Morphomics via next-generation electron microscopy.

The living body is composed of innumerable fine and complex structures. Although these structures have been studied in the past, a vast amount of information pertaining to them still remains unknown. When attempting to observe these ultra-structures, the use of electron microscopy (EM) has become indispensable. However, conventional EM settings are limited to a narrow tissue area, which can bias observations. Recently, new trends in EM research have emerged, enabling coverage of far broader, nano-scale fields of view for two-dimensional wide areas and three-dimensional large volumes. Moreover, cutting-edge bioimage informatics conducted via deep learning has accelerated the quantification of complex morphological bioimages. Taken together, these technological and analytical advances have led to the comprehensive acquisition and quantification of cellular morphology, which now arises as a new omics science termed 'morphomics'.

Heterozygous loss-of-function DHX9 variants are associated with neurodevelopmental disorders: Human genetic and experimental evidences.

DExH-box helicases are involved in unwinding of RNA and DNA. Among the 16 DExH-box genes, monoallelic variants of DHX16, DHX30, DHX34, and DHX37 are known to be associated with neurodevelopmental disorders. In particular, DHX30 is well established as a causative gene for neurodevelopmental disorders. Germline variants of DHX9, the closest homolog of DHX30, have not been reported until now as being associated with congenital disorders in humans, except that one de novo heterozygous variant, p.(Arg1052Gln) of the gene was identified during comprehensive screening in a patient with autism; unfortunately, the phenotypic details of this individual are unknown. Herein, we report a patients with a heterozygous de novo missense variant, p.(Gly414Arg) of DHX9 who presented with a short stature, intellectual disability, and ventricular non-compaction cardiomyopathy. The variant was located in the glycine codon of the ATP-binding site, G-C-G-K-T. To assess the pathogenicity of these variants, we generated transgenic Drosophila lines expressing human wild-type and mutant DHX9 proteins: 1) the mutant proteins showed aberrant localization both in the nucleus and the cytoplasm; 2) ectopic expression of wild-type protein in the visual system led to the rough eye phenotype, whereas expression of the mutant proteins had minimal effect; 3) overexpression of the wild-type protein in the retina led to a reduction in axonal numbers, whereas expression of the mutant proteins had a less pronounced effect. Furthermore, in a gene-editing experiment of Dhx9 G416 to R416, corresponding to p.(Gly414Arg) in humans, heterozygous mice showed a reduced body size, reduced emotionality, and cardiac conduction abnormality. In conclusion, we established that heterozygosity for a loss-of-function variant of DHX9 can lead to a new neurodevelopmental disorder.

Comprehensive ECG reference intervals in C57BL/6N substrains provide a generalizable guide for cardiac electrophysiology studies in mice.

Reference ranges provide a powerful tool for diagnostic decision-making in clinical medicine and are enormously valuable for understanding normality in pre-clinical scientific research that uses in vivo models. As yet, there are no published reference ranges for electrocardiography (ECG) in the laboratory mouse. The first mouse-specific reference ranges for the assessment of electrical conduction are reported herein generated from an ECG dataset of unprecedented scale. International Mouse Phenotyping Consortium data from over 26,000 conscious or anesthetized C57BL/6N wildtype control mice were stratified by sex and age to develop robust ECG reference ranges. Interesting findings include that heart rate and key elements from the ECG waveform (RR-, PR-, ST-, QT-interval, QT corrected, and QRS complex) demonstrate minimal sexual dimorphism. As expected, anesthesia induces a decrease in heart rate and was shown for both inhalation (isoflurane) and injectable (tribromoethanol) anesthesia. In the absence of pharmacological, environmental, or genetic challenges, we did not observe major age-related ECG changes in C57BL/6N-inbred mice as the differences in the reference ranges of 12-week-old compared to 62-week-old mice were negligible. The generalizability of the C57BL/6N substrain reference ranges was demonstrated by comparison with ECG data from a wide range of non-IMPC studies. The close overlap in data from a wide range of mouse strains suggests that the C57BL/6N-based reference ranges can be used as a robust and comprehensive indicator of normality. We report a unique ECG reference resource of fundamental importance for any experimental study of cardiac function in mice.

A male-killing gene encoded by a symbiotic virus of Drosophila.

In most eukaryotes, biparentally inherited nuclear genomes and maternally inherited cytoplasmic genomes have different evolutionary interests. Strongly female-biased sex ratios that are repeatedly observed in various arthropods often result from the male-specific lethality (male-killing) induced by maternally inherited symbiotic bacteria such as Spiroplasma and Wolbachia. However, despite some plausible case reports wherein viruses are raised as male-killers, it is not well understood how viruses, having much smaller genomes than bacteria, are capable of inducing male-killing. Here we show that a maternally inherited double-stranded RNA (dsRNA) virus belonging to the family Partitiviridae (designated DbMKPV1) induces male-killing in Drosophila. DbMKPV1 localizes in the cytoplasm and possesses only four genes, i.e., one gene in each of the four genomic segments (dsRNA1-dsRNA4), in contrast to ca. 1000 or more genes possessed by Spiroplasma or Wolbachia. We also show that a protein (designated PVMKp1; 330 amino acids in size), encoded by a gene on the dsRNA4 segment, is necessary and sufficient for inducing male-killing. Our results imply that male-killing genes can be easily acquired by symbiotic viruses through reassortment and that symbiotic viruses are hidden players in arthropod evolution. We anticipate that host-manipulating genes possessed by symbiotic viruses can be utilized for controlling arthropods.

Tertiary lymphoid tissues: a regional hub for kidney inflammation.

Tertiary lymphoid tissues (TLTs) are inducible ectopic lymphoid tissues that develop at sites of chronic inflammation in nonlymphoid organs. As with lymph nodes, TLTs initiate adaptive immune responses and coordinate local tissue immunity. Although virtually ignored for decades, TLTs have recently received a great deal of attention for their ability to influence disease severity, prognosis and response to therapy in various diseases, including cancer, autoimmune disorders and infections. TLTs are also induced in kidneys of patients with chronic kidney diseases such as immunoglobulin A nephropathy and lupus nephritis. Nevertheless, TLTs in the kidney have not been extensively investigated and their mechanism of development, functions and clinical relevance remain unknown, mainly because of the absence of adequate murine kidney TLT models and limited availability of human kidney samples containing TLTs. We recently found that aged kidneys, but not young kidneys, exhibit multiple TLTs after injury. Interestingly, although they are a minor component of TLTs, resident fibroblasts in the kidneys diversify into several distinct phenotypes that play crucial roles in TLT formation. Furthermore, the potential of TLTs as a novel kidney injury/inflammation marker as well as a novel therapeutic target for kidney diseases is also suggested. In this review article we describe the current understanding of TLTs with a focus on age-dependent TLTs in the kidney and discuss their potential as a novel therapeutic target and kidney inflammation marker.

Essential Roles of Exocyst Complex Component 3-like 2 on Cardiovascular Development in Mice.

Angiogenesis is a process to generate new blood vessels from pre-existing vessels and to maintain vessels, and plays critical roles in normal development and disease. However, the molecular mechanisms underlying angiogenesis are not fully understood. This study examined the roles of exocyst complex component (Exoc) 3-like 2 (Exoc3l2) during development in mice. We found that Exoc3l1, Exoc3l2, Exoc3l3 and Exoc3l4 are expressed abundantly in endothelial cells at embryonic day 8.5. The generation of Exoc3l2 knock-out (KO) mice showed that disruption of Exoc3l2 resulted in lethal in utero. Substantial numbers of Exoc3l2 KO embryos exhibited hemorrhaging. Deletion of Exoc3l2 using Tie2-Cre transgenic mice demonstrated that Exoc3l2 in hematopoietic and endothelial lineages was responsible for the phenotype. Taken together, these findings reveal that Exoc3l2 is essential for cardiovascular and brain development in mice.

SIRT7 Deficiency Protects against Aging-Associated Glucose Intolerance and Extends Lifespan in Male Mice.

Sirtuins (SIRT1-7 in mammals) are evolutionarily conserved nicotinamide adenine dinucleotide-dependent lysine deacetylases/deacylases that regulate fundamental biological processes including aging. In this study, we reveal that male Sirt7 knockout (KO) mice exhibited an extension of mean and maximum lifespan and a delay in the age-associated mortality rate. In addition, aged male Sirt7 KO mice displayed better glucose tolerance with improved insulin sensitivity compared with wild-type (WT) mice. Fibroblast growth factor 21 (FGF21) enhances insulin sensitivity and extends lifespan when it is overexpressed. Serum levels of FGF21 were markedly decreased with aging in WT mice. In contrast, this decrease was suppressed in Sirt7 KO mice, and the serum FGF21 levels of aged male Sirt7 KO mice were higher than those of WT mice. Activating transcription factor 4 (ATF4) stimulates Fgf21 transcription, and the hepatic levels of Atf4 mRNA were increased in aged male Sirt7 KO mice compared with WT mice. Our findings indicate that the loss of SIRT7 extends lifespan and improves glucose metabolism in male mice. High serum FGF21 levels might be involved in the beneficial effect of SIRT7 deficiency.

Role of Scl39a13/ZIP13 in cardiovascular homeostasis.

Zinc plays a critical role in many physiological processes, and disruption of zinc homeostasis induces various disorders, such as growth retardation, osteopenia, immune deficiency, and inflammation. However, how the imbalance in zinc homeostasis leads to heart disease is not yet fully understood. Cardiovascular diseases are a major cause of death worldwide, and the development of novel therapeutic targets to treat it is urgently needed. We report that a zinc transporter, ZIP13, regulates cardiovascular homeostasis. We found that the expression level of Zip13 mRNA was diminished in both primary neonatal cardiomyocytes and mouse heart tissues treated with the cardiotoxic agent doxycycline. Primary neonatal cardiomyocytes from Zip13 gene-knockout (KO) mice exhibited abnormal irregular arrhythmic beating. RNA-seq analysis identified 606 differentially expressed genes in Zip13-KO mouse-derived primary neonatal cardiomyocytes and Gene ontology (GO) analysis revealed that both inflammation- and cell adhesion-related genes were significantly enriched. In addition, telemetry echocardiography analysis suggested that arrhythmias were likely to occur in Zip13-KO mice, in which elevated levels of the cardiac fibrosis marker Col1a1, vascular inflammation-related gene eNOS, and Golgi-related molecule GM130 were observed. These results indicate the physiological importance of ZIP13-it maintains cardiovascular homeostasis by resolving inflammation and stress response. Our findings suggest that optimizing ZIP13 expression and/or function may improve cardiovascular disease management.

Compact laboratory-based X-ray microscope enabling nondestructive 3D structure acquisition of mouse nephron with high speed and better user accessibility.

X-ray microscopes adopting computed tomography enable nondestructive 3D visualization of biological specimens at micron-level resolution without conventional 2D serial sectioning that is a destructive/laborious method and is routinely used for analyzing renal biopsy in clinical diagnosis of kidney diseases. Here we applied a compact commercial system of laboratory-based X-ray microscope to observe a resin-embedded osmium-stained 1-mm strip of a mouse kidney piece as a model of renal biopsy, toward a more efficient diagnosis of kidney diseases. A reconstructed computed tomography image from several hours of data collection using CCD detector allowed us to unambiguously segment a single nephron connected to a renal corpuscle, which was consistent with previous reports using serial sectioning. Histogram analysis on the segmented nephron confirmed that the proximal and distal tubules were distinguishable on the basis of their X-ray opacities. A 3D rendering model of the segmented nephron visualized a convoluted structure of renal tubules neighboring the renal corpuscle and a branched structure of efferent arterioles. Furthermore, another data collection using scientific complementary metal-oxide semiconductor detector with a much shorter data acquisition time of 15 min provided similar results from the same samples. These results suggest a potential application of the compact laboratory-based X-ray microscope to analyze mouse renal biopsy.

Deficiency of protocadherin 9 leads to reduction in positive emotional behaviour.

Protocadherin 9 (Pcdh9) is a member of the cadherin superfamily and is uniquely expressed in the vestibular and limbic systems; however, its physiological role remains unclear. Here, we studied the expression of Pcdh9 in the limbic system and phenotypes of Pcdh9-knock-out mice (Pcdh9 KO mice). Pcdh9 mRNA was expressed in the fear extinction neurons that express protein phosphatase 1 regulatory subunit 1 B (Ppp1r1b) in the posterior part of the basolateral amygdala (pBLA), as well as in the Cornu Ammonis (CA) and Dentate Gyrus (DG) neurons of the hippocampus. We show that the Pcdh9 protein was often localised at synapses. Phenotypic analysis of Pcdh9 KO mice revealed no apparent morphological abnormalities in the pBLA but a decrease in the spine number of CA neurons. Further, the Pcdh9 KO mice were related to features such as the abnormal optokinetic response, less approach to novel objects, and reduced fear extinction during recovery from the fear. These results suggest that Pcdh9 is involved in eliciting positive emotional behaviours, possibly via fear extinction neurons in the pBLA and/or synaptic activity in the hippocampal neurons, and normal optokinetic eye movement in brainstem optokinetic system-related neurons.

Establishment of mouse line showing inducible priapism-like phenotypes.

Purpose: Penile research is expected to reveal new targets for treatment and prevention of the complex mechanisms of its disorder including erectile dysfunction (ED). Thus, analyses of the molecular processes of penile ED and continuous erection as priapism are essential issues of reproductive medicine. Methods: By performing mouse N-ethyl-N-nitrosourea mutagenesis and exome sequencing, we established a novel mouse line displaying protruded genitalia phenotype (PGP; priapism-like phenotype) and identified a novel Pitpna gene mutation for PGP. Extensive histological analyses on the Pitpna mutant and intracavernous pressure measurement (ICP) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS)/MS analyses were performed. Results: We evaluated the role of phospholipids during erection for the first time and showed the mutants of inducible phenotypes of priapism. Moreover, quantitative analysis using LC-ESI/MS/MS revealed that the level of phosphatidylinositol (PI) was significantly lower in the mutant penile samples. These results imply that PI may contribute to penile erection by PITPα. Conclusions: Our findings suggest that the current mutant is a mouse model for priapism and abnormalities in PI signaling pathways through PITPα may lead to priapism providing an attractive novel therapeutic target in its treatment.

Loss of the N-acetylgalactosamine side chain of the GPI-anchor impairs bone formation and brain functions and accelerates the prion disease pathology.

Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrPC, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)-galactose-sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrPC GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases.

Characterization of single nucleotide polymorphisms for a forward genetics approach using genetic crosses in C57BL/6 and BALB/c substrains of mice.

Forward genetics is a powerful approach based on chromosomal mapping of phenotypes and has successfully led to the discovery of many mouse mutations in genes responsible for various phenotypes. Although crossing between genetically remote strains can produce F2 and backcross mice for chromosomal mapping, the phenotypes are often affected by background effects from the partner strains in genetic crosses. Genetic crosses between substrains might be useful in genetic mapping to avoid genetic background effects. In this study, we investigated single nucleotide polymorphisms (SNPs) available for genetic mapping using substrains of C57BL/6 and BALB/c mice. In C57BL/6 mice, 114 SNP markers were developed and assigned to locations on all chromosomes for full utilization for genetic mapping using genetic crosses between the C57BL/6J and C57BL/6N substrains. Moreover, genetic differences were identified in the 114 SNP markers among the seven C57BL/6 substrains from five production breeders. In addition, 106 SNPs were detected on all chromosomes of BALB/cAJcl and BALB/cByJJcl substrains. These SNPs could be used for genotyping in BALB/cJ, BALB/cAJcl, BALB/cAnNCrlCrlj, and BALB/cCrSlc mice, and they are particularly useful for genetic mapping using crosses between BALB/cByJJcl and other BALB/c substrains. The SNPs characterized in this study can be utilized for genetic mapping to identify the causative mutations of the phenotypes induced by N-ethyl-N-nitrosourea mutagenesis and the SNPs responsible for phenotypic differences between the substrains of C57BL/6 and BALB/c mice.

Mouse resources at the RIKEN BioResource Research Center and the National BioResource Project core facility in Japan.

The RIKEN BioResource Research Center (BRC) was established in 2001 as a comprehensive biological resource center in Japan. The Experimental Animal Division, one of the BRC infrastructure divisions, has been designated as the core facility for mouse resources within the National BioResource Project (NBRP) by the Japanese government since FY2002. Our activities regarding the collection, preservation, quality control, and distribution of mouse resources have been supported by the research community, including evaluations and guidance on advancing social and research needs, as well as the operations and future direction of the BRC. Expenditure for collection, preservation, and quality-control operations of the BRC, as a national core facility, has been funded by the government, while distribution has been separately funded by users' reimbursement fees. We have collected over 9000 strains created mainly by Japanese scientists including Nobel laureates and researchers in cutting-edge fields and distributed mice to 7000 scientists with 1500 organizations in Japan and globally. Our users have published 1000 outstanding papers and a few dozen patents. The collected mouse resources are accessible via the RIKEN BRC website, with a revised version of the searchable online catalog. In addition, to enhance the visibility of useful strains, we have launched web corners designated as the "Mouse of the Month" and "Today's Tool and Model." Only high-demand strains are maintained in live colonies, while other strains are cryopreserved as embryos or sperm to achieve cost-effective management. Since 2007, the RIKEN BRC has built up a back-up facility in the RIKEN Harima branch to protect the deposited strains from disasters. Our mice have been distributed with high quality through the application of strict microbial and genetic quality control programs that cover a globally accepted pathogens list and mutated alleles generated by various methods. Added value features, such as information about users' publications, standardized phenotyping data, and genome sequences of the collected strains, are important to facilitate the use of our resources. We have added and disseminated such information in collaboration with the NBRP Information Center and the NBRP Genome Information Upgrading Program. The RIKEN BRC has participated in international mouse resource networks such as the International Mouse Strain Resource, International Mouse Phenotyping Consortium, and Asian Mouse Mutagenesis and Resource Association to facilitate the worldwide use of high-quality mouse resources, and as a consequence it contributes to reproducible life science studies and innovation around the globe.

Effects of Pparγ1 deletion on late-stage murine embryogenesis and cells that undergo endocycle.

Peroxisome proliferator-activated receptor (PPAR) γ1, a nuclear receptor, is abundant in the murine placenta during the late stage of pregnancy (E15-E16), although its functional roles remain unclear. PPARγ1 is encoded by two splicing isoforms, namely Pparγ1canonical and Pparγ1sv, and its embryonic loss leads to early (E10) embryonic lethality. Thus, we generated knockout (KO) mice that carried only one of the isoforms to obtain a milder phenotype. Pparγ1sv-KO mice were viable and fertile, whereas Pparγ1canonical-KO mice failed to recover around the weaning age. Pparγ1canonical-KO embryos developed normally up to 15.5 dpc, followed by growth delays after that. The junctional zone of Pparγ1canonical-KO placentas severely infiltrated the labyrinth, and maternal blood sinuses were dilated. In the wild-type, PPARγ1 was highly expressed in sinusoidal trophoblast giant cells (S-TGCs), peaking at 15.5 dpc. Pparγ1canonical-KO abolished PPARγ1 expression in S-TGCs. Notably, the S-TGCs had unusually enlarged nuclei and often occupied maternal vascular spaces, disturbing the organization of the fine labyrinth structure. Gene expression analyses of Pparγ1canonical-KO placentas indicated enhanced S-phase cell cycle signatures. EdU-positive S-TGCs in Pparγ1canonical-KO placentas were greater in number than those in wild-type placentas, suggesting that the cells continued to endoreplicate in the mutant placentas. These results indicate that PPARγ1, a known cell cycle arrest mediator, is involved in the transition of TGCs undergoing endocycling to the terminal differentiation stage in the placentas. Therefore, PPARγ1 deficiency, induced through genetic manipulation, leads to placental insufficiency.

Targeting oxytocin receptor (Oxtr)-expressing neurons in the lateral septum to restore social novelty in autism spectrum disorder mouse models.

Autism spectrum disorder (ASD) is a continuum of neurodevelopmental disorders and needs new therapeutic approaches. Recently, oxytocin (OXT) showed potential as the first anti-ASD drug. Many reports have described the efficacy of intranasal OXT therapy to improve the core symptoms of patients with ASD; however, the underlying neurobiological mechanism remains unknown. The OXT/oxytocin receptor (OXTR) system, through the lateral septum (LS), contributes to social behavior, which is disrupted in ASD. Therefore, we selectively express hM3Dq in OXTR-expressing (OXTR+) neurons in the LS to investigate this effect in ASD mouse models developed by environmental and genetic cues. In mice that received valproic acid (environmental cue), we demonstrated successful recovery of impaired social memory with three-chamber test after OXTR+ neuron activation in the LS. Application of a similar strategy to Nl3R451C knock-in mice (genetic cue) also caused successful recovery of impaired social memory in single field test. OXTR+ neurons in the LS, which are activated by social stimuli, are projected to the CA1 region of the hippocampus. This study identified a candidate mechanism for improving core symptoms of ASD by artificial activation of DREADDs, as a simulation of OXT administration to activate OXTR+ neurons in the LS.

Pathogenic POGZ mutation causes impaired cortical development and reversible autism-like phenotypes.

Pogo transposable element derived with ZNF domain (POGZ) has been identified as one of the most recurrently de novo mutated genes in patients with neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), intellectual disability and White-Sutton syndrome; however, the neurobiological basis behind these disorders remains unknown. Here, we show that POGZ regulates neuronal development and that ASD-related de novo mutations impair neuronal development in the developing mouse brain and induced pluripotent cell lines from an ASD patient. We also develop the first mouse model heterozygous for a de novo POGZ mutation identified in a patient with ASD, and we identify ASD-like abnormalities in the mice. Importantly, social deficits can be treated by compensatory inhibition of elevated cell excitability in the mice. Our results provide insight into how de novo mutations on high-confidence ASD genes lead to impaired mature cortical network function, which underlies the cellular pathogenesis of NDDs, including ASD.

Soft windowing application to improve analysis of high-throughput phenotyping data.

MOTIVATION: High-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximizes analytic power while minimizing noise from unspecified environmental factors. RESULTS: Here we introduce 'soft windowing', a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant P-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft-windowed and non-windowed approaches, respectively, from a set of 2082 mutant mouse lines. Our method is generalizable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources. AVAILABILITY AND IMPLEMENTATION: The method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mice doubly deficient in Six4 and Six5 show ventral body wall defects reproducing human omphalocele.

Omphalocele is a human congenital anomaly in ventral body wall closure and may be caused by impaired formation of the primary abdominal wall (PAW) and/or defects in abdominal muscle development. Here, we report that mice doubly deficient in homeobox genes Six4 and Six5 showed the same ventral body wall closure defects as those seen in human omphalocele. SIX4 and SIX5 were localized in surface ectodermal cells and somatic mesoderm-derived mesenchymal and coelomic epithelial cells (CECs) in the PAW. Six4-/-;Six5-/- fetuses exhibited a large omphalocele with protrusion of both the liver and intestine, or a small omphalocele with protrusion of the intestine, with complete penetrance. The umbilical ring of Six4-/-;Six5-/- embryos was shifted anteriorly and its lateral size was larger than that of normal embryos at the E11.5 stage, before the onset of myoblast migration into the PAW. The proliferation rates of surface ectodermal cells in the left and right PAW and somatic mesoderm-derived cells in the right PAW were lower in Six4-/-;Six5-/- embryos than those of wild-type embryos at E10.5. The transition from CECs of the PAW to rounded mesothelial progenitor cells was impaired and the inner coelomic surface of the PAW was relatively smooth in Six4-/-;Six5-/- embryos at E11.25. Furthermore, Six4 overexpression in CECs of the PAW promoted ingression of CECs. Taken together, our results suggest that Six4 and Six5 are required for growth and morphological change of the PAW, and the impairment of these processes is linked to the abnormal positioning and expansion of the umbilical ring, which results in omphalocele.

Synchronized mesenchymal cell polarization and differentiation shape the formation of the murine trachea and esophagus.

Tube morphogenesis is essential for internal-organ development, yet the mechanisms regulating tube shape remain unknown. Here, we show that different mechanisms regulate the length and diameter of the murine trachea. First, we found that trachea development progresses via sequential elongation and expansion processes. This starts with a synchronized radial polarization of smooth muscle (SM) progenitor cells with inward Golgi-apparatus displacement regulates tube elongation, controlled by mesenchymal Wnt5a-Ror2 signaling. This radial polarization directs SM progenitor cell migration toward the epithelium, and the resulting subepithelial morphogenesis supports tube elongation to the anteroposterior axis. This radial polarization also regulates esophageal elongation. Subsequently, cartilage development helps expand the tube diameter, which drives epithelial-cell reshaping to determine the optimal lumen shape for efficient respiration. These findings suggest a strategy in which straight-organ tubulogenesis is driven by subepithelial cell polarization and ring cartilage development.