DOI： 10.1242/dev.202079

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

OBJECTIVE: Cambodia is located within the distribution range of the red junglefowl, the common ancestor of domestic chickens. Although a variety of indigenous chickens have been reared in Cambodia since ancient times, their genetic characteristics have yet to be sufficiently defined. Here, we conducted a large-scale population genetic study to investigate the genetic diversity and population genetic structure of Cambodian indigenous chickens and their phylogenetic relationships with other chicken breeds and native chickens worldwide. METHODS: A Bayesian phylogenetic tree was constructed based on 625 mitochondrial DNA D-loop sequences, and Bayesian clustering analysis was performed for 666 individuals with 23 microsatellite markers, using samples collected from 28 indigenous chicken populations in 24 provinces and three commercial chicken breeds. RESULTS: A total of 92 haplotypes of mitochondrial D-loop sequences belonging to haplogroups A to F and J were detected in Cambodian chickens; in the indigenous chickens, haplogroup D (44.4%) was the most common, and haplogroups A (21.0%) and B (13.2%) were also dominant. However, haplogroup J, which is rare in domestic chickens but abundant in Thai red junglefowl, was found at a high frequency (14.5%), whereas the frequency of haplogroup E was considerably lower (4.6%). Population genetic structure analysis based on microsatellite markers revealed the presence of three major genetic clusters in Cambodian indigenous chickens. Their genetic diversity was relatively high, which was similar to findings reported for indigenous chickens from other Southeast Asian countries. CONCLUSION: Cambodian indigenous chickens are characterized by mitochondrial D-loop haplotypes that are common to indigenous chickens throughout Southeast Asia, and may retain many of the haplotypes that originated from wild ancestral populations. These chickens exhibit high population genetic diversity, and the geographical distribution of three major clusters may be attributed to inter-regional trade and poultry transportation routes within Cambodia or international movement between Cambodia and other countries.

DOI： 10.5713/ab.21.0351

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uteri of birds, such as Japanese quail, Coturnix japonica, which has brown-speckled eggshells; however, the molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis. We also performed genetic linkage analysis to identify the causative genomic region of the white eggshell phenotype. We found that 11 genes, including 5'-aminolevulinate synthase 1 (ALAS1) and hephaestin-like 1 (HEPHL1), were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, while their expression levels were downregulated in the cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5'-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on these findings, we discussed the molecular basis of PPIX production in the uteri of Japanese quails.

DOI： 10.1371/journal.pone.0265008

PubMed

Authorship：Lead author,　Last author   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/dvdy.323

Other URL： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/dvdy.323

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)-containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide- and 5-hydroxythalidomide-induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell-free protein synthesis system, PLZF was identified as a thalidomide-dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5-hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5-hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide-dependent CRBN substrate SALL4, was induced by thalidomide or 5-hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide-induced phenotypes. Our findings implicate PLZF as an important thalidomide-induced CRBN neosubstrate involved in thalidomide teratogenicity.

DOI： 10.15252/embj.2020105375

PubMed

Other URL： https://onlinelibrary.wiley.com/doi/full-xml/10.15252/embj.2020105375

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

<title>Abstract</title>In this study, we aimed to elucidate the origin of domestic chickens and their evolutionary history over the course of their domestication. We conducted a large-scale genetic study using mitochondrial DNA D-loop sequences and 28 microsatellite DNA markers to investigate the diversity of 298 wild progenitor red junglefowl (<italic>Gallus gallus</italic>) across two subspecies (<italic>G. g. gallus</italic> and <italic>G. g. spadiceus</italic>) from 12 populations and 138 chickens from 10 chicken breeds indigenous to Thailand. Twenty-nine D-loop sequence haplotypes were newly identified: 14 and 17 for Thai indigenous chickens and red junglefowl, respectively. Bayesian clustering analysis with microsatellite markers also revealed high genetic diversity in the red junglefowl populations. These results suggest that the ancestral populations of Thai indigenous chickens were large, and that a part of the red junglefowl population gene pool was not involved in the domestication process. In addition, some haplogroups that are distributed in other countries of Southeast Asia were not observed in either the red junglefowls or the indigenous chickens examined in the present study, suggesting that chicken domestication occurred independently across multiple regions in Southeast Asia.

DOI： 10.1038/s41598-021-81589-7

PubMed

Other URL： http://www.nature.com/articles/s41598-021-81589-7

Publishing type：Research paper (scientific journal)  

Background: The phalanges are the final skeletal elements to form in the vertebrate limb and their identity is regulated by signaling at the phalanx forming region (PFR) located at the tip of the developing digit ray. Here, we seek to explore the relationship between PFR activity and phalanx morphogenesis, which define the most distal limb skeletal elements, and signals associated with termination of limb outgrowth. Results: As Grem1 is extinguished in the distal chick limb mesoderm, the chondrogenesis marker Aggrecan is up-regulated in the metatarsals and phalanges. Fate mapping confirms that subridge mesoderm cells contribute to the metatarsal and phalanges when subridge Grem1 is down-regulated. Grem1 overexpression specifically blocks chick phalanx development by inhibiting PFR activity. PFR activity and digit development are also disrupted following overexpression of a Gli3 repressor, which results in Grem1 expression in the distal limb and downregulation of Bmpr1b. Conclusions: Based on expression and fate mapping studies, we propose that downregulation of Grem1 in the distal limb marks the transition from metatarsal to phalanx development. This suggests that downregulation of Grem1 in the distal limb mesoderm is necessary for phalanx development. Grem1 downregulation allows for full PFR activity and phalanx progenitor cell commitment to digit fate.

DOI： 10.1002/dvdy.431

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

BACKGROUND: Hedgehog signaling has various regulatory functions in tissue morphogenesis and differentiation. To investigate its involvement in anterior pituitary precursor development and the lens precursor potential for anterior pituitary precursors, we investigated Talpid mutant Japanese quail embryos, in which hedgehog signaling is defective. RESULTS: Talpid mutants develop multiple pituitary precursor-like pouches of variable sizes from the oral ectoderm (OE). The ectopic pituitary pouches initially express the pituitary-associated transcription factor (TF) LHX3 similarly to Rathke's pouch, the genuine pituitary precursor. The pouches coexpress the TFs SOX2 and PAX6, a signature of lens developmental potential. Most Talpid mutant pituitary pouches downregulate LHX3 expression and activate the lens-essential TF PROX1, leading to the development of small lens tissue expressing α-, β-, and δ-crystallins. In contrast, mutant Rathke's pouches express a lower level of LHX3, which is primarily localized in the cytoplasm, and activate the lens developmental pathway. CONCLUSIONS: Hedgehog signaling in normal embryos regulates the development of Rathke's pouch in two steps. First, by confining Rathke's pouch development in a low hedgehog signaling region of the OE. Second, by sustaining LHX3 activity to promote anterior pituitary development, while inhibiting ectopic lens development.

DOI： 10.1002/dvdy.222

PubMed

Other URL： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/dvdy.222

Authorship：Lead author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

<title>Abstract</title>The Creeper (<italic>Cp</italic>) chicken is characterized by chondrodystrophy in <italic>Cp</italic>/+ heterozygotes and embryonic lethality in <italic>Cp</italic>/<italic>Cp</italic> homozygotes. However, the genes underlying the phenotypes have not been fully known. Here, we show that a 25 kb deletion on chromosome 7, which contains the Indian hedgehog (<italic>IHH</italic>) and non-homologous end-joining factor 1 (<italic>NHEJ1</italic>) genes, is responsible for the <italic>Cp</italic> trait in Japanese bantam chickens. <italic>IHH</italic> is essential for chondrocyte maturation and is downregulated in the <italic>Cp/+</italic> embryos and completely lost in the <italic>Cp</italic>/<italic>Cp</italic> embryos. This indicates that chondrodystrophy is caused by the loss of <italic>IHH</italic> and that chondrocyte maturation is delayed in <italic>Cp</italic>/<italic>+</italic> heterozygotes. The <italic>Cp</italic>/<italic>Cp</italic> homozygotes exhibit impaired DNA double-strand break (DSB) repair due to the loss of <italic>NHEJ1</italic>, resulting in DSB accumulation in the vascular and nervous systems, which leads to apoptosis and early embryonic death.

DOI： 10.1038/s42003-020-0870-z

PubMed

Other URL： http://www.nature.com/articles/s42003-020-0870-z

Authorship：Last author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Japanese indigenous chickens have a long breeding history, possibly beginning 2000 years ago. Genetic characterization of Japanese indigenous chickens has been performed using mitochondrial D-loop region and microsatellite DNA markers. Their phylogenetic relationships with chickens worldwide and genetic variation within breeds have not yet been examined. In this study, the genetic characteristics of 38 Japanese indigenous chicken breeds were assessed by phylogenetic analyses of mitochondrial D-loop sequences compared with those of indigenous chicken breeds overseas. To evaluate the genetic relationships among Japanese indigenous chicken breeds, a STRUCTURE analysis was conducted using 27 microsatellite DNA markers. D-loop sequences of Japanese indigenous chickens were classified into five major haplogroups, A–E, among 15 haplogroups found in chickens worldwide. The haplogroup composition suggested that Japanese indigenous chickens originated mainly from China, with some originating from Southeast Asia. The STRUCTURE analyses revealed that Japanese indigenous chickens are genetically differentiated from chickens overseas; Japanese indigenous chicken breeds possess distinctive genetic characteristics, and Jidori breeds, which have been reared in various regions of Japan for a long time, are genetically close to each other. These results provide new insights into the history of chickens around Asia in addition to novel genetic data for the conservation of Japanese indigenous chickens.

DOI： 10.3390/ani10112074

PubMed

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Hybrid incompatibility, such as sterility and inviability, prevents gene flow between closely-related populations as a reproductive isolation barrier. F1 hybrids between chickens and Japanese quail (hereafter, referred to as quail), exhibit a high frequency of developmental arrest at the preprimitive streak stage. To investigate the molecular basis of the developmental arrest at the preprimitive streak stage in chicken-quail F1 hybrid embryos, we investigated chromosomal abnormalities in the hybrid embryos using molecular cytogenetic analysis. In addition, we quantified gene expression in parental species and chicken- and quail-derived allele-specific expression in the hybrids at the early blastoderm and preprimitive streak stages by mRNA sequencing. Subsequently, we compared the directions of change in gene expression, including upregulation, downregulation, or no change, from the early blastoderm stage to the preprimitive streak stage between parental species and their hybrids. Chromosome analysis revealed that the cells of the hybrid embryos contained a fifty-fifty mixture of parental chromosomes, and numerical chromosomal abnormalities were hardly observed in the hybrid cells. Gene expression analysis revealed that a part of the genes that were upregulated from the early blastoderm stage to the preprimitive streak stage in both parental species exhibited no upregulation of both chicken- and quail-derived alleles in the hybrids. GO term enrichment analysis revealed that these misregulated genes are involved in various biological processes, including ribosome-mediated protein synthesis and cell proliferation. Furthermore, the misregulated genes included genes involved in early embryonic development, such as primitive streak formation and gastrulation. These results suggest that numerical chromosomal abnormalities due to a segregation failure does not cause the lethality of chicken-quail hybrid embryos, and that the downregulated expression of the genes that are involved in various biological processes, including translation and primitive streak formation, mainly causes the developmental arrest at the preprimitive streak stage in the hybrids.

DOI： 10.1371/journal.pone.0240183

PubMed

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/dvg.23388

Other URL： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/dvg.23388

Authorship：Last author   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/dgd.12682

Other URL： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/dgd.12682

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Haploinsufficiency of NSD1, which dimethylates histone H3 lysine 36 (H3K36), causes Sotos syndrome (SoS), an overgrowth syndrome. DNMT3A and DNMT3B recognizes H3K36 trimethylation (H3K36me3) through PWWP domain to exert de novo DNA methyltransferase activity and establish imprinted differentially methylated regions (DMRs). Since decrease of H3K36me3 and genome-wide DNA hypomethylation in SoS were observed, hypomethylation of imprinted DMRs in SoS was suggested. We explored DNA methylation status of 28 imprinted DMRs in 31 SoS patients with NSD1 defect and found that hypomethylation of IGF2-DMR0 and IG-DMR in a substantial proportion of SoS patients. Luciferase assay revealed that IGF2-DMR0 enhanced transcription from the IGF2 P0 promoter but not the P3 and P4 promoters. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) revealed active enhancer histone modifications at IGF2-DMR0, with high enrichment of H3K4me1 and H3 lysine 27 acetylation (H3K27ac). CRISPR-Cas9 epigenome editing revealed that specifically induced hypomethylation at IGF2-DMR0 increased transcription from the P0 promoter but not the P3 and P4 promoters. NSD1 knockdown suggested that NSD1 targeted IGF2-DMR0; however, IGF2-DMR0 DNA methylation and IGF2 expression were unaltered. This study could elucidate the function of IGF2-DMR0 as a DNA methylation dependent, P0 promoter-specific enhancer. NSD1 may play a role in the establishment or maintenance of IGF2-DMR0 methylation during the postimplantation period.

DOI： 10.1096/fj.201901757r

PubMed

Other URL： https://onlinelibrary.wiley.com/doi/full-xml/10.1096/fj.201901757R

ニワトリ胚へのin ovoエレクトロポレーション(EP)で注入部位に関する指標の代替色素として、Brilliant Blueが適していることを見出した。Brilliant Blueは0.2%でDNA注入部位の追跡に十分であった。この化学物質ではEP後に赤色蛍光が観察されなかった。これらの結果から、Brilliant BlueはEPによってニワトリ胚に導入された赤色蛍光蛋白質を検出するために利用可能と考えられた。また、レポーター遺伝子EGFPとmCherryを一緒にEPした後、2種の蛍光強度を正確に測定するためBrilliant Blueを使用したところ、蛍光強度の平均を算出するために有用なツールであると考えられた。

レンチウイルス感染により作成したEGFP遺伝子導入ニワトリ株における、遺伝子の統合部位と発達中の器官の蛍光強度を解析した。導入遺伝子はMED27のエクソン3と4の間に局在した。一部のホモ接合体とヘテロ接合体は胚の早期段階で致死的であった。パラフィン切片の抗GFP抗体による免疫組織化学法により、St.14、17、24の導入遺伝子胚のEGFP発現の組織学的分析を行った。次に凍結切片を作成し、それぞれの組織における導入遺伝子から直接EGFP蛍光強度を測定した。その結果、胚の発達段階や組織において、EGFP発現は特徴がみられた。野生型細胞と一緒に微量培養したEGFP発現細胞は、生細胞画像を介してはっきりと識別することができた。

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Abstract

Chick embryo electroporation is a powerful tool for the introduction of transgenes into tissues of interest for the study of developmental biology. This method often uses Fast Green to visualize the injected area by staining the solution containing DNA green. Here, we show that Fast Green fluoresces in a red color after electroporation, suggesting that researchers need to be cautious when detecting red fluorescence. Fast Green solution did not show any fluorescence before injection into chick embryos, but fluoresced red within 3 min post‐injection into chick embryos. We identified Brilliant Blue as suitable alternative dye for use as an indicator of injection sites in ovo electroporation. We found that 0.2% of Brilliant Blue was sufficient to track the area of DNA injection. In addition, this chemical did not show red fluorescence after electroporation. Our findings demonstrate that Brilliant Blue can be used for detecting red fluorescent proteins introduced into chick embryos by electroporation. Our study also shows useful examples for the application of Brilliant Blue for the precise quantification of two fluorescence intensities after EGFP and mCherry co‐electroporation.

DOI： 10.1111/dgd.12629

Other URL： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/dgd.12629

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Elucidating how body parts from different primordia are integrated during development is essential for understanding the nature of morphological evolution. In tetrapod evolution, while the position of the hindlimb has diversified along with the vertebral formula, the mechanism responsible for this coordination has not been well understood. However, this synchronization suggests the presence of an evolutionarily conserved developmental mechanism that coordinates the positioning of the hindlimb skeleton derived from the lateral plate mesoderm with that of the sacral vertebrae derived from the somites. Here we show that GDF11 secreted from the posterior axial mesoderm is a key factor in the integration of sacral vertebrae and hindlimb positioning by inducing Hox gene expression in two different primordia. Manipulating the onset of GDF11 activity altered the position of the hindlimb in chicken embryos, indicating that the onset of Gdf11 expression is responsible for the coordinated positioning of the sacral vertebrae and hindlimbs. Through comparative analysis with other vertebrate embryos, we also show that each tetrapod species has a unique onset timing of Gdf11 expression, which is tightly correlated with the anteroposterior levels of the hindlimb bud. We conclude that the evolutionary diversity of hindlimb positioning resulted from heterochronic shifts in Gdf11 expression, which led to coordinated shifts in the sacral-hindlimb unit along the anteroposterior axis.

DOI： 10.1038/s41559-017-0247-y

PubMed

Publishing type：Research paper (scientific journal)  

To understand organ morphogenetic mechanisms, it is essential to clarify how spatiotemporally-regulated molecular/cellular dynamics causes physical tissue deformation. In the case of vertebrate limb development, while some of the genes and oriented cell behaviors underlying morphogenesis have been revealed, tissue deformation dynamics remains incompletely understood. We here introduce our recent work on the reconstruction of tissue deformation dynamics in chick limb development from cell lineage tracing data. This analysis has revealed globally-aligned anisotropic tissue deformation along the proximo-distal axis not only in the distal region but also in the whole limb bud. This result points to a need, as a future challenge, to find oriented molecular/cellular behaviors for realizing the observed anisotropic tissue deformation in both proximal and distal regions, which will lead to systems understanding of limb morphogenesis.

DOI： 10.1016/j.gde.2017.04.005

PubMed

DOI： 10.1016/j.mod.2017.04.060

Publishing type：Research paper (scientific journal)  

Background: The organizing center, which serves as a morphogen source, has crucial functions in morphogenesis in animal development. The center is necessarily located in a certain restricted area in the morphogenetic field, and there are several ways in which an organizing center can be restricted. The organizing center for limb morphogenesis, the ZPA (zone of polarizing activity), specifically expresses the Shh gene and is restricted to the posterior region of the developing limb bud.Results: The pre-pattern along the limb anteroposterior axis, provided by anterior Gli3 expression and posterior Hand2 expression, seems insufficient for the initiation of Shh expression restricted to a narrow, small spot in the posterior limb field. Comparison of the spatiotemporal patterns of gene expression between Shh and some candidate genes (Fgf8, Hoxd10, Hoxd11, Tbx2, and Alx4) upstream of Shh expression suggested that a combination of these genes' expression provides the restricted initiation of Shh expression.Conclusions: Taken together with results of functional assays, we propose a model in which positive and negative transcriptional regulatory networks accumulate their functions in the intersection area of their expression regions to provide a restricted spot for the ZPA, the source of morphogen, Shh. Developmental Dynamics 246:417-430, 2017. (c) 2017 Wiley Periodicals, Inc.

DOI： 10.1002/dvdy.24493

PubMed

Publishing type：Part of collection (book)  

Electroporation enables delivering bionanomolecules, such as DNAs, RNAs, siRNAs, and morpholinos, into chick embryos in a spatially and temporally restricted fashion. Recent advances in electroporation techniques allowed us to deliver transgenes into the restricted area of the limb bud and to analyze the function of the enhancers in the limb field. Here, we describe the introduction of transgenes by electroporation in the limb field and its application on enhancer analysis.

DOI： 10.1007/978-1-4939-7216-6_13

PubMed

Reptile development is an intriguing research target for understating the unique morphogenesis of reptiles as well as the evolution of vertebrates. However, there are numerous difficulties associated with studying development in reptiles. The number of available reptile eggs is usually quite limited. In addition, the reptile embryo is tightly adhered to the eggshell, making it a challenge to isolate reptile embryos intact. Furthermore, there have been few reports describing efficient procedures for isolating intact embryos especially prior to pharyngula stage. Thus, the aim of this review is to present efficient procedures for obtaining early-stage reptilian embryos intact. We first describe the method for isolating early-stage embryos of the Japanese striped snake. This is the first detailed method for obtaining embryos prior to oviposition in oviparous snake species. Second, we describe an efficient strategy for isolating early-stage embryos of the soft-shelled turtle.

DOI： 10.1111/dgd.12278

PubMed

International / domestic magazine：International journal  

Hereditary Multiple Malformation (HMM) is a naturally occurring, autosomal recessive, homozygous lethal mutation found in Japanese quail. Homozygote embryos (hmm-/-) show polydactyly similar to talpid2 and talpid3 mutants. Here we characterize the molecular profile of the hmm-/- limb bud and identify the cellular mechanisms that cause its polydactyly. The hmm-/- limb bud shows a severe lack of sonic hedgehog (SHH) signaling, and the autopod has 4 to 11 unidentifiable digits with syn-, poly-, and brachydactyly. The Zone of Polarizing Activity (ZPA) of the hmm-/- limb bud does not show polarizing activity regardless of the presence of SHH protein, indicating that either the secretion pathway of SHH is defective or the SHH protein is dysfunctional. Furthermore, mesenchymal cells in the hmm-/- limb bud do not respond to ZPA transplanted from the normal limb bud, suggesting that signal transduction downstream of SHH is also defective. Since primary cilia are present in the hmm-/- limb bud, the causal gene must be different from talpid2 and talpid3. In the hmm-/- limb bud, a high amount of GLI3A protein is expressed and GLI3 protein is localized to the nucleus. Our results suggest that the regulatory mechanism of GLI3 is disorganized in the hmm-/- limb bud.

DOI： 10.3389/fcell.2016.00149

PubMed

Publishing type：Research paper (scientific journal)  

The species-specific morphology of digits in the tetrapod limb, including the length and number of metacarpal, metatarsal, and phalangeal bones, suggests that a common developmental mechanism for digit formation is modified in a species-specific manner. Here, we examined the function of the AP-2 beta transcription factor in regulating digit length in the chicken autopod. Mutations in the gene encoding AP-2 beta are are associated with Char syndrome, a human autosomal dominant disorder. Char syndrome patients exhibit autopod skeletal defects, including loss of phalanges and shortened fingers, suggestive of a function for AP-2 beta in normal digit development. The ectopic expression of two different dominant-negative forms of chick AP-2 beta, equivalent to mutant forms associated with human Char syndrome, in the developing chick hindlimb bud resulted in defective digit formation, including reductions in the number and length of phalanges and metatarsals. A detailed analysis of the AP-2 beta expression pattern in the limb bud indicated a correlation between the pattern/duration of AP-2 beta expression in the limb mesenchyme and digit length in three amniote species, the chicken, mouse and gecko. In addition, we found that AP-2 beta expression expression was downstream of Fgf signals from the apical ectodermal ridge, which is crucial in digit morphogenesis, and that excessive AP-2 beta function resulted in dysregulated digit length. Taken together, these results suggest that AP-2 beta functions as a novel transcriptional regulator for digit morphogenesis. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2015.08.006

PubMed

Publishing type：Research paper (scientific journal)  

Tissue-level characterization of deformation dynamics is crucial for understanding organ morphogenetic mechanisms, especially the interhierarchical links among molecular activities, cellular behaviors and tissue/organ morphogenetic processes. Limb development is a well-studied topic in vertebrate organogenesis. Nevertheless, there is still little understanding of tissue-level deformation relative to molecular and cellular dynamics. This is mainly because live recording of detailed cell behaviors in whole tissues is technically difficult. To overcome this limitation, by applying a recently developed Bayesian approach, we here constructed tissue deformation maps for chick limb development with high precision, based on snapshot lineage tracing using dye injection. The precision of the constructed maps was validated with a clear statistical criterion. From the geometrical analysis of the map, we identified three characteristic tissue growth modes in the limb and showed that they are consistent with local growth factor activity and cell cycle length. In particular, we report that SHH signaling activity changes dynamically with developmental stage and strongly correlates with the dynamic shift in the tissue growth mode. We also found anisotropic tissue deformation along the proximal-distal axis. Morphogenetic simulation and experimental studies suggested that this directional tissue elongation, and not local growth, has the greatest impact on limb shaping. This result was supported by the novel finding that anisotropic tissue elongation along the proximal-distal axis occurs independently of cell proliferation. Our study marks a pivotal point for multi-scale system understanding in vertebrate development.

DOI： 10.1242/dev.109728

PubMed

Publishing type：Research paper (scientific journal)  

The morphogenesis of snake embryos is an elusive yet fascinating research target for developmental biologists. However, few data exist on development of early snake embryo due to limited availability of pregnant snakes, and the need to harvest early stage embryos directly from pregnant snakes before oviposition without knowing the date of fertilization. We established an ex vivo culture method for early snake embryos using the Japanese striped snake, Elaphe quadrivirgata. This method, which we named sausage-style (SS) culture, allows us to harvest snake embryos at specific stages for each experiment. Using this SS culture system, we calculated somite formation rate at early stages before oviposition. The average somite formation rate between 6/7 and 12/13 somite stages was 145.9min, between 60/70 and 80/91 somite stages 42.4min, and between 113-115 and 126/127 somite stages 71min. Thus, somite formation rate that we observed during early snake embryogenesis was changed over time. We also describe a developmental staging series for E.quadrivirgata. This is the first report of a developmental series of early snake embryogenesis prior to oviposition by full-color images with high-resolution. We propose that the SS culture system is an easy method for treating early snake embryos ex vivo.

DOI： 10.1111/dgd.12157

PubMed

Publishing type：Research paper (scientific journal)  

To understand the morphogenetic mechanisms of organ development and regeneration, it is essential to clarify the inter-hierarchical relationship between microscopic, molecular/cellular activities and organ-level tissue deformation dynamics. While the former have been studied for several decades, the latter macroscopic geometrical information about physical tissue deformation - is often missing, especially for many vertebrates. This is mainly because live recording of detailed cell behaviors in whole tissues during vertebrate organogenesis is technically difficult. In this study, we have developed a novel method that combines snapshot lineage tracing with Bayesian statistical estimation to construct whole-organ deformation maps from landmark data on limited numbers of space-time points. Following the validation of the method using artificially generated data sets, we applied it to the analysis of tissue deformation dynamics in chick limb development. A quantitative tissue deformation map for St.23-St.24 has been constructed, and its precision has been proven by evaluating its predictive performance. Geometrical analyses of the map have revealed a spatially heterogeneous volume growth pattern that is consistent with the expression pattern of a major morphogen and anisotropic tissue deformation along an axis. Thus, our method enables deformation dynamics analysis in organogenesis using practical lineage marking techniques. (C) 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license

DOI： 10.1016/j.jtbi.2014.04.027

Publishing type：Research paper (scientific journal)  

Background: The forelimb-specific gene tbx5 is highly conserved and essential for the development of forelimbs in zebrafish, mice, and humans. Amongst birds, a single order, Dinornithiformes, comprising the extinct wingless moa of New Zealand, are unique in having no skeletal evidence of forelimb-like structures.
Results: To determine the sequence of tbx5 in moa, we used a range of PCR-based techniques on ancient DNA to retrieve all nine tbx5 exons and splice sites from the giant moa, Dinornis. Moa Tbx5 is identical to chicken Tbx5 in being able to activate the downstream promotors of fgf10 and ANF. In addition we show that missexpression of moa tbx5 in the hindlimb of chicken embryos results in the formation of forelimb features, suggesting that Tbx5 was fully functional in wingless moa. An alternatively spliced exon 1 for tbx5 that is expressed specifically in the forelimb region was shown to be almost identical between moa and ostrich, suggesting that, as well as being fully functional, tbx5 is likely to have been expressed normally in moa since divergence from their flighted ancestors, approximately 60 mya.
Conclusions: The results suggests that, as in mice, moa tbx5 is necessary for the induction of forelimbs, but is not sufficient for their outgrowth. Moa Tbx5 may have played an important role in the development of moa's remnant forelimb girdle, and may be required for the formation of this structure. Our results further show that genetic changes affecting genes other than tbx5 must be responsible for the complete loss of forelimbs in moa.

DOI： 10.1186/1471-2148-14-75

PubMed

Digit identity has been studied using the chick embryo as a model system for more than 40 years. Using this model system, several milestone findings have been reported, such as the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), the Shh gene, and the theory of morphogen and positional information. These experimental results and models provided context for understanding pattern formation in developmental biology. The focus of this review is on the determination of digit identity during limb development. First, the history of studies on digit identity determination is described, followed by descriptions of the molecular mechanisms and current models for determination of digit identity. Finally, future questions and remarkable points will be discussed.

DOI： 10.1111/dgd.12022

PubMed

Publishing type：Research paper (scientific journal)  

The evolutionarily conserved, non-coding similar to 800-base-pair (bp) zone of polarizing activity (ZPA) regulatory sequence (ZRS) controls Shh expression in the posterior limb. We report that the chicken mutant oligozeugodactyly (ozd), which lacks limb Shh expression, has a large deletion within the ZRS. Furthermore, the preaxial polydactylous, Silkie Breed chicken, which develops ectopic anterior limb Shh expression, has a single bp change within the ZRS. Using an in vivo reporter assay to examine enhancer function in the chick limb, we demonstrate that the wild-type ZRS drives beta-galactosidase reporter expression in the ZPA of both wild-type and ozd limbs. The Silkie ZRS drives beta-galactosidase in both posterior and anterior Shh domains in wild-type limb buds. These results support the hypothesis that the ZRS integrates positive and negative prepatterned regulatory inputs in the chicken model system and demonstrate the utility of the chicken limb as an efficient genetic system for gene regulatory studies. Developmental Dynamics 240: 1212-1222, 2011. (C) 2011 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.22634

PubMed

Publishing type：Research paper (scientific journal)  

Half a century ago, thalidomide was widely prescribed to pregnant women as a sedative but was found to be teratogenic, causing multiple birth defects. Today, thalidomide is still used in the treatment of leprosy and multiple myeloma, although how it causes limb malformation and other developmental defects is unknown. Here, we identified cereblon (CRBN) as a thalidomide-binding protein. CRBN forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1) and Cul4A that is important for limb outgrowth and expression of the fibroblast growth factor Fgf8 in zebrafish and chicks. Thalidomide initiates its teratogenic effects by binding to CRBN and inhibiting the associated ubiquitin ligase activity. This study reveals a basis for thalidomide teratogenicity and may contribute to the development of new thalidomide derivatives without teratogenic activity.

DOI： 10.1126/science.1177319

PubMed

Publishing type：Part of collection (book)  

Limb development has been studied for over 100 years by several generations of developmental biologists. The developing limb is one of the best models with which to study pattern formation in vertebrates. We have used chick limb development to answer a simple but basic question, namely, why heterogeneous tissues are formed at correct positions and times from a homogeneous population of cells (Pearse &amp
Tabin, 1998). Limb development starts as two pairs of tissue bulges in the lateral plate meso-derm (LPM). These are called the forelimb and hindlimb fields (Fig. 9.1). After limb initiation, one can clearly identify three-dimensional axes in the limb buds: the proximal-distal (PD
from shoulder to fingers), dorso-ventral (DV
from back to palm), and antero-posterior (AP
from thumb to little fingers) axes. Morphological changes and differences along these three axes are determined by pattern formation during limb bud stages. Following establishment of these axes, one can visually recognize condensation of cartilages. Muscles, tendons, and neurons migrate and differentiate after cartilage formation. Because the stages and events are easily recognized morphologically and in detail, it is therefore the limb bud is an excellent model with which to study the molecular mechanisms of embryonic patterning and tissue differentiation in vertebrates. © 2009 Springer Japan.

DOI： 10.1007/978-4-431-09427-2_9

Electroporation is a powerful tool with which to study limb development. Limb development, however, remains an intricate series of events, requiring the precise dissection of developmental processes using relevant transgenes. In this review, we describe the anatomy of the limb field as the basis of targeted electroporation, and specific expression vectors are discussed. We share a useful protocol for electroporation of chick limb buds, and the expression pattern of enhanced green fluorescent protein in the limb buds is used to demonstrate relevant embryonic patterning. Finally, useful trouble-shooting techniques are described.

DOI： 10.1111/j.1440-169x.2008.01054.x

PubMed

Publishing type：Research paper (scientific journal)  

The zone of polarizing activity is the primary signaling center controlling anterior-posterior patterning of the amniote limb bud. The autopodial interdigits (IDs) are secondary signaling centers proposed to determine digit identity by acting on the cells of the digital ray. Here, we focus on events accompanying digital fate determination and define a region of the digital ray that expresses Sox9 and Bmpr1b and is phosphorylated-SMAD1/5/8 (p-SMAD1/5/8) positive. We name this region the phalanx-forming region (PFR), and show that the PFR cells arise from the distal subridge mesenchyme of digital ray. This phalanx-forming cell lineage is subsequently committed to the cartilage lineage; the fate of these cells is initially labile but becomes fixed as they are incorporated into the condensed cartilage of the digit primordium. Using an in vivo reporter assay, we establish that each digital PFR has a unique p-SMAD1/5/8 activity signature. In addition, we show that changes in this activity correlate with the identity of the digit that forms after experimental manipulation, supporting the idea that threshold signaling levels can lead to different developmental outcomes in a morphogenetic field. Our data define the molecular profile of the PFR, and we propose a model for understanding formation and variation of digits during autopodial development.

DOI： 10.1073/pnas.0707899105

PubMed

Publishing type：Research paper (scientific journal)  

Based on recent data, a new view is emerging that vertebrate Dachshund (Dach) proteins are components of Six1/6 transcription factor-dependent signaling cascades. Although Drosophila data strongly suggest a tight link between Dpp signaling and the Dachshund gene, a functional relationship between vertebrate Dach and BMP signaling remains undemonstrated. We report that chick Dach1 interacts with the Smad complex and the corepressor mouse Sin3a, thereby acting as a repressor of BMP-mediated transcriptional control. In the limb, this antagonistic action regulates the formation of the apical ectodermal ridge (AER) in both the mesenchyme and the AER itself, and also controls pattern formation along the proximodistal axis of the limb. Our data introduce a new paradigm of BMP antagonism during limb development mediated by Dach1, which is now proven to function in different signaling cascades with distinct interacting partners.

DOI： 10.1242/dev.01252

PubMed

Publishing type：Research paper (scientific journal)  

Despite extensive studies on the anterior-posterior (AP) axis formation of limb buds, mechanisms that specify digit identities along the AP axis remain obscure. Using the four-digit chick leg as a model, we report here that Tbx2 and Tbx3 specify the digit identities of digits IV and III, respectively. Misexpression of Tbx2 and Tbx3 induced posterior homeotic transformation of digit III to digit IV and digit II to digit III, respectively. Conversely, misexpression of their mutants VP16DeltaTbx2 and VP16DeltaTbx3 induced anterior transformation. In both cases, alterations in the expression of several markers (e.g., BMP2, Shh, and HoxD genes) were observed. In addition, Tbx2 and Tbx3 rescued Noggin-mediated inhibition of interdigital BMP signaling, signaling which is pivotal in establishing digit identities. Hence, we conclude that Tbx3 specifies digit III, and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade.

PubMed

Publishing type：Research paper (scientific journal)  

A tight loop between members of the fibroblast growth factor and the Wnt families plays a key role in the initiation of vertebrate limb development. We show for the first time that Tbx5 and Tbx4 are directly involved in this process. When dominant-negative forms of these Tbx genes were misexpressed in the chick prospective limb fields, a limbless phenotype arose with repression of both Wnt and Fgf genes By contrast, when Tbx5 and Tbx4 were misexpressed in the flank, an additional wing-like and an additional leg-like limbs were induced, respectively. This additional limb formation was accompanied by the induction of both Wnt and Fgf genes These results highlight the pivotal roles of Tbx5 and Tbx4 during limb initiation, specification of forelimb/hindlimb and evolution of tetrapod limbs, placing Tbx genes at the center of a highly conserved genetic program.

DOI： 10.1242/dev.00474

PubMed

Publishing type：Research paper, summary (international conference)  

DOI： 10.1016/j.ydbio.2008.05.516

Presentation type：Oral presentation (invited, special)  

Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (general)  

Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (invited, special)