eri

ph

ea

rl b

loo

d G

FP

(

40

60

80

100

d G

FP

(-)

B-l

ym

ph

oc

yte

s


B

5 10 150

20

40


% P

eri

ph

era

l blo

od

C

20

40

60

80

100

al b

loo

d G

FP

(-)

T-ly

mp

ho

cy

tes


5 10 150


% P

eri

ph

era

1 0

1.1

1.2

1.3

10

7 c

ells

/mic

rolit

er)

Sec23b-/-Sec23b+/+

glo

bin

(g/d

l)

12

14

16

18 Sec23b-/-Sec23b+/+

A B

2 m

onths

5 m

onths

0.8

0.9

1.0

Months post-transplant

RB

C c

ou

nts

(x


He

mo

g

2 m

onths

5 m

onths

8

10

12

C D

He

ma

toc

rit (

%)

45

50

55

60

65

70Sec23b-/-Sec23b+/+

Band 3β-actin

Sec23b+/+ Sec23b-/-

D


2 m

onths

5 m

onths

40

Sec23b+/+ Sec23b-/-

80

100

e m

arr

ow

Sec23b+/+

Sec23b-/-

E F

X 34,000 X 34,000c23b+/+

ec23b-/-

0

20

40

60

80

% e

ryth

roid

ce

lls in

bo

ne

500 nm 500 nm Sec2 Se

c

Transplant recipients

6

8

10

RB

C p

rec

urs

ors

0 74 yth

roid

ce

lls

40

60Sec23b+/+

Sec23b-/-

G H

Sec23b+/+

Sec23b-/-

0

2

4

6

% b

i/mu

lti-n

uc

lea

ted

R p=0.74

Erythroid stage

% o

ut o

f to

tal e

ry

I II III IV V0

20

EC23B-GFP

C23A-GFP

EC23A-GFP

SEC23B-GFP

A B

SEC23AGAPDHSSE S SSEC23BGAPDH

lsC D E

Sec23b

+/+

FLCSec23b

-/-FLC

Sec23b

+/+

Ter119+ cells

Sec23b

-/-FLC Te

r119+ cell

50

100

% e

xp

res

sio

n

C D E

SEC23BGAPDH SEC23BGAPDHS

WT T

er11

9+ c

ells

Sec23

b null

Ter11

9+ c

ells

0

G

100

150

200

xp

res

sio

n

100

150

200

t ex

pre

ss

ion

WT Ter119+ cells

Sec23b null Ter119+ cells

F G

SEC23A/A

ctin

SEC23A/R

alA

0

50

100

Pe

rce

nt e

x

WT Ter119+ cells

SEC23B deficient Ter119+ cells

Sec24

a

Sec24

b

Sec24

c

Sec24

d

Sar1a

Sar1b

TRAPPC30

50

Pe

rce

nt

mcb accepts, published online ahead of print on 28 july

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