Isoleucyl-tRNA synthetase 1 is essential for embryogenesis in mice

Abstract

Isoleucyl-tRNA synthetase 1 (IARS1) is essential for protein synthesis. Although IARS1 mutations cause growth and metabolic disorders in humans, its role in embryonic development remains unclear. We generated Iars1 knockout (KO) mice and observed complete absence of homozygous KOs among 44 pups, indicating embryonic lethality. A χ² test confirmed significant deviation from Mendelian ratios. At E12.5, multiple resorbed implantation sites were observed, and histology at E9.5 showed embryos lacking recognizable structures. Evidence of implantation (decidual swellings with residual trophoblast and maternal vasculature) indicates post-implantation survival but failure to progress beyond early organogenesis. These results demonstrate that IARS1 is indispensable for early mouse development and provide a useful model to study the link between IARS1 deficiency and disrupted organogenesis.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that charge specific amino acids to their cognate tRNAs, thereby ensuring the fidelity of protein translation [11]. Beyond this canonical role, aaRSs also execute diverse noncanonical functions, including regulation of signal transduction, immune responses, and cellular stress adaptation [7].

Pathogenic variants in aaRS genes have been implicated in a wide spectrum of human disorders, ranging from neurodevelopmental syndromes to metabolic diseases [16, 18]. Isoleucyl-tRNA synthetase 1 (IARS1) is the cytoplasmic isoleucyl-tRNA synthetase. Pathogenic variants in IARS1 have been reported in patients presenting with intrauterine growth restriction, developmental delay, hypotonia, and infantile liver disease [13]. Functional studies in cells and animal models support the pathogenicity of IARS1 mutations by demonstrating impaired enzymatic activity, growth defects, and secondary mitochondrial abnormalities [9, 14, 15]. Our recent work using Iars1 V79L mutant mice have revealed postnatal phenotypes such as growth retardation and fatty liver [20]. However, reports on Iars1-null mice are scarce, and the requirement for IARS1 during embryogenesis remains unclear. Because embryonic development critically depends on accurate protein synthesis and metabolic regulation [17], disruption of aaRS function is expected to cause severe developmental defects [1]. Indeed, embryonic lethality has been documented for several aaRS genes, including Dars2, Fars2, and Tars1 [2, 4, 22]. To address this gap, we generated Iars1 knockout mice using CRISPR/Cas9 and assessed embryonic viability in vivo. Our findings provide direct evidence that IARS1 is indispensable for embryogenesis, establishing a foundation for elucidating the molecular pathology of IARS1-related disorders.

All animal experiments were conducted in accordance with the ARRIVE guidelines and the Act on Welfare and Management of Animals in Japan. All procedures complied with the Regulations for the Care and Use of Laboratory Animals of Kitasato University. The study protocol was approved by the Animal Care and Use Committee of Kitasato University and authorized by the President of Kitasato University (Approval No. 19-152).

A knockout allele was generated by introducing a 2-bp deletion in exon 3 of Iars1 using CRISPR/Cas9-mediated genome editing. A crRNA guide sequence targeting exon 3 of Iars1 (NCBI accession number: NM_172015.3) was designed (5′-TGTCAACGTGAAACCCGCTC-3′). Both crRNA and tracrRNA were synthesized by Fasmac (Tokyo, Japan). Recombinant Cas9 protein (60 ng/µL; New England Biolabs, Ipswich, MA, USA), crRNA (0.61 pmol/µL), tracrRNA (0.61 pmol/µL) were microinjected into fertilized C57BL/6JJcl (CLEA Japan, Tokyo, Japan) eggs. After overnight culture, 2-cell stage embryos were transferred into pseudopregnant females. Founder mice carrying the 2-bp deletion were identified by DNA sequencing using the following primers: forward 5′-CGTCCTGGATGTCTGCCATT-3′ and reverse 5′-GGTTATGCTAACAGCCACACC-3′. The product size is 524 bp. As shown in Fig. 1, the deletion (AG) occurs within exon 3 and leads to a frameshift mutation in the coding region. Comparison of wild-type and mutant genomic sequences revealed that the frameshift alters the codon usage starting at position 74. This mutation results in the replacement of the normal amino acid sequence (…VTRYAHQSGFHVDRRF…) with a short aberrant sequence (…VTRYAHQRVSR*) and introduces a premature stop codon at residue 79. Consequently, the Iars1 mutant allele produces a severely truncated protein, lacking the essential catalytic domain of IARS1. This truncation is predicted to eliminate enzymatic activity, thus establishing a null allele. The altered amino acid sequence and early termination are depicted in the lower panel of Fig. 1. Mice were maintained in an air-conditioned facility at 22 ± 2°C and 40–60% relative humidity with a 12-hr light/dark cycle, and were provided standard chow (CE-2; CLEA Japan, Tokyo, Japan) and water ad libitum.

Fig. 1.

CRISPR/Cas9 editing of the Iars1 locus generates a frameshift and premature termination. Top, schematic of the Iars1 locus with exons highlighting exon 3. Middle, genomic sequence alignment showing the wild-type and mutant alleles; a 2-bp deletion in exon 3 produces a reading-frame shift. Bottom, predicted amino-acid sequences: the wild-type IARS1 N-terminus versus the mutant translation, which shifts out of frame and terminates prematurely at residue 79 (red asterisk).

Iars1^+/– mice confirmed by DNA sequencing were intercrossed. Vaginal plugs were checked daily at 9:00 AM and the day of plug detection was designated as embryonic day 0.5 (E0.5). Embryos (with intact uteri) were collected at E9.5 and E12.5. E9.5 embryos underwent histological examination for morphological abnormalities and resorption. Newborns were genotyped at birth to assess genotype segregation.

Embryonic and uterine samples at E9.5 and E12.5 were collected and fixed in 10% neutral buffered formalin at room temperature. After fixation, tissues were processed by standard paraffin embedding, and consecutive sections 4–5 µm thick were prepared. Sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE) according to standard procedures [21]. Stained sections were examined by light microscopy, and representative images were captured.

Embryonic lethality at E9.5 was evaluated by microscopic examination of HE-stained sections. Resorbed embryos were defined as specimens lacking recognizable embryonic structures. To exclude technical artifacts or misidentification, entire conceptuses were serially sectioned and internal structures were inspected throughout; this confirmed that the identified sites represented embryos undergoing resorption. E9.5 corresponds to a critical window of organogenesis—encompassing neural tube closure, cardiac morphogenesis, and somite segmentation—which provides reliable morphological landmarks of normal progression [3, 19]. Accordingly, E9.5 was selected to allow detection of both normally developing and resorbing embryos, providing a sensitive readout of early embryonic lethality in Iars1-deficient mice.

Genotyping of 44 newborns from Iars1^+/– intercrosses yielded 14 wild-type (Iars1^+/+), 30 heterozygous (Iars1^+/–), and 0 homozygous knockout (Iars1^–/–) pups. This deviated significantly from the expected Mendelian ratio of 1:2:1 (χ²=14.73, df=2, P=6.3 × 10⁻⁴). The expected number of homozygous knockout offspring was approximately 11 (44 × 0.25), and the complete absence of this genotype indicates that homozygous Iars1-null embryos do not survive to birth (Table 1).

Table 1. Genotype distribution of newborn mice from Iars1 heterozygous intercrosses.

	+/+	+/–	–/–	Total
Male	4	17	0	21
Female	10	13	0	23
Total (%)	14 (31.8)	30 (68.2)	0 (0)	44 (100)
Expected (%)	25	50	25	100

Values represent the number of pups observed. Homozygous KO mice were not recovered from the offspring. The observed distribution significantly deviated from the expected Mendelian ratio of 1:2:1 (χ²=14.73, df=2, P=6.3 × 10⁻⁴).

This finding strongly supports the conclusion that IARS1 is indispensable for embryonic viability. To investigate the timing of embryonic lethality, embryos were analyzed at E12.5 and E9.5. Macroscopic examination of the uterus revealed multiple resorption sites in the E12.5 uteri (Fig. 2a, arrowheads), whereas no remarkable differences were observed in the E9.5 uteri (Fig. 2b). These findings suggest that IARS1 deficiency leads to embryonic death before midgestation. Histological analysis at E9.5 confirmed the presence of resorbed embryos lacking identifiable structures in HE-stained sections (Fig. 3). These findings demonstrate that IARS1-deficient embryos undergo embryonic lethality by this stage. Detailed histological evaluation of resorbed conceptuses at E9.5 revealed a complete loss of recognizable embryonic structures.

Fig. 2.

Increased number of resorbed embryos at E12.5 and E9.5. Representative uterus at embryonic day 12.5 (E12.5) (a), and E9.5 (b) from a litter produced by intercrossing Iars1^+/– mice. Sites labeled N denote normal conceptuses; sites labeled R denote resorbed implantation sites. Scale bar in millimeters.

Fig. 3.

Representative histological images of embryos at embryonic day 9.5 (E9.5). Representative histological images at embryonic day 9.5 (E9.5). (a) Normally developing embryo (Em). The nt indicates the neural tube; arrows indicate somites. Trophoblast giant cells (TGCs) are labeled on the left. (b) Resorbed embryo (dashed outline) lacking distinguishable embryonic structures. The arrow indicates remnants of maternal blood vessels, and the arrowhead indicates hypertrophied trophoblast giant cells. Both images are typical examples from multiple embryos examined. Scale bars: 200 μm.

Hallmark features of normal E9.5 development—such as the neural tube, somites, optic vesicles, and limb buds—were absent. Instead, the remaining tissue appeared as disorganized cell masses lacking defined body axes or compartmentalization (Fig. 3b). Despite this embryonic regression, the uterine decidua remained hypertrophied (Fig. 2b), and remnants of trophoblastic cells and maternal vasculature were occasionally observed, confirming that implantation had occurred (≥E5.5).

These findings suggest failure to progress beyond early organogenesis. In addition, some resorbed embryos displayed morphological signs consistent with necrotic degeneration, characterized by eosinophilic debris and nuclear pyknosis. While not definitive, the presence of macrophage-like foamy cells in the surrounding decidual tissue may reflect ongoing phagocytic clearance of degenerating embryonic material.

Based on these observations, embryonic death most likely occurred between E8.5 and E9.5, followed by progressive resorption over the next 1–2 days [12]. Implantation appeared normal—as indicated by prominent decidual swellings and residual trophoblast/maternal vasculature—placing survival at least through E5.5–E6.5. By contrast, serial sections at E9.5 revealed no identifiable embryonic structures and, in several sites, histologic features compatible with advanced resorption, which accords with the documented delay between demise and overt resorption in mice [5, 8]. Taken together, these findings indicate failure to progress beyond early organogenesis and provide direct evidence that IARS1 is indispensable for mouse embryogenesis. Moreover, the inferred lethal window and morphologic progression parallel early embryonic lethality reported for other aminoacyl-tRNA synthetase gene models (e.g., Dars2, Fars2, Tars1) that used comparable criteria to assign an E8.5–E9.5 window [2, 4, 22].

Notably, while previous reports on IARS1 mutant mice have primarily focused on postnatal phenotypes such as growth retardation and fatty liver, our study establishes that complete loss of IARS1 causes embryonic lethality. This discrepancy underscores the importance of allelic dosage and mutation type in determining disease severity and onset. Furthermore, our results highlight the critical role of accurate protein translation during early development, consistent with the concept that disruption of aaRS function compromises both cellular viability and tissue morphogenesis at stages prior to midgestation [1].

The mechanistic basis of embryonic lethality in Iars1-null mice likely involves multiple cellular vulnerabilities. One plausible cause is impaired translation due to defective isoleucyl-tRNA charging, which would compromise the synthesis of essential proteins required for cell proliferation and differentiation. In support of this, other aminoacyl-tRNA synthetase (aaRS) gene knockouts also exhibit early embryonic arrest. Consistent with this notion, knockout or editing-defective models of other aaRS genes—such as Aars2 and Dars2—also exhibit early embryonic arrest associated with mitochondrial mistranslation and respiratory deficiency [6, 10]. Nevertheless, early-stage development up to gastrulation (~E7.5–8.5) appears to proceed in Iars1-null embryos, likely due to maternally inherited mRNA and protein pools, which may transiently compensate for zygotic loss. As zygotic transcription assumes control during early organogenesis, the loss of IARS1 may cause a sharp decline in translational capacity, leading to cellular stress, apoptosis, or failed tissue morphogenesis. In addition, aaRS deficiency has been shown to activate integrated stress responses and disrupt proteostasis [7, 16], which could further exacerbate embryonic vulnerability. Moreover, IARS1 deficiency has been linked to secondary mitochondrial dysfunction and abnormal lipid metabolism in patient-derived cells, raising the possibility that energy deficits or metabolic derangements contribute to embryonic demise [20]. Finally, emerging evidence suggests that cytoplasmic aaRSs, including IARS1, can function as scaffolding proteins in stress-response signaling pathways, and disruption of such noncanonical functions may further compromise embryonic viability.

Taken together, these results establish IARS1 as a critical gene for early embryonic development and support the role of protein synthesis fidelity during organogenesis. Notably, most reported pathogenic IARS1 variants in humans are hypomorphic missense mutations that retain partial enzyme function.

Homozygous or compound heterozygous null alleles have not been identified, suggesting that complete loss of IARS1 is embryonically lethal in humans as well. Our findings in mice support this hypothesis and position the Iars1-null model as a valuable tool to study the developmental consequences of IARS1 deficiency.

CONFLICT OF INTEREST

There are no conflicts of interest to declare.