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Introduction

Rab proteins are a subfamily of the Ras superfamily of small GTPases that are key regulators of intracellular membrane trafficking, from the formation of transport vesicles to their fusion with membranes. These proteins can be detected in an inactive or active conformation, depending on the nucleotide-bound status, and are considered switches that cycle between an active, membrane-associated status and an inactive cytosolic status. To date, >70 mammalian Rab proteins have been identified (1). Some Rab proteins exhibit a regulated expression, tissue-specific expression, or developmental-specific expression, while others are ubiquitously expressed (1). Each Rab protein exhibits a characteristic subcellular distribution (2).

Rab1a regulates vesicular protein transport from the endoplasmic reticulum to the Golgi apparatus (3,4) and to the cell surface (5). It also plays a role in secretion of interleukin (IL)-8 and growth hormones. Rab1a function has been implicated in neuronal differentiation (6) and cardiac development (7). The overexpression of Rab1a in transgenic mice has been shown to be associated with an increased cardiac mass and cardiac hypertrophy, leading to cardiomyopathy (7). Rab1a activity is also targeted by bacterial (8-10) and viral pathogens (11). Additionally, Rab1a regulates the mTORC1 pathway in glucose homeostasis (12), colorectal cancer (13), liver cancer development (14) and breast cancer cells (15).

The guanosine nucleotide diphosphate (GDP) dissociation inhibitor (GDI) proteins regulate the Rab family GTPase function by binding to Rab GTPase in its GDP-bound inactive form to retrieve it from the cell membrane and to maintain a soluble pool of inactive proteins ready to be re-used (16). The GDI family includes the GDI1 and GDI2 proteins. GDI1 is expressed primarily in neural and sensory tissues, whereas GDI2 is ubiquitously expressed (17).

In a recent study, it was demonstrated by the authors that GDI2 binds to the immunoreceptor tyrosine-based inhibitory motif (ITIM) domain of sialic acid-binding immunoglobulin-type lectin G (Siglec-G) in hematopoietic cells, such as B-1a cells under conditions of normal homeostasis, whereas Rab1a is recruited to the ITIM domain during bacterial infection (18). Therefore, it was hypothesized that GDI2 and Rab1a may regulate the immune response through interaction with the ITIM domain during bacterial infection. The present study thus aimed to explore the function of Rab1a in vivo by generating a Rab1a null mutant model with a trapped Rab1a gene. The homozygous deletion of the Rab1a gene resulted in early embryonic lethality. Rab1a protein was expressed from the trapped gene during early post-implantation development, suggesting a critical role of Rab1a in the transport of materials between organelles in eukaryotic cells.

Materials and methods

Reagents

Rabbit anti-mouse Rab1a antibodies (cat. no. sc-311) were obtained from Santa Cruz Biotechnology, Inc. and lipopolysaccharide (LPS; from Escherichia coli (E. coli) 055:B5 strain] were purchased from MillliporeSigma. Goat anti-mouse β-actin (cat. no. sc-1615) and horse- radish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibodies (cat. no. sc-2004) were purchased from Santa Cruz Biotechnology, Inc. 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) was obtained from Thermo Fisher Scientific, Inc.

Generation of Rab1a mutant mice

A male chimeric mouse generated from the ES cell line, XB498, was obtained from Bay Genomics, LLC. The ES cell line, XB498, was generated by using a gene trap protocol with the trapping construct pGT0pfs containing the intron from the engrailed 2 homeobox gene upstream of the gene encoding the β-galactosidase/neomycin-resistance fusion protein (please see Fig. 1 and https://igtc.org/cgi-bin/annotation.py?cellline=XB498). Genotyping was determined by the polymerase chain reaction (PCR) analysis of DNA from tail biopsies, as previously described (19).

Figure 1 Localization of the gene trap vector interrupting the Rab1a gene. Genomic exon organization of the murine Rab1a gene. The numbers above the exons correspond to the Rab1a cDNA. The second row presents a diagram of the mutated allele of Rab1a gene in which a β-galactosidase gene was inserted between exons 2 and 3. The four primers (P1, P2, P3 and P4) used for genotyping are shown. Ex, exon.

PCR-based genotyping of mice

Aliquots of 0.1 μg (10 μl) DNA were mixed with 10 μl of 2X GoTaq Green Master Mix buffer (Promega Corporation) and 10 pmol of each primer, as previously described (19). PCR amplification was carried out at 96°C for 2 min, with 35 cycles of 96°C for 10 sec, 55°C for 30 sec, and 72°C for 60 sec. To screen for the homologous recombination of DNA, the following primers were used: P1, 5′-ACT GAG TAT CCC TGG CTG GC-3′ and P2, 5′-AAG AGT AGG CTA GCC AGT CA-3′. The wild-type (WT) allele was not amplified (no band was detected), while the mutant allele produced a 300-bp band corresponding to the amplification product. The following primers were also used to confirm the presence or absence of the WT allele: P3, 5′-AGC ACA GAC AAG CAC AGT AG-3′ and P4, 5′-GTT ATC AGG CTT GGC AGC AG-3′. The amplification of the WT allele produced a 485-bp band, while the mutant allele was not amplified and therefore produced no band. Therefore, WT mice (Rab1a+/+) produced a WT 485-band and homozygous mice (Rab1a−/−) produced a 300-bp band, while heterozygous mice (Rab1a+/−) produced both a WT 485-bp band and a mutant 300-bp band. The PCR products were separated by agarose gel electrophoresis, stained with SYBR® Safe DNA Gel Stain (Thermo Fisher Scientific, Inc.) and visualized using Axygen Gel Documentation System (Corning, Inc.).

X-gal staining of mouse tissue and mouse embryos

The X-gal staining of tissues (frozen sections for E7 and E15 embryos and adult small intestine samples were prepared from WT or Rab1a+/− mice) was performed using standard procedures, as previously described (20). Embryos were embedded in optimal cutting temperature (OCT) compound and subjected to cryo-sectioning to generate slices that were 8-μm-thick. The cryosections were fixed in X-gal fixation buffer (0.1 M phosphate buffer, pH 7.3, 5 mM EGTA, 2 mM MgCl2, 0.2% glutaraldehyde) for 15 min, washed three times with X-gal wash buffer (0.1 M phosphate buffer, pH 7.3, 2 mM MgCl2), and stained overnight at 37°C in X-gal staining buffer [0.1 M phosphate buffer, pH 7.3, 2 mM MgCl2, 5 mM K4Fe(CN)6, 3H2O, 5 mM K3Fe(CN)6, 1 mg/ml X-gal]. The stained sections were washed three times with X-gal wash buffer and mounted in Aquatex® aqueous mounting medium (MilliporeSigma). Images were acquired using an EVOS FL Auto Imaging System (Thermo Fisher Scientific, Inc.).

Experimental animal models

The Rab1a mutant male mouse was from the Mutant Mouse Regional Resource Center (MMRRC) at the University of California, Davis (UC Davis). For backcrossing, two WT C57BL/6 female were obtained from Jackson Laboratory. A total of 159 (male, 53; female, 106) adult mice (Rab1a+/+, 16; Rab1a+/−, 16 for LPS treatment experiments and Rab1a+/−, 127 for producing pups and embryos) (6-8 weeks of age; weight, 20-25 g), as well as 250 pups and 77 embryos were produced in the laboratory of Dr GYC and used in the present study. The mice were maintained in individually ventilated cages (25°C and 55-65% humidity) with a 12/12-h light/dark cycle and free access to standard laboratory mouse chow and water. Age- and sex-matched WT littermates were used as controls for heterozygous Rab1a+/− or homozygous Rab1a−/− mice. All procedures involving animals were approved by the University of Tennessee Health Science Center (UTHSC) Animal Care and Use Committee (IACUC), protocol nos. 17-117 (approved January 29, 2018) and 20-0211 (approved January 26, 2021). At the end of each experiment, adults and neonates >10 days of age were euthanized with CO2 followed by cervical dislocation. The CO2 displacement rate for a euthanasia chamber is 30-70% per min (as a percentage of the chamber volume per minute). To assess the period of developmental failure, pregnant mice were euthanized with CO2 followed by cervical dislocation and embryos from heterozygote hybridization were collected on embryonic day (E)10.5, E11.5, E12.5 and E14.5, and genotyped using PCR as stated above. Neonates <10 days of age were euthanized with 5% isoflurane followed by decapitation with scissors. For the mouse model of endotoxemia, age, sex and weight-matched WT and Rab1a+/− mice were injected (i.p.) with 10 mg/kg LPS in PBS. The mice were monitored for up to 5 days.

Western blot analysis

Embryo lysates were prepared by incubation in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, pH 7.6, including protease inhibitors, 1 μg/ml leupeptin, 1 μg/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride), sonication, centrifugation was performed at 4°C and at 16,200 × g for 5 min to remove cell debris. Proteins in the lysates were determined by BCA and separated on 10% SDS-PAGE gels, transferred to PVDF membranes, and then examined by western blotting, as previously described (21). After blocking with 5% skimmed milk in PBS-T (PBS with 0.01% Tween-20) at room temperature for 1 h, the blots were incubated with goat anti-mouse β-actin primary antibodies (1:1,000 dilution) at 4°C overnight. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibodies (1:5,000 dilution) at room temperature for 2 h and the signal was detected using a luminol-based enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology, Inc.).

Histological analysis and immunohistochemistry

Tissues from WT or mutant mice were fixed in 4% paraformaldehyde at room temperature for 24 h, dehydrated and embedded in paraffin according to the standard procedure and as previously described (19). Sections at a thickness of 5 μm were cut, dewaxed in xylene, dehydrated in 100% ethanol and then stained with hematoxylin and eosin [H&E; hematoxylin (cat. no. SH26-500D, Fisher Chemical; Thermo Fisher Scientific, Inc.) for 3 min and eosin (cat. no. S176-16OZ, Poly Scientific R&D Corp.) for 30 sec] at room temperature or reacted with anti-mouse Rab1a antibody (1:1,000; cat. no. sc-311, Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. The sections were washed in phosphate-buffered saline (PBS) and subsequently incubated with HRP-conjugated goat anti-rabbit secondary antibodies (1:1,000; cat. no. sc-2004, Santa Cruz Biotechnology, Inc.) at room temperature for 30 min. After being washed in PBS, slides were developed with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin for 10 sec at room temperature. For the control, immunohistochemical staining was performed by omitting the primary antibody. No significant staining was observed upon control staining (data not shown). Images were acquired using an EVOS FL Auto Imaging System (Thermo Fisher Scientific, Inc.).

Measurement of inflammatory cytokine levels

Blood samples were obtained at the indicated time points and cytokines in the serum and measured using a mouse cytokine bead array designed for inflammatory cytokines (cat. no. 552364; BD Biosciences), as previously described (21-24). The kit quantitatively detects the levels of the cytokines, IL-6, IL-10, monocyte chemoattractant protein-1 (MCP-1), interferon-γ (IFN-γ), tumor necrosis factor (TNF) and the IL-12 heterodimer (IL-12p70).

Statistical analysis

The differences in cytokine concentrations were analyzed using two-tailed t-tests in single pairwise comparisons calculated with Excel (Microsoft). Data are presented as the mean ± SD. A value of P<0.05 was considered to indicate a statistically significant difference.

Results

Generation of Rab1a mutant mice

To determine the function of Rab1a in vivo, we obtained a Rab1a mutant mouse from MMRRC which was generated by the blastocyst injection of a Rab1a trapped embryonic stem cell clone (XB498, Bay Genomics). Gene disruption was caused by the insertion of the retroviral gene trap vector pGT0pfs containing a promoterless β-galactosidase reporter gene. Selection for the expression of the gene requires transcription from an endogenous cellular promoter and consequently, a mutation in a cellular gene. The expression of the tagged gene can be examined by staining for β-galactosidase. The methods used for gene trap mutagenesis have been previously reported (25-27). In the XB498 ES cell line, the gene trap vector pGT0pfs was inserted between exons 2 and 3 of Rab1a (Fig. 1 and Data S1); the point of insertion was confirmed by PCR and DNA sequencing (Fig. 2 and Data S2). Offspring were genotyped by PCR analyses using primers P1 and P2 for the knockout (KO) PCR and primers P3 and P4 for the WT PCR (Fig. 1).

Figure 2 DNA sequence of the Rab1a insertion site. The nucleotide sequence near the splice junction joining the Rab1a exon 2 splice donor to the splice acceptor of the vector is indicated. The red arrow indicates the insertion site between Rab1a genomic DNA sequence and vector DNA sequence.

Chimeric male offspring were mated with WT C57Bl/6 mice to test for the germline transmission of the disrupted Rab1a allele. Heterozygous Rab1a+/− mice were viable and displayed no obvious abnormality in weight or fertility during a 12-month observation period (data not shown). To remove contaminating background heterozygosity, Rab1a+/− mice were backcrossed >10 generations with C57BL/6 mice.

Expression of Rab1a in mice

The expression of β-galactosidase is controlled by the endogenous Rab1a gene promoter. Thus, β-galactosidase expression was used in Rab1a+/− mice to document the pattern of Rab1a expression in mouse embryos. To visualize the expression pattern of Rab1a, X-gal staining of cryosections of Rab1a+/+ and Rab1a+/− E7 embryos was performed; sections of WT embryos served as the negative controls. Rab1a was ubiquitously expressed in whole embryos (Fig. 3). To investigate endogenous Rab1a protein expression, WT E7 embryos were collected, sectioned and immunostained with anti-Rab1a antibodies. Similar to the Rab1a gene, Rab1a protein was ubiquitously expressed in the whole embryo (Fig. 3). Based on these findings, it was concluded that Rab1a protein expression was consistent with Rab1a β-galactosidase activity.

Figure 3 Expression of Rab1a in embryos. (A) X-gal staining of cryosections of Rab1a−/− KO and WT embryos. Corresponding WT sections served as negative controls for specific X-gal staining in Rab1a−/− knockout mice. (B) Immunohistochemical staining for the detection of Rab1a. Slices from WT embryos were stained with anti-Rab1a antibody and counterstained with hematoxylin. Tissue sections were stained with hematoxylin and eosin. The experiments shown in this figure were reproduced twice and representative images are shown. Scale bars: A, 2,000 μm; B, 400 μm. Higher magnification images are also shown in panel B (for higher magnification images scale bar, 200 μm). The blue arrows indicate positive staining. The red arrows indicate non-specific staining. X-gal, 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside; WT, wild-type.

In addition, the expression of Rab1a during embryo development was examined using X-gal staining. At E15 embryo, Rab1a was mainly expressed in the intestine (Fig. 4) and a small amount of Rab1a was also detected in the brain (Fig. 4), as previously reported (28,29). In adult mice, Rab1a was expressed in the small intestine (Fig. 5).

Figure 4 Expression of Rab1a in embryos at embryonic day 15. X-gal staining of cryosections of Rab1a−/− KO and WT embryos. Corresponding WT sections served as negative controls for specific X-gal staining in Rab1a−/− KO mice. Higher magnification images are also shown. The experiments in this figure were reproduced twice and representative images are shown. Scale bar, 500 μm; scale bar in higher magnification images, 400 μm. X-gal, 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside; WT, wild-type; KO, knockout. The blue arrow indicates intestinal staining. The red arrow indicates brain staining.

Figure 5 Expression of Rab1a in the small intestine of adult mice. X-gal staining of cryosections from the small intestine of Rab1a−/− KO and WT mice. Corresponding WT sections served as negative controls for specific X-gal staining in Rab1a−/− KO mice. Higher magnification images are also shown. Experiments in this figure were reproduced twice and representative images are shown (bottom panel). Scale bar, 500 μm; scale bar in higher magnification images, 100 μm. X-gal, 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside; WT, wild-type; KO, knockout.

Rab1a deficiency causes embryonic lethality

To generate Rab1a−/− mice, heterozygous Rab1a+/− mice were intercrossed. The genotypes of the offspring were identified at 2 weeks after birth. None of the 250 offspring were homozygous mutants (Rab1a−/−) (total, 250; Rab1a+/+, 94; Rab1a+/−, 156; Rab1a−/−, 0), and no increase in neonatal mortality was observed in the initial 2 weeks after birth. The ratio between the WT and heterozygote mice was 0.60, in accordance with Mendel's law. These results thus suggest that Rab1a is essential for embryonic development: one functional Rab1a allele is sufficient for murine embryonic development; however, a double mutant leads to embryonic lethality.

To characterize the effect of Rab1a mutation on embryonic development, timed matings (breeding we set up at 5 p.m. and the following morning the presence of a copulatory plug was examined at 7 a.m. If the presence of a copulatory plug was confirmed, this day was recorded as day 0.5) were performed between mice heterozygous for Rab1a. Embryos were collected at E12.5 and E14.5 from Rab1a+/− breeding mice and genotyped using PCR analysis with genomic DNA. No viable Rab1a−/− embryos were recovered (Fig. 6). The developmental retardation of Rab1a−/− embryos was apparent at E12.5, suggesting that embryonic lethality occurred prior to E12.5 (Fig. 6). Embryos at E10.5 and E11.5 were also collected and it was found that viable Rab1a−/− embryos were recovered at E10.5, whereas no viable Rab1a−/− embryos were recovered at E11.5; the data for viable embryos in different embryonic stages are summarized in Table I, the different numbers in the table indicate the viable embryos found in the different embryonic genotypes. Thus, Rab1a mutation-induced embryonic lethality occurred between E10.5 and E11.5.

Figure 6 Rab1a mutant results in embryonic lethality. (A) Representative images of E12.5 embryos of the indicated genotypes. (B) Polymerase chain reaction genotyping results for the embryos in panel A using the indicated allele-specific primers. (C) Representative images of embryos at E14.5 of the indicated genotypes. Nos. 1-6 correspond to the same embryos in panels A and B. Scale bar, 1.0 cm. E12.5, embryonic day 12.5; E14.5, embryonic day 14.5.

Table I Effects of Rab1a mutation on the number of viable embryos.

Table I

Effects of Rab1a mutation on the number of viable embryos.

Stage	Total	Rab1a+/+	Rab1a+/−	Rab1a−/−
E10.5	21	6	10	5
E11.5	23	9	14	0
E12.5	18	7	11	0
E14.5	15	5	10	0

[i] E, embryonic day.

Rab1a protein deficiency in Rab1a−/− KO mice

WT and mutant alleles were assessed using PCR of genomic DNA isolated from mice (Fig. 7A). Western blot analysis was also performed to test the successful disruption of the Rab1a gene in Rab1a−/− mice. E10.5 embryos were collected and genotyped using PCR. Rab1a+/+ and Rab1a−/− embryos were lysed and used for western blot analysis with anti-Rab1a antibody. As shown in Fig. 7B, Rab1a was completely absent in Rab1a−/− embryos, indicating the functional loss of Rab1a; β-actin was used as a loading control.

Figure 7 Analyses to demonstrate the deletion of the Rab1a gene. (A) Representative polymerase chain reaction results illustrating the mouse genotypes. (B) Loss of Rab1a in mice generated using the XB498 cell line. Proteins were extracted from embryonic tissues, separated by SDS/PAGE, and subjected to western blot analysis. The representative blots demonstrate the lack of Rab1a protein in embryo lysates of Rab1a−/− knockout mice in comparison to WT control mice. Actin levels were used as sample loading controls. Data are representative of two independent experiments. WT, wild-type.

One Rab1a allele is sufficient for resistance to LPS-induced sepsis

The loss of GDI2 in tumor cells alters the crosstalk between tumor cells and tumor-associated macrophages to enhance both local inflammation and tumor cell invasion and growth, resulting in inflammatory cytokine secretion by macrophages to promote metastatic growth (30). Moreover, Rab1a is required for NLRP3 inflammasome activation and inflammatory lung injury (31). Recently, the authors demonstrated that Rab1a bound to the ITIM domain of Siglec-G under normal homeostasis. By contrast, Rab1a was recruited to the ITIM domain during bacterial infection, suggesting that GDI2 and Rab1a may regulate immune response through interaction with the ITIM domain during bacterial infection (18). In the present study, to investigate whether Rab1a plays a role during bacterial infection, WT and Rab1a+/− mice were challenged with 10 mg/kg LPS and collected serum from the mice as previously described (21,22,24). As shown in Fig. 8, both WT and Rab1a+/− mice produced similar levels of inflammatory cytokines following LPS stimulation. Moreover, after 120 h, 50% (8/16, Rab1a+/+) and 56% (9/16, Rab1a+/−) of the mice did not survive; from data pooled from two independent experiments, a similar percentage of death was observed following LPS treatment (32) (data not shown); no significant differences were observed between the WT and Rab1a+/− mice as regards survival following the LPS challenge.

Figure 8 One Rab1a allele is sufficient for the resistance to LPS challenge. Serum concentrations of cytokines TNF-α, IL-6, IL-12, IL-10, MCP-1 and IFN-γ at 16 h following the lipopolysaccharide (LPS) challenge (mean ± SD, n=8) were measured by using a mouse cytokine bead array designed for inflammatory cytokines. The experiments in this figure were reproduced twice. n.s., no significant differences were found. TNF-α, tumor necrosis factor α; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; IFN-γ, interferon γ.

Discussion

The transfer of material between organelles in eukaryotic cells is predominantly mediated by vesicular transport. GTP binding proteins play key roles in the regulation of vesicular traffic at several stages of the exocytic and endocytic transport pathways. Rab GTPases are small GTP-binding proteins in the Ras superfamily. Following a vesicle fusion event, Rab is returned to its membrane of origin by GDI. GDI proteins regulate the GDP-GTP exchange reaction of Rab family members that are involved in the vesicular trafficking of molecules between cellular organelles. GDIs decrease the rate of dissociation of GDP from Rab proteins and release GDP from membrane bound Rabs (1,33).

The authors have previously demonstrated that Rab1a may regulate the immune response through interaction with the ITIM domain during bacterial infection in vitro (18). To further investigate the function of Rab1a in vivo, the present study generated mice with a trapped Rab1a gene and uncovered a novel role for Rab1a during early embryonic development in mice. None of the 250 genotyped pups from Rab1a heterozygous mating pairs exhibited the homozygous deletion of the Rab1a allele. The present study was also unable to detect any viable Rab1a null embryos after E11.5, indicating a severe early embryonic defect caused by the complete loss of Rab1a function. However, one functional Rab1a allele is sufficient for murine embryo development, as the frequency of heterozygote offspring was as predicted.

Although Rab1a interacts with the ITIM domain during bacterial infection, there was no significant difference in cytokine production and survival between the WT and Rab1a−/− KO mice after the LPS challenge. These data suggest that one Rab1a allele is sufficient to maintain function. The conditional KO strategy is a useful method which may be used to solve the problem of embryonic lethality observed in conventional gene KOs (34). Therefore, to explore the function of Rab1a in hematopoietic cells in bacterial infection and to further investigate the role of Rab1a in embryonic development, a Rab1a conditional KO mouse model is needed (34).

Rab8 is reportedly necessary for the proper localization of apical proteins and the absorption and digestion of various nutrients in the small intestine (35). Previous research has indicated that Rab11a is essential for the proper localization of apical proteins in the intestine and that the loss of Rab11 leads to the mislocalization of apical proteins in the small intestine, as demonstrated using Rab11a intestine-specific knockout (IKO) mice (36). Rab25 KO mice exhibit increased numbers of intestinal neoplasms when crossed with APCmin/+ mice (37). With the use of X-gal staining, the present study found that Rab1a was mainly expressed in the small intestine in E15 embryos (Fig. 4) and in adult mice (Fig. 5). It would be of interest to determine whether Rab1a also plays an important role in controlling the proper localization of apical proteins or the absorption and digestion of various nutrients in the small intestine. However, intestine specific Rab1a conditional KO mice are required to further investigate the function of Rab1a in the small intestine.

Although the present study demonstrates that Rab1a is essential for embryonic development and homozygotes die between E10.5 and E11.5, the mechanisms underlying the regulatory effects of Rab1a on embryonic development remain unclear. Moreover, while it was found that Rab1a was mainly expressed in the small intestine in E15 embryos and in adult mice, it is not yet clear whether Rab1a plays a critical role in the small intestine. Further experiments using Rab1a conditional KO mice are thus required to provide further insight into this matter.

Supplementary Data

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

GYC designed the experiments. YW, DY and GYC conducted the experiments. GC wrote the manuscript. YW and GYC confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

All procedures involving animals were approved by the University of Tennessee Health Science Center (UTHSC) Animal Care and Use Committee (IACUC), protocol nos. 17-117 (approved January 29, 2018) and 20-0211 (approved January 26, 2021).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

Not applicable.

Funding

The present study was supported by Grant R01AI137255 from the National Institutes of Health.

References