microfocus-x-ray-ct-microct-imaging-actinia-equina-cnidaria-harmothoe

Microfocus X-ray CT (microCT) Imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha)

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. ...

National Institute of Genetics

Consomic strains, also known as chromosome substitution strains, are powerful tools for assigning polygenes that control quantitative complex traits to specific chromosomes. Here, we report generation of a full set of mouse consomic strains, in which each chromosome of the common laboratory strain C57BL/6J (B6) is replaced by its counterpart from the inbred strain MSM/Ms, which is derived from Japanese wild mouse, Mus musculus molossinus. The genome sequence of MSM/Ms is divergent from that of B6, whose genome is predominantly derived from Western European wild mouse, Mus musculus domesticus. MSM/Ms exhibits a number of quantitative complex traits markedly different from those of B6. We systematically determined phenotypes of these inter-subspecific consomic strains, focusing on complex traits related to reproduction, growth, and energy metabolism. We successfully detected more than 200 statistically significant QTLs affecting 26 traits. Furthermore, phenotyping of the consomic strains revealed that the measured values for quantitative complex traits often far exceed the range between B6 host and MSM/Ms donor strains; this may result from segregation of alleles or nonadditive interactions among multiple genes derived from the two mouse subspecies (that is, epistasis). Taken together, the results suggest that the inter-subspecific consomic strains will be very useful for identification of latent genetic components underlying quantitative complex traits.

Mouse inter-subspecific consomic strains for genetic dissection of quantitative complex traits.

Partial trisomy distal 4q (denoted 4q+) is a human chromosomal disorder caused by duplication of the distal end of the long arm of chromosome 4 (Chr4). This disorder manifests typical phenotypes, including craniofacial, renal, heart and thumb developmental defects. Although these clinical features are likely caused by a dosage imbalance in the gene network involving the trisomic region, the causative gene or genes and the molecular bases are largely unknown. Here, we report mouse Recombination-induced mutation 4 (Rim4) as a model animal of 4q+. The Rim4 genome contains an insertion of a 6.5 Mb fragment from mouse chromosome 8 into chromosome 6. This insertion fragment contains 17 genes, including Hand2, that encode the basic helix-loop-helix transcription factor and is syntenic to the distal end of human Chr4, 4q32.3 to 4q34.1, which is responsible for 4q+. A comparison of phenotypes between patients with Rim4 and 4q+ revealed that Rim4 shows direct parallels with many phenotypes of 4q+ such as craniofacial, heart, cervical vertebra and limb deformities. Rebalancing the gene dosage by a genetic cross with Hand2 knockout mice ameliorated symptoms of the heart and limb deformities of Rim4. Conversely, an increase in copy number of Hand2 in wild-type mice recaptures the heart and limb deformities of Rim4. Our results collectively demonstrate that overdosage of Hand2 is a major cause for at least the limb and heart phenotypes of 4q+ and that mouse Rim4 provides a unique animal model for understanding the molecular bases underlying the complex phenotypes of 4q+.

Overdosage of Hand2 causes limb and heart defects in the human chromosomal disorder partial trisomy distal 4q.

Taking good care of elderly is a major challenge of our society, and thus identification of potential drug targets to reduce age-associated disease burden is desirable. α-klotho(-/-) (α-kl) is a short-lived mouse model that displays multiple phenotypes resembling human aging-related syndromes. Such ageing phenotype of α-kl(-/-) mice is associated with activation of a proteolytic enzyme, Calpain-1. We hypothesized that uncontrolled activation of calpain-1 might be causing age-related phenotypes in α-kl-deficient mice. We found that daily administration of BDA-410, a calpain-1 inhibitor, strikingly ameliorated multiple aging-related phenotypes. Treated mice showed recovery of reproductive ability, increased body weight, reduced organ atrophy, and suppression of ectopic calcifications, bone mineral density reduction, pulmonary emphysema and senile atrophy of skin. We also observed ectopic expression of FGF23 in calcified arteries of α-kl(-/-) mice, which might account for the clinically observed association of increased FGF23 level with increased risk of cardiovascular mortality. These findings allow us to propose that modulation of calpain-1 activity is a potential therapeutic option for delaying age-associated organ pathology, particularly caused by the dysregulation of mineral ion homeostasis. 

Calpain 1 inhibitor BDA-410 ameliorates α-klotho-deficiency phenotypes resembling human aging-related syndromes.

The spectra of phenotypes associated with aging and mitochondrial diseases sometimes appear to overlap with each other. We used aged mice and a mouse model of mitochondrial diseases (transmitochondrial mito-miceΔ with deleted mtDNA) to study whether premature aging phenotypes observed in mtDNA mutator mice are associated with aging or mitochondrial diseases. Here, we provide convincing evidence that all the mice examined had musculoskeletal disorders of osteoporosis and muscle atrophy, which correspond to phenotypes prevalently observed in the elderly. However, precise investigation of musculoskeletal disorders revealed that the spectra of osteoporosis and muscle atrophy phenotypes in mtDNA mutator mice were very close to those in mito-miceΔ, but different from those of aged mice. Therefore, mtDNA mutator mice and mito-miceΔ, but not aged mice, share the spectra of musculoskeletal disorders. 

Transmitochondrial mito-miceΔ and mtDNA mutator mice, but not aged mice, share the same spectrum of musculoskeletal disorders.

Shh signalling plays a crucial role for endoderm development. A Shh endoderm enhancer, MACS1, is well conserved across terrestrial animals with lungs. Here, we first show that eliminating mouse MACS1 causes severe defects in laryngeal development, indicating that MACS1-directed Shh signalling is indispensable for respiratory organogenesis. Extensive phylogenetic analyses revealed that MACS1 emerged prior to the divergence of cartilaginous and bony fishes, and even euteleost fishes have a MACS1 orthologue. Meanwhile, ray-finned fishes evolved a novel conserved non-coding sequence in the neighbouring region. Transgenic assays showed that MACS1 drives reporter expression ventrally in laryngeal epithelium. This activity has been lost in the euteleost lineage, and instead, the conserved non-coding sequence of euteleosts acquired an enhancer activity to elicit dorsal epithelial expression in the posterior pharynx and oesophagus. These results implicate that evolution of these two enhancers is relevant to the morphological transition from ventral lungs to dorsal gas bladder.

Evolution of Shh endoderm enhancers during morphological transition from ventral lungs to dorsal gas bladder.

Monocot stems lack the vascular cambium and instead have characteristic structures in which intercalary meristems generate internodes and veins remain separate and scattered. However, developmental processes of these unique structures have been poorly described. BELL1-like homeobox (BLH) transcription factors (TFs) are known to heterodimerize with KNOTTED1-like homeobox TFs to play crucial roles in shoot meristem maintenance, but their functions are elusive in monocots. We found that maize () BLH12 and BLH14 have redundant but important roles in stem development. BLH12/14 interact with KNOTTED1 (KN1) in vivo and accumulate in overlapping domains in shoot meristems, young stems, and provascular bundles. Similar to  loss-of-function mutants,  () double mutants fail to maintain axillary meristems. Unique to  is an abnormal tassel branching and precocious internode differentiation that results in dwarfism and reduced veins in stems. Micro-computed tomography observation of vascular networks revealed that  double mutants had reduced vein number due to fewer intermediate veins in leaves and precocious anastomosis in young stems. Based on these results, we propose two functions of BLH12/14 during stem development: (1) maintaining intercalary meristems that accumulate KN1 and prevent precocious internode differentiation and (2) preventing precocious anastomosis of provascular bundles in young stems to ensure the production of sufficient independent veins.

KNOTTED1 Cofactors, BLH12 and BLH14, Regulate Internode Patterning and Vein Anastomosis in Maize.

Trabecular bone structure has an important influence on bone strength, but little is known about its genetic regulation. To elucidate the genetic factor(s) regulating trabecular bone structure, we compared the trabecular bone structures of two genetically remote mouse strains, C57BL/6J and Japanese wild mouse-derived MSM/Ms. Phenotyping by X-ray micro-CT revealed that MSM/Ms has structurally more fragile trabecular bone than C57BL/6J. Toward identification of genetic determinants for the difference in fragility of trabecular bone between the two mouse strains, we employed phenotype screening of consomic mouse strains in which each C57BL/6J chromosome is substituted by its counterpart from MSM/Ms. The results showed that many chromosomes affect trabecular bone structure, and that the consomic strain B6-Chr15, carrying MSM/Ms-derived chromosome 15 (Chr15), has the lowest values for the parameters BV/TV, Tb.N, and Conn.D, and the highest values for the parameters Tb.Sp and SMI. Subsequent phenotyping of subconsomic strains for Chr15 mapped four novel trabecular bone structure-related QTL () on mouse Chr15. These results collectively indicate that genetic regulation of trabecular bone structure is highly complex, and that even in the single Chr15, the combined action of the four s controls the fragility of trabecular bone. Given that  is syntenic to human Chr 12q12-13.3, where several bone-related SNPs are assigned, further study of  should facilitate our understanding of the genetic regulation of bone formation in humans.

Genetic Dissection of Trabecular Bone Structure with Mouse Intersubspecific Consomic Strains.

Interaction between the epithelium and mesenchyme coordinates patterning and differentiation of oral cavity structures including teeth, palatal rugae and tongue papillae. SHH is one of the key signaling molecules for this interaction. Epithelial expression of Shh in the tooth buds and tongue papillae is regulated by at least two enhancers, MRCS1 and MFCS4. However, it is unclear how the two enhancers cooperate to regulate Shh. Here, we found that simultaneous deletion of MRCS1 and MFCS4 results in the formation of a supernumerary tooth in front of the first molar. Since deletion of either single enhancer barely affects tooth development, MRCS1 and MFCS4 evidently act in a redundant fashion. Binding motifs for WNT signaling mediators are shared by MRCS1 and MFCS4, and play a central role in regulating Shh expression, indicating that the two redundant enhancers additively exert their Shh regulation by responding to WNT signal input.

SHH signaling directed by two oral epithelium-specific enhancers controls tooth and oral development.

Age-related systemic environments influence neurogenesis and organ regeneration of heterochronic parabiotic partners; however, the difficulty of manipulating small embryos prevents the effects of aged systemic environments on primitive organs at the developmental stage from being analysed. Here, we describe a novel transplantation system to support whole living embryos/larvae as grafts in immunodeficient zebrafish by the intrusion of host blood vessels into the grafts, allowing bodies similar to those of heterochronic parabiosis to be generated by subcutaneous grafting. Although grafted embryos/larvae formed most organs, not all organogenesis was supported equally; although the brain, eyes and the intestine usually developed, the liver, testes and heart developed insufficiently or even occasionally disappeared. Removal of host germ cells stimulated testis development in grafted embryos. These results indicate that primitive testes are susceptible to the systemic environments that originated from the germ cells of aged hosts and imply that the primitive liver and heart are similar. Upon applying this method to embryonic lethal mutants, various types of organs, including testes that developed in germ-cell-removed recipients, and viable offspring were obtained from the mutants. This unique transplantation system will lead to new insights into the age-related systemic environments that are crucial for organogenesis in vertebrates.

Development and growth of organs in living whole embryo and larval grafts in zebrafish.

Xenoturbella is a group of marine benthic animals lacking an anus and a centralized nervous system. Molecular phylogenetic analyses group the animal together with the Acoelomorpha, forming the Xenacoelomorpha. This group has been suggested to be either a sister group to the Nephrozoa or a deuterostome, and therefore it may provide important insights into origins of bilaterian traits such as an anus, the nephron, feeding larvae and centralized nervous systems. However, only five Xenoturbella species have been reported and the evolutionary history of xenoturbellids and Xenacoelomorpha remains obscure.

A new species of Xenoturbella from the western Pacific Ocean and the evolution of Xenoturbella.

Acquisition of new -regulatory elements (CREs) can cause alteration of developmental gene regulation and may introduce morphological novelty in evolution. Although structural variation in the genome generated by chromosomal rearrangement is one possible source of new CREs, only a few examples are known, except for cases of retrotransposition. In this study, we show the acquisition of novel regulatory sequences as a result of large genomic insertion in the spontaneous mouse mutation Hammer toe ().  mice exhibit syndactyly with webbing, due to suppression of interdigital cell death in limb development. We reveal that, in the  genome, a 150-kb noncoding DNA fragment from chromosome 14 is inserted into the region upstream of the Sonic hedgehog () promoter in chromosome 5. Phenotyping of mouse embryos with a series of CRISPR/Cas9-aided partial deletion of the 150-kb insert clearly indicated that two different regions are necessary for the syndactyly phenotype of  We found that each of the two regions contains at least one enhancer for interdigital regulation. These results show that a set of enhancers brought by the large genomic insertion elicits the interdigital  expression and the  phenotype. Transcriptome analysis indicates that ectopic expression of  up-regulates Chordin () that antagonizes bone morphogenetic protein signaling in the interdigital region. Indeed, overexpressing transgenic mice recapitulated syndactyly with webbing. Thus, the  mutation provides an insight into enhancer acquisition as a source of creation of novel gene regulation.

Enhancer adoption caused by genomic insertion elicits interdigital  expression and syndactyly in mouse.

Melanocytes develop from the vertebrate embryo-specific neural crest, migrate, and localize in various organs, including not only the skin but also several extracutaneous locations such as the heart, inner ear and choroid. Little is known about the functions of extracutaneous melanocytes except for cochlear melanocytes, which are essential for hearing ability. In this study, we focused on the structure of the choroid, in which melanocytes are abundant around the well-developed blood vascular system. By comparing structural differences in the choroid of wild-type and melanocyte-deficient Mitf/Mitf mutant mice, our observations suggest that choroidal melanocytes contribute to the morphogenesis and/or maintenance of the normal vasculature structure of that tissue.

Melanocytes contribute to the vasculature of the choroid.

After publication of Nakano et al. (2017) [1], the authors became aware of the fact that the new species-group name erected for the two specimens of a Japanese xenoturbellid species in the article is not available because Nakano et al. (2017) [1] does not meet the requirement of the amendment of Article 8.5.3 of the International Code of Zoological Nomenclature (the Code) [2]. The authors therefore describe the two xenoturbellids as a new species again in this correction article. Methods for morphological observation, DNA extraction and sequencing were as described in Nakano et al. (2017) [1]. The holotype and paratype specimens are deposited in the National Museum of Nature and Science, Tsukuba (NSMT), Japan. The DNA sequences obtained were deposited in the International Nucleotide Sequence Database (INSD).

Correction to: A new species of Xenoturbella from the western Pacific Ocean and the evolution of Xenoturbella.

Inflorescence architecture is diverse in angiosperms, and is mainly determined by the arrangement of the branches and flowers, known as phyllotaxy. In rice (Oryza sativa), the main inflorescence axis, called the rachis, generates primary branches in a spiral phyllotaxy, and flowers (spikelets) are formed on these branches. Here, we have studied a classical mutant, named verticillate rachis (ri), which produces branches in a partially whorled phyllotaxy. Gene isolation revealed that RI encodes a BELL1-type homeodomain transcription factor, similar to Arabidopsis PENNYWISE/BELLRINGER/REPLUMLESS, and is expressed in the specific regions within the inflorescence and branch meristems where their descendant meristems would soon initiate. Genetic combination of an ri homozygote and a mutant allele of RI-LIKE1 (RIL1) (designated ri ril1/+ plant), a close paralog of RI, enhanced the ri inflorescence phenotype, including the abnormalities in branch phyllotaxy and rachis internode patterning. During early inflorescence development, the timing and arrangement of primary branch meristem (pBM) initiation were disturbed in both ri and ri ril1/+ plants. These findings suggest that RI and RIL1 were involved in regulating the phyllotactic pattern of the pBMs to form normal inflorescences. In addition, both RI and RIL1 seem to be involved in meristem maintenance, because the ri ril1 double-mutant failed to establish or maintain the shoot apical meristem during embryogenesis.

BELL1-like homeobox genes regulate inflorescence architecture and meristem maintenance in rice.

Many scarab beetles have sexually dimorphic exaggerated horns that are an evolutionary novelty. Since the shape, number, size, and location of horns are highly diverged within Scarabaeidae, beetle horns are an attractive model for studying the evolution of sexually dimorphic and novel traits. In beetles including the Japanese rhinoceros beetle Trypoxylus dichotomus, the sex differentiation gene doublesex (dsx) plays a crucial role in sexually dimorphic horn formation during larval-pupal development. However, knowledge of when and how dsx drives the gene regulatory network (GRN) for horn formation to form sexually dimorphic horns during development remains elusive. To address this issue, we identified a Trypoxylus-ortholog of the sex determination gene, transformer (tra), that regulates sex-specific splicing of the dsx pre-mRNA, and whose loss of function results in sex transformation. By knocking down tra function at multiple developmental timepoints during larval-pupal development, we estimated the onset when the sex-specific GRN for horn formation is driven. In addition, we also revealed that dsx regulates different aspects of morphogenetic activities during the prepupal and pupal developmental stages to form appropriate morphologies of pupal head and thoracic horn primordia as well as those of adult horns. Based on these findings, we discuss the evolutionary developmental background of sexually dimorphic trait growth in horned beetles.

Precise staging of beetle horn formation in Trypoxylus dichotomus reveals the pleiotropic roles of doublesex depending on the spatiotemporal developmental contexts.

Article	Year
Mouse inter-subspecific consomic strains for genetic dissection of quantitative complex traits. Genome research\| PubMed ID: 18256237	2008
Overdosage of Hand2 causes limb and heart defects in the human chromosomal disorder partial trisomy distal 4q. Human molecular genetics\| PubMed ID: 23449628	2013
Calpain 1 inhibitor BDA-410 ameliorates α-klotho-deficiency phenotypes resembling human aging-related syndromes. Scientific reports\| PubMed ID: 25080854	2014
Transmitochondrial mito-miceΔ and mtDNA mutator mice, but not aged mice, share the same spectrum of musculoskeletal disorders. Biochemical and biophysical research communications\| PubMed ID: 25511702	2015
Evolution of Shh endoderm enhancers during morphological transition from ventral lungs to dorsal gas bladder. Nature communications\| PubMed ID: 28155855	2017
KNOTTED1 Cofactors, BLH12 and BLH14, Regulate Internode Patterning and Vein Anastomosis in Maize. The Plant cell\| PubMed ID: 28381444	2017
Genetic Dissection of Trabecular Bone Structure with Mouse Intersubspecific Consomic Strains. G3 (Bethesda, Md.)\| PubMed ID: 28855285	2017
SHH signaling directed by two oral epithelium-specific enhancers controls tooth and oral development. Scientific reports\| PubMed ID: 29021530	2017
Development and growth of organs in living whole embryo and larval grafts in zebrafish. Scientific reports\| PubMed ID: 29184141	2017
A new species of Xenoturbella from the western Pacific Ocean and the evolution of Xenoturbella. BMC evolutionary biology\| PubMed ID: 29249199	2017
Enhancer adoption caused by genomic insertion elicits interdigital expression and syndactyly in mouse. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29255029	2018
Melanocytes contribute to the vasculature of the choroid. Genes & genetic systems\| PubMed ID: 29607881	2018
Correction to: A new species of Xenoturbella from the western Pacific Ocean and the evolution of Xenoturbella. BMC evolutionary biology\| PubMed ID: 29879905	2018
BELL1-like homeobox genes regulate inflorescence architecture and meristem maintenance in rice. The Plant journal : for cell and molecular biology\| PubMed ID: 30657229	2019
Precise staging of beetle horn formation in Trypoxylus dichotomus reveals the pleiotropic roles of doublesex depending on the spatiotemporal developmental contexts. PLoS genetics\| PubMed ID: 30969957	2019

Akiteru Maeno

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