Arl8b is required for lysosomal degradation of maternal proteins in the visceral yolk sac endoderm of mouse embryos

The visceral yolk sac (VYS) is a membranous sac derived from embryonic tissues and envelopes the embryo. The VYS endoderm (VYSE), which forms an apical cell layer facing the uterine fluid, plays important roles in the nutritional supply to embryos before placentation: maternal proteins are absorbed from the uterine fluid in the VYSE, degraded by lysosomes and supplied to embryos (Zohn and Sarkar, 2010). The importance of endosomal and lysosomal functions of the VYSE for early embryogenesis has been suggested using an ex vivo and in vivo studies. For example, rat whole embryos cultured with protease inhibitors showed developmental defects such as decreased embryo body size, somite numbers and neural tube volume (Daston et al., 1991; Freeman and Lloyd, 1983). Mouse mutants for genes involved in the endocytic pathways also show various developmental defects, including defective neural tube closure and growth retardation (Schwarz et al., 2002; Wallingford and Giachelli, 2014; Zheng et al., 2006). Despite these studies, the importance of the endosomal and lysosomal functions of the VYSE for early embryogenesis remains obscure, because protease inhibitors and the systemic knockout of the genes can act directly on the embryo proper to affect embryonic development. It is thus necessary to develop a novel tool to investigate the precise role(s) for endosomal and lysosomal functions of the VYSE in early embryogenesis.

Arl8, which exists in two isoforms (Arl8a and Arl8b) in mammals, is the first small GTPase that has been shown to primarily localize to lysosomes (Hofmann and Munro, 2006). Previous studies have shown that Arl8 is involved in lysosomal motility along microtubules (Guardia et al., 2016; Pu et al., 2015; Rosa-Ferreira and Munro, 2011) and lysosomal fusion with late endosomes and phagosomes (Garg et al., 2011; Nakae et al., 2010; Sasaki et al., 2013). Although these studies indicated that Arl8 is crucial for lysosomal function, the in vivo role of Arl8 in mammals remains to be determined. In the present study, we generated and analyzed Arl8b-deleted mice to more clearly elucidate the precise role(s) of Arl8 and its involvement in lysosomal function in vivo, especially in the VYSE.

To investigate the physiological function of Arl8b, we generated an Arl8b-deficient mouse using a gene-trap embryonic stem (ES) cell line, and found that these mice died either just before birth or shortly after birth. Western blotting analysis and immunofluorescence of embryonic day 8.5 (E8.5) embryos showed that Arl8b was below detectable levels in mice homozygous for the gene-trap allele (see below). In addition, Arl8b mRNA was not detected by reverse transcription (RT)-PCR analysis of E9.0 embryos (Fig. S1A). These results confirm that the gene-trap insertion resulted in a null mutation of Arl8b (hereinafter referred to as Arl8b^−/−). Arl8b^−/− embryos were smaller than Arl8b^+/+ and Arl8b^+/− (hereinafter collectively referred to as control) embryos at E10.5, as evident in their crown–rump length (Fig. 1A,B,E). Moreover, the VYS of Arl8b^−/− embryos was relatively opaque and rough compared with that of control embryos (Fig. 1C,D). The VYS consists of several cell layers: the VYSE, endothelial cells, blood cells, and mesenchyme (Fig. 2A). Several studies suggested that the VYS mediates nutrient supply from the mother to the developing embryo, thereby contributing to embryogenesis (Baron et al., 2012; Zohn and Sarkar, 2010). Because Arl8b was highly expressed and partially colocalized with lysosomal-associated membrane protein 1 (Lamp1) in the VYSE (Fig. 2B–D), we focused on the role of Arl8b in the VYS in the present study.

The Arl8b^−/− VYS was also opaque at E8.5 (similar to E10.5) compared with the control VYS (Fig. 3A,B). Histological analysis of Hematoxylin and Eosin (HE)-stained sections of E8.5 embryos revealed that the cytoplasm of the VYSE was hypertrophic in Arl8b^−/− embryos (Fig. 3C–F). The hypertrophic cytoplasm of the Arl8b^−/− VYSE was observed at E7.5, from which the VYS starts to develop and envelop the embryo proper (Fig. S1B–E). These results suggest that the defect(s) of Arl8b^−/− embryos starts from at least E7.5. Intriguingly, the VYSE of Arl8b^−/− embryos was filled with enlarged vesicular structures, most of which were stained positive for Lamp1 (Fig. 3J). Immunolocalization analysis showed that Arl8b immunoreactivity was abolished in the Arl8b^−/− VYS (Fig. 3I,J). To better characterize the enlarged vesicular structures in the Arl8b^−/− VYSE, we conducted transmission electron microscopic analysis. The control VYSE contained large vacuolar structures with low electron density beneath the apical plasma membrane (Fig. 3G, arrows). In contrast, the Arl8b^−/− VYSE had no such structures, but instead exhibited large numbers of highly electron-dense globular structures, which probably corresponded to Lamp1-positive vesicular structures (Fig. 3H; Fig. S1F,G). Taken together, these results suggest that formation of late endocytic organelles was affected in the Arl8b^−/− VYSE.

Because Arl8 is involved in endocytic/phagocytic transport to lysosomes (Garg et al., 2011; Nakae et al., 2010; Sasaki et al., 2013), we evaluated endocytosis in the Arl8b^−/− VYSE using a pulse-chase endocytosis assay with fluorescent dextrans (Aoyama et al., 2012; Kawamura et al., 2012). Isolated embryos with the VYS at E8.5 were incubated sequentially with Alexa Fluor 546- and Alexa Fluor 488-conjugated dextrans, and then incubated in the absence of the dextrans (Fig. 4A). In the VYSE of control embryos, after 60 min of incubation, endocytosed Alexa Fluor 546- and Alexa Fluor 488-conjugated dextrans were detected in vacuolar compartments, most of which contained both fluorescent dextrans, suggesting that both were trafficked to lysosomes (Fig. 4B). Similarly, fluorescent dextrans were endocytosed in the VYSE of Arl8b^−/− embryos; however, the two dextran types mostly did not colocalize, but were rather localized in distinct vesicular compartments (Fig. 4C). These results suggest that endocytic trafficking to lysosomes was impaired in the Arl8b^−/− VYSE.

The VYSE actively absorbs materials from maternal uterine fluid by endocytosis and degrades them in lysosomes to supply the breakdown products to the embryo as nutrients (Zohn and Sarkar, 2010). We thus investigated whether loss of Arl8b affects the lysosomal degradation of maternal proteins in the VYSE. We first analyzed the total protein profiles of E8.5 VYS lysates by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining and found that band intensities of some proteins were increased in the Arl8b^−/− VYS (Fig. 4D). A prominent band of ∼70 kDa (arrowhead, Fig. 4D) was identified as albumin by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. The increased albumin level in Arl8b^−/− VYS lysates compared with control VYS lysates was confirmed by western blot analysis (Fig. 4E). We next analyzed levels of immunoglobulin G (IgG), which together with albumin is a representative uterine fluid protein (Tunon et al., 1998). IgG levels were also increased in the Arl8b^−/− VYS lysates compared with the control VYS lysates (Fig. 4E). Immunofluorescence staining revealed that IgG localized to Lamp1-positive vesicles in the control and Arl8b^−/− VYSE at E8.5 (Fig. 4F,G). Given that neither albumin nor IgG are produced in E8.5 embryos (Jochheim et al., 2004; Raff et al., 1976) and that endocytic traffic to lysosomes was impaired in the Arl8b^−/− VYSE (Fig. 4C), these results suggest that endocytosed maternal albumin and IgG accumulated in late endocytic organelles and failed to be degraded in lysosomes in the Arl8b^−/− VYSE.

Cathepsin L, together with cathepsin B, plays central roles in proteolysis in lysosomes in the VYSE (Daston et al., 1991). Cathepsin L is synthesized as precursor (of ∼38 kDa), converted to intermediate form (of ∼28 kDa) in late endosomes and subsequently cleaved into its mature form (comprising polypeptides of ∼21 kDa and 7 kDa linked by disulfide bonds) in lysosomes (Collette et al., 2004; Ishidoh and Kominami, 2002). The processing of cathepsin L from the intermediate to the mature form is retarded by inhibiting transport from late endosomes to lysosomes (Punnonen et al., 1994). In addition, cathepsin-L-deficient mouse embryos display accumulation of Lamp1-positive vesicles (Tholen et al., 2014), as observed in the Arl8b^−/− VYSE. We therefore analyzed the effect of loss of Arl8b on the amount and maturation stage of cathepsin L and found that levels of the mature form of cathepsin L were decreased in Arl8b^−/−VYS lysates compared with control (Fig. 4E). These results suggest that cathepsin L maturation was impaired due to defective endocytic traffic to lysosomes in the Arl8b^−/− VYSE.

Following degradation of maternal proteins by lysosomes in the VYSE, the breakdown products are transported from the VYSE to the embryo to be reused for protein synthesis (Freeman and Lloyd, 1983). In rat embryos, inhibition of this process by protease inhibitors leads to abnormal development, such as decreased crown–rump length (Daston et al., 1991). We thus speculated that the defects in lysosomal degradation in the VYSE may be responsible for the decreased crown–rump length in Arl8b^−/− embryos. To test this hypothesis, we generated an Arl8b floxed mouse line (Fig. 5A,B) and crossed it with Transthyretin (Ttr)-Cre mice expressing Cre recombinase specifically in the VYSE during early embryogenesis (Kwon and Hadjantonakis, 2009; Kwon et al., 2008). We crossed male mice of Ttr-Cre;Arl8b^flox/+ with female mice of Arl8b^flox/flox to obtain Ttr-Cre;Arl8b^flox/flox embryos, which showed abolished Arl8b immunoreactivity in the VYSE (Fig. 5C,D). E10.5 embryos of the resulting Ttr-Cre;Arl8b^flox/flox mice were significantly smaller than littermate controls (Fig. 6A,B,E) and exhibited opaque VYS (Fig. 6C,D). At E8.5, Ttr-Cre;Arl8b^flox/flox exhibited opaque VYS and hypertrophic VYSE (Fig. 6F–I), and accumulation of Lamp1-positive vesicles (Fig. 6J,K) similar to those observed in Arl8b^−/− embryos. Accumulation of albumin and IgG was also observed in the Ttr-Cre;Arl8b^flox/flox VYS (Fig. 6L), and IgG was detected in Lamp1-positive vesicles (Fig. 6J,K). Furthermore, levels of the mature form of cathepsin L were decreased in the Ttr-Cre;Arl8b^flox/flox VYS compared with the control VYS (Fig. 6L). These results suggest that loss of Arl8b in the VYSE causes embryonic growth defects presumably due to the defective lysosomal degradation of maternal proteins.

Because the breakdown products of maternal proteins in the VYSE are supplied to the embryo as nutrition, we hypothesized that defective maternal protein degradation in VYSE leads to reduced amounts of amino acids in the embryo proper and delayed growth. To test this hypothesis, we conducted amino acid quantification by liquid chromatography (LC)- and ion chromatography (IC)-MS analyses at E9.5 and found that the amounts of some amino acids in the Ttr-Cre;Arl8b^flox/flox embryo proper were significantly decreased compared with control embryos (Fig. 7). In particular, in the embryo proper, most essential amino acids (EAAs) were decreased, whereas glycine and serine, which can be produced from glucose via glycolysis, were not affected. Central metabolic pathways including glycolysis and tricarboxylic acid (TCA)-cycle metabolites were not impaired in the embryo proper (Fig. S3 and S4). By contrast, almost all amino acids as well as the metabolites of the central metabolic pathways were largely decreased in the VYS (Figs S2, S5 and S6). Collectively, these results suggest that the defective lysosomal degradation in the Ttr-Cre;Arl8b^flox/flox VYSE causes reduction of the amino acid pool in the embryo proper, which may lead to embryonic growth defects.

This study demonstrates that Arl8b is important for mouse early embryogenesis. Systemic knockout of Arl8b resulted in embryonic growth defects and impaired lysosomal degradation of maternal proteins such as albumin in the VYSE. Ttr-Cre;Arl8b^flox/flox mice, in which Arl8b was deleted specifically in the VYSE, also showed these phenotypes. Furthermore, metabolomic analysis of Ttr-Cre;Arl8b^flox/flox embryos revealed reduction of the free amino acid pool in the embryos. Considering a role for the VYSE in the uptake and transport of nutrients to the developing embryo (Zohn and Sarkar, 2010), these results suggest that Arl8b mediates efficient lysosomal degradation of maternal proteins to provide nutrition to the embryo, thereby contributing to normal embryonic growth (Fig. 8).

The importance of the VYSE in nutrition uptake and delivery during mammalian embryogenesis has been proposed in a variety of experiments, mainly in rodents. Disruption of the VYSE functions by Trypan Blue, which is incorporated into and accumulates in the VYSE when injected into pregnant rats, causes several developmental defects, including neural tube defects (Lloyd, 1997). Ex vivo culture of rat whole embryos in the presence of protease inhibitors leads to abnormal development (Daston et al., 1991; Freeman and Lloyd, 1983). Consistent with these studies, our findings support the idea that lysosomal function in the VYSE is important for mouse embryonic development, especially for embryo body size. In addition, using Ttr-Cre;Arl8b^flox/flox mice, we have directly shown that lysosomal dysfunction in the VYSE causes reduction of the amino acid pool in the embryo proper. To the best of our knowledge, this is the first report providing empirical evidence for the influence of specific inhibition of lysosomal function in the VYSE on the metabolite levels of the embryo in vivo. The Ttr-Cre;Arl8b^flox/flox mouse would thus serve as a useful model system to further study the roles of the lysosomal functions of the VYSE in mouse embryonic development.

For normal embryonic growth, the embryo must fulfil the metabolic demands needed to support massive cell proliferation by acquiring all components required for doubling cellular mass from extracellular or endogenously synthesized sources. In rat embryos, the main source of amino acids used for de novo protein synthesis in the embryo proper are derived from the digestion of maternal proteins in the VYS (Beckman et al., 1997). Thus, it is likely that the defects in lysosomal degradation of maternal proteins in the VYSE of Arl8b mutants result in a decreased amino acid pool (especially the EAA pool) in the embryo proper, which may cause reduction of protein synthesis and the small body size phenotype. mTORC1 (the mammalian target of rapamycin complex 1) is a key regulator of cell growth and is activated by amino acids (Saxton and Sabatini, 2017). Although our metabolomic analysis revealed the decrease in the free amino acid pool in the Ttr-Cre;Arl8b^flox/flox embryo proper, the phosphorylation level of S6K (pS6K, an indicator of mTORC1 activation) in the mutant embryo proper was comparable to that in control (not shown). Thus, reduction of the free amino acid pool, even if it does not lead to decrease in pS6K levels, could impair the embryonic growth.

In addition to the reduced amino acid levels, other factor(s) may contribute to the small body size of Arl8b mutant embryos. Our metabolomic analysis revealed that various metabolites of central metabolic pathways were decreased in the Ttr-Cre;Arl8b^flox/flox VYSE. A recent study also demonstrated that rat embryos cultured with protease inhibitors showed a reduction of glutathione levels in the VYSE (Harris et al., 2015). Thus, the functional integrity of the VYSE may be impaired when lysosomal function is defective, thereby failing to support the embryonic growth via unknown mechanisms. Further studies are required to elucidate the molecular mechanism underlying the growth control of the embryo via the VYSE, and Ttr-Cre;Arl8b^flox/flox mice may provide a useful tool for these analyses.

It should be noted that while most of Arl8b^−/− mice died around birth, substantial numbers of viable postnatal Ttr-Cre;Arl8b^flox/flox mice were obtained (not shown). Arl8b^−/− embryos also showed abnormal brain development: the mutant embryos were defective in the development of the neural ridge, which gives rise to the roof plate, and showed an abnormal pattern of apoptosis in the roof plate (Hashimoto K.H., Y.Y., Y. Kishi, Y. Kikko, K. Takasaki, Y. Maeda, Y. Matsumoto, M.O., M.M., S.O., T.K, K.K., unpublished observations). In contrast, Ttr-Cre;Arl8b^flox/flox embryos did not show these phenotypes observed in Arl8b^−/− embryos (not shown). These results suggest that loss of Arl8b in the VYSE is not an exclusive cause of lethality of Arl8b^−/− mice, and that Arl8b plays important roles not only in the VYSE but also in the embryo proper for normal development.

Consistent with previous studies using cultured mammalian cells and Caenorhabditis elegans (Garg et al., 2011; Nakae et al., 2010), our results indicate that Arl8b is required for endocytic traffic to lysosomes. How Arl8b functions in the endocytic transport of maternal proteins to lysosomes in the VYSE remains to be determined. Previous studies showed that Arl8b interacts with Vps41 (Garg et al., 2011; Khatter et al., 2015; Sasaki et al., 2013). Vps41 is a component of the homotypic fusion and vacuole protein sorting complex (HOPS complex), which acts as a tethering factor for vacuole and lysosome membrane fusion. Although Vps41/mVam2-deleted mouse embryos exhibit developmental arrest at E7.0, before VYS formation, E6.0 mutant embryos display abnormally fragmented late endosomal compartments in the visceral endoderm (VE), a subpopulation of which differentiates into the VYSE (Aoyama et al., 2012). Additionally, the late stages of the endocytic pathway are defective in the VE of the mutant embryos (Aoyama et al., 2012). Arl8b may thus interact with Vps41/mVam2 to facilitate endocytic traffic to lysosomes in the VYSE. Alternatively, given that Arl8b can interact with the kinesin adaptor SKIP/PLEKHM2 (Rosa-Ferreira and Munro, 2011) and also with kinesin-3 KIF1A (Niwa et al., 2016; Wu et al., 2013), Arl8b may facilitate endocytic transport to lysosomes via controlling kinesin-dependent lysosomal motility. Future analysis is required to clarify the functional role of Arl8b in endocytic traffic to lysosomes in the VYSE.

In summary, our findings highlight the physiological importance of Arl8b in mouse embryogenesis. Lysosomes play important roles not only in metabolism but also in controlling the activity of various signaling pathways. Little is known of how lysosomal function is regulated to meet the constantly changing cellular demands that occur during development or under various other physiological conditions. Further studies using mice with Arl8b deleted in a spatiotemporally controlled manner could reveal the physiological importance of lysosomal functions in different cells and tissues during development and homeostasis.

All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Tokyo, Japan. Arl8b gene-trap mice were generated using a gene-trap ES cell line (MMRRC, SIGTR ES cell line AK0793) (Saitoh et al., in press). These mice were backcrossed at least 10 times with C57BL/6J mice. The pEZ FrtLox DT vector (kindly provided by Dr Klaus Rajewsky) was used for construction of a conditional knockout targeting vector. Arl8b genomic fragments were amplified by PCR using pBAC clone RP23-404C7 (CHORI, Oakland, CA, USA) as template DNA. The primers used for the amplification are listed in Table S1. The PCR products of these amplifications were inserted at XhoI, SalI and NotI sites in pEZ FrtLox DT. The targeting vector was linearized with NotI and introduced into JM8A1.N3 ES cells by electroporation for homologous recombination. Ttr-Cre transgenic mice were generously provided by Dr Anna-Katerina Hadjantonakis (Kwon and Hadjantonakis, 2009; Kwon et al., 2008).

Total RNAs from E9.0 embryos were obtained using RNAiso Plus (TaKaRa, Shiga, Japan) and reverse-transcribed into cDNAs using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan). These cDNAs were amplified by PCR using primers listed in Table S2.

The following primary antibodies were used: anti-Lamp1 (1D4B, the Developmental Studies Hybridoma Bank, Iowa City, IA, USA), anti-albumin (ab106582, Abcam, Cambridge, UK), anti-cathepsin L (AF1515, R&D, Minneapolis, MN, USA), anti-actin (MAB1501, Merck Millipore, Billerica, MA, USA) and anti-Gapdh (MAB374, Merck Millipore). Anti-Arl8 antibody was purified from whole rabbit serum as described previously (Okai et al., 2004). Secondary antibodies were from Thermo Fisher Scientific (Waltham, MA, USA; Alexa Fluor 488- and 568-conjugated donkey-anti-mouse IgG, goat anti-rabbit IgG and rabbit anti-goat IgG) and Jackson ImmunoResearch (West Grove, PA, USA; Alexa Fluor 647-conjugated donkey-anti-Rat IgG). POD-conjugated secondary antibodies were from Jackson ImmunoResearch (goat anti-mouse IgG, donkey anti-goat IgG), Merck Millipore (goat anti-rabbit IgG) and Thermo Fisher Scientific (rabbit anti-chicken IgY). Nuclei were stained using Hoechst (Thermo Fisher Scientific) and DRAQ5 (Biostatus, Loughborough, UK). The working dilutions of the antibodies are listed in Table S3.

Embryos were dissected in ice-cold phosphate-buffered saline (PBS) and immediately fixed in 4% paraformaldehyde (PFA)/PBS for 2 h at 4°C. Samples were then soaked in 20% sucrose/PBS overnight at 4°C, washed in PBS, immersed in optimal cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan), frozen in liquid N₂ and cut into 8 μm sections. For immunofluorescence staining, the sections were blocked with 3% BSA/PBS containing 0.1% Tween 20 at room temperature for 30 min. Incubations with primary antibodies and then secondary antibodies were performed overnight at 4°C. For HE staining, the sections were stained with Mayer's Hematoxylin solution (Wako, Osaka, Japan) and Eosin Y (Nakalai Tesque, Kyoto, Japan). For transmission electron microscopy, E8.5 embryos were fixed in 4% PFA and 2% glutaraldehyde in 0.1 M phosphate buffer overnight at 4°C and post-fixed with 2% osmium tetroxide in 0.1 M phosphate buffer for 2 h. The samples were dehydrated in graded ethanol solutions, infiltrated with propylene oxide, embedded in resin (Quetol-812; Nisshin EM Co., Tokyo, Japan) and cut into 70 nm ultra-thin sections.

VYS was lysed in radioimmunoprecipitation buffer containing pepstatin A (10 μg ml^–1, Nakalai Tesque), leupeptin (10 μg ml^–1, Peptide Institute, Osaka, Japan), NaF (1 mM) and Na₃VO₄ (1 mM). The extracts were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After blocking with 5% skimmed milk/Tris-buffered saline containing 0.2% Tween 20, the membranes were probed with antibodies and the bound antibodies were detected using Immobilon Western Chemiluminescent HRP substrate (Merck Millipore).

An endocytosis assay using fluorescent dextrans was performed as described previously (Kawamura et al., 2012) with minor modifications. Embryos were dissected in Tyrode's buffer and cultured in Dulbecco's modified Eagle medium supplemented with 50% rat serum (Charles River, Wilmington, MA, USA) under 5% CO₂ and 95% air at 37°C for 15 min. The embryos were incubated with Alexa Fluor 546-conjugated dextran (0.5 mg ml^–1; Thermo Fisher Scientific, D22911) for 15 min and chased for 15 min. Samples were then pulse labeled with Alexa Fluor 488-conjugated dextran (0.5 mg ml^–1, Thermo Fisher Scientific, D22910) for 15 min, chased for 60 min and fixed in 4% PFA/PBS overnight.

Fluorescent images were obtained using confocal laser scanning microscopes (SP5; Leica, Wetzlar, Germany; and LSM700; Zeiss, Oberkochen, Germany), and transmission electron microscopic images were acquired using a JEM-1400Plus (JEOL Ltd) at an acceleration voltage of 80 kV.

Using ImageJ software (NIH), a threshold for electron density was set so that the electron signal intensity of the cytoplasm was under the threshold. Vesicles were classified into three groups on the basis of their electron densities: vesicles with suprathreshold intensity across their whole area were classified as ‘high-density’, those with subthreshold intensity across their whole area as ‘low density’, and others as ‘middle density’.

Metabolite extraction from embryos for metabolome analyses was performed as described previously (Miyazawa et al., 2017). Briefly, one frozen E9.5 embryo (21–24-somite stage) together with internal standard (IS) compounds (see below) was homogenized in ice-cold methanol (500 μl) using a manual homogenizer [Finger Masher (AM79330), Sarstedt, Nümbrecht, Germany], followed by the addition of an equal volume of chloroform and 0.4 times the volume of ultrapure water (LC/MS grade, Wako). The suspension was then centrifuged at 15,000 g for 15 min at 4°C. After centrifugation, the aqueous phase was ultrafiltered using an ultrafiltration tube (Ultrafree MC-PLHCC, Human Metabolome Technologies, Yamagata, Japan). The filtrate was concentrated with a vacuum concentrator (SpeedVac, Thermo Fisher Scientific). The concentrated filtrate was dissolved in 50 μl of ultrapure water and used for LC-MS/MS and IC-MS analyses.

We used 2-morpholinoethanesulfonic acid and 1,3,5-benzenetricarboxylic acid (trimesate) as ISs for anionic metabolites. These compounds are not present in the tissues, thus they serve as ideal standards. Loss of endogenous metabolites during sample preparation was corrected by calculating the recovery rate (%) for each sample measurement.

For metabolome analysis focused on glucose metabolic central pathways, namely glycolysis, TCA cycle and pentose phosphate pathway, the anionic metabolites were measured using an orbitrap-type MS (Q-Exactive Focus, Thermo Fisher Scientific) connected to an high-performance IC system (ICS-5000+, Thermo Fisher Scientific) that enables highly selective and sensitive metabolite quantification owing to the IC separation and Fourier Transfer MS principle (Hu et al., 2015). The IC was equipped with an anion electrolytic suppressor (Thermo Scientific Dionex AERS 500) to convert the potassium hydroxide gradient into pure water before the sample enters the mass spectrometer. The separation was performed using a Thermo Scientific Dionex IonPac AS11-HC, 4 μm particle size column. The IC flow rate was 0.25 ml min^–1 supplemented post-column with 0.18 ml min^–1 makeup flow of MeOH. The potassium hydroxide gradient conditions for IC separation were as follows: from 1 mM to 100 mM (0–40 min), 100 mM (40–50 min), and 1 mM (50.1–60 min), at a column temperature of 30°C. The Q-Exactive Focus mass spectrometer was operated under an electrospray ionization (ESI)-negative mode for all detections. Full mass scan (m/z 70−900) was used at a resolution of 70,000. The automatic gain control target was set at 3×10⁶ ions, and maximum ion injection time was 100 ms. Source ionization parameters were optimized with the spray voltage at 3 kV and other parameters were as follows: transfer temperature at 320°C, S-Lens level at 50, heater temperature at 300°C, Sheath gas at 36, and Aux gas at 10.

The amount of amino acids in the embryonic tissues was quantified using LC-MS/MS. Briefly, a triple-quadrupole mass spectrometer equipped with an ESI ion source (LCMS-8040, Shimadzu Corporation, Kyoto, Japan) was used in the positive- and negative-ESI and multiple reaction monitoring (MRM) modes. The samples were resolved on a Discovery HS F5-3 column [2.1 mm internal diameter, 150 mm long, 3 μm particle size, Sigma-Aldrich], using a step gradient with mobile phase A (0.1% formate) and mobile phase B (0.1% acetonitrile) at ratios of 100:0 (0–5 min), 75:25 (5–11 min), 65:35 (11–15 min), 5:95 (15–20 min) and 100:0 (20–25 min), at a flow rate of 0.25 ml min^–1 and a column temperature of 40°C. MRM conditions for each amino acid are listed in Table S4.

The data of the relative Arl8b protein levels were analyzed by two-tailed t test. The data of crown–rump length and amino acid concentrations were analyzed using one-way analysis of variance with Tukey's post hoc test. Relative Arl8b protein levels were analyzed by two-tailed t test. A value of P<0.05 was considered significant.

We thank Dr Anna-Katerina Hadjantonakis for generously providing the Ttr-Cre transgenic mice; Dr Yoshiteru Sasaki for providing the pEZ FrtLox DT vector; Dr G. Sun-Wada and Dr Y. Wada for technical advice and useful suggestions; and the members of M.M., T.K. and K.K.'s laboratories for useful discussions. The monoclonal antibody (1D4B) developed by J. T. August, was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242, USA.