Homeolog-specific targeted mutagenesis in Xenopus laevis using TALENs.

Transcription activator-like effector nucleases (TALENs) have previously been used for targeted genome editing in various organisms including Xenopus laevis. However, because of genomic polyploidization, X. laevis usually possess homeologous genes (homeologs) with quite similar sequences that make the analysis of gene function difficult. In the present study, we show methodological examples of targeted gene modification of X. laevis homeologs. The X. laevis cytoglobin gene (cygb) consists of two homeologs (xlcygba and xlcygbb), and molecular phylogenetic analysis suggested that they have potentially different functions. Thus, there is a need to establish a method of homeolog-specific gene disruption to clarify gene functions in detail. Here, we show successful examples of homeolog-specific and simultaneous gene disruption for xlcygba and xlcygbb. We found that selective digestion can be performed with at least three mismatches in TALEN target sites in both homeologs. This report paves the way for the functional analyses of X. laevis homeologs, even those containing nearly identical sequences.

Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9.

Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) mediated by microhomology-mediated end-joining, termed the PITCh (Precise Integration into Target Chromosome) system. TALEN-mediated PITCh, termed TAL-PITCh, enables efficient integration of exogenous donor DNA in human cells and animals, including silkworms and frogs. We further demonstrate that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence. Thus, our PITCh-ing strategies will be useful for a variety of applications, not only in cultured cells, but also in various organisms, including invertebrates and vertebrates.

Production of a non-triple helical collagen alpha chain in transgenic silkworms and its evaluation as a gelatin substitute for cell culture.

We generated transgenic silkworms that synthesized human type I collagen alpha1 chain [alpha1(I) chain] in the middle silk glands and secreted it into cocoons. The initial content of the recombinant alpha1(I) chain in the cocoons of the transgenic silkworms was 0.8%. The IE1 gene, a trans-activator from the baculovirus, was introduced into the transgenic silkworm to increase the content of the chain. We also generated silkworms homozygous for the transgenes. These manipulations increased the alpha1(I) chain content to 8.0% (4.24 mg per cocoon). The alpha1(I) chain was extracted and purified from the cocoons using a very simple method. The alpha1(I) chain contained no hydroxyprolines due to the absence of prolyl-hydroxylase activity in the silk glands. Circular dichroism analysis showed that the secondary structure of the alpha1(I) chain is similar to that of denatured type I collagen, demonstrating the absence of the triple helical structure. Human skin fibroblasts were seeded on the alpha1(I) chain-coated dishes. The cells attached and spread, although at decreased chain concentrations the spreading rate was lower than that of the collagen and gelatin. Cynomolgus monkey embryonic stem cells cultured on the alpha1(I) chain-coated dishes maintained an undifferentiated state after 30 passages, and their pluripotency was confirmed by teratoma formation in severe combined immunodeficient mice. These results show that the recombinant human alpha1(I) chain is a promising candidate biomaterial as a high-quality and safe gelatin substitute for cell culture.

The third type III module of human fibronectin mediates cell adhesion and migration.

Fibronectin (FN) is a major extracellular matrix protein involved in various biological events. This study demonstrated that the third FN type III repeat (FnIII3) and several fragments containing the repeat promote cell spreading and migration of human dermal fibroblasts (HDFs), whereas the fourth repeat (FnIII4) did not. A variety of cell types also spread on FnIII3 in a cell-type-specific manner, but not on FnIII4. Immunofluorescence assays revealed that FnIII3 induced the organization of focal contacts and stress fibres in HDFs. Cyclic [RGDFV] peptides with a D-Phe residue, which are selective inhibitors of cell adhesion to vitronectin, inhibited HDF spreading on FnIII3 equally with GRGDS, indicating little involvement of alphaV-integrins in FnIII3 spreading. An anti-beta1 integrin mAb inhibited cell spreading on FnIII3 and FN. To our knowledge, this is the first demonstration that a novel domain of FnIII3 functions in cell spreading and migration through an interaction with unresolved beta1 integrin(s) in an RGD-dependent manner.

Gene expression and tissue distribution of cytoglobin and myoglobin in the Amphibia and Reptilia: possible compensation of myoglobin with cytoglobin in skeletal muscle cells of anurans that lack the myoglobin gene.

Cytoglobin (Cygb), a recently discovered vertebrate cytoplasmic heme-binding globin, is considered to be in a clade with vertebrate myoglobin (Mb), which is exclusively distributed in the cytoplasm of cardiac and skeletal muscles as an oxygen storage protein. GenBank databases (NCBI and JGI) and gene synteny analyses showed the absence of the Mb gene (mb) in two anuran amphibians, Xenopus laevis and X. tropicalis. Here we conducted comparative studies on the gene expression and tissue distribution of Cygb and Mb in anuran and reptilian tissues. Cygb and Mb genes were cloned from a reptile, iguana (Iguana iguana). Two types of cygb (cygb-1 and -2) were cloned, with lengths of 1066 and 1034 bp, and 196 and 193 amino acid residues, respectively. Their nucleotide and amino acid sequence identities were 90 and 87%, respectively. The Mb gene covered 1416 bp with an open reading frame of 465 bp, giving rise to a 154 amino acid protein. The distal ligand-binding histidine at E7, the proximal heme-binding histidine at F8, and the phenylalanine residue at CD1 were conserved in Mb and Cygb. The nucleotide and amino acid sequence identity of I. iguana cygb-1 and cygb-2 against X. laevis cygb were approximately 67% and 65%, respectively. RT-PCR demonstrated that X. laevis cygb was uniquely expressed in the heart and skeletal muscles, and faintly in the liver and spleen, which was quite contrasted with Iguana and the other vertebrates, where mb is exclusively expressed in the heart and skeletal muscles. Immunohistochemical analyses showed the distribution of Cygb in the cytoplasm of skeletal muscle cells. Interestingly, Cygb in the heart was localized in the nuclei. Considering the absence of mb in the Anura, we hypothesize that Cygb in muscle cells of anurans compensates for the lack of Mb for the storage and intracellular transportation of oxygen.

Characterization of histone lysine-specific demethylase in relation to thyroid hormone-regulated anuran metamorphosis.

The thyroid hormone receptor (THR) is a member of the nuclear transcription factor and plays a central role in regulating anuran metamorphosis. Previous studies with mammalian cells have suggested that THR is involved in chromatin remodeling through histone methylation. In the present study, we cloned cDNA of lysine-specific demethylase gene, xLSD1, from Xenopus laevis and examined its expression in relation to metamorphosis. Overexpression of xLSD1 in A6 cells, a Xenopus laevis cell line, resulted in the decrease of methylation status of lysine residues of histone H3, indicating that the protein of cloned xLSD1 was functionally active. The expression of LSD1 at mRNA levels was up-regulated in the body skin and the intestine during natural and thyroid hormone-induced metamorphosis. Larval epidermal basal cells and intestinal epithelial cells at the premetamorphic stage were identified as the xLSD1-expressing cells. At the metamorphic climax stage the progenitor cells of adult epidermal basal cells also expressed xLSD1, whereas those of the adult intestinal epithelial cells did not. We propose that LSD1 participates in the regulation of metamorphosis through THR- or another transcriptional factor-induced chromatin remodeling.

Identification of vitamin A-free cells in a stellate cell-enriched fraction of normal rat liver as myofibroblasts.

Myofibroblasts (MFs) as well as hepatic stellate cells (HSCs) are known to be involved in liver fibrogenesis. Quiescent HSCs (qHSCs) in culture have been thought to differentiate to replicative activated HSCs (aHSCs). In this study a qHSC-enriched fraction isolated by Nycodenz-isodensity centrifugation was separated with a fluorescence-activated cell sorter, which revealed the presence of a small fraction (occupancy rate=0.4%) of cells that did not show vitamin A-autofluorescence under ultraviolet (UV)-irradiation (UV- cells). The remaining vitamin A-containing cells were autofluorescent (UV+) and originally expressed markers of qHSCs, and, in culture, did not grow, lost vitamin A, and expressed markers of aHSCs. UV- cells showed morphology of MFs, and, in culture, grew to form colonies and expressed markers of MFs. These results indicated that UV+ and UV- cells represent qHSCs and MFs, respectively, and that aHSCs have no growth potential and are a cell-type distinct from proliferative MFs. Gene expression profiles of UV- cells (MFs) newly identified gremlin as one of MF-preferential genes and its proteins were localized around fibrotic septa in rat and human livers. In addition, we suggested that the qHSC-enriched fraction included approximately 6% of liver MFs.

Typing of hepatic nonparenchymal cells using fibulin-2 and cytoglobin/STAP as liver fibrogenesis-related markers.

Fibulin-2 and cytoglobin/stellate cell activation-associated protein (Cygb/STAP) are considered to be markers of hepatic myofibroblasts (MFs) and stellate cells (HSCs), respectively. The aim of the present study was to characterize the nonparenchymal cells (NPCs) of normal rat livers and carbon tetrachloride-induced fibrotic rat livers with respect to the expression of these two proteins. NPCs in normal (Glisson's capsules) and fibrotic (fibrotic septa) connective tissues were immunohistochemically categorized into four cell types in terms of the expression of fibulin-2 and Cygb/STAP: fibulin-2 and Cygb/STAP double-positive (Fib(+)/STAP(+)); fibulin-2-positive and Cygb/STAP-negative (Fib(+)/STAP(-)); Fib(-)/STAP(+); and Fib(-)/STAP(-). The Glisson's capsules had Fib(+)/STAP(+) and Fib(-)/STAP(-) cell occupancy rates of 45.5% and 54.5%, respectively, but did not contain Fib(+)/STAP(-) or Fib(-)/STAP(+) cells. On the other hand, the fibrotic septa contained Fib(+)/STAP(+), Fib(-)/STAP(+), and Fib(-)/STAP(-) cells at occupancy rates of 35.0%, 50.5%, and 9.1%, respectively, but did not contain Fib(+)/STAP(-) cells. Thus, fibrosis is characterized by a dramatic increase in Fib(-)/STAP(+) NPCs, and a dramatic decrease in Fib(-)/STAP(-) NPCs. Fib(+)/STAP(+) NPCs are located uniformly in Glisson's capsules and peripherally in fibrotic septa. The present study strongly suggests that Fib(+)/STAP(+) and Fib(-)/STAP(+) NPCs correspond to MFs and activated HSCs, respectively, both of which may contribute to liver fibrogenesis.

Unique expression patterns of matrix metalloproteinases in regenerating newt limbs.

The process of regeneration of urodele limbs includes a drastic remodeling of extracellular matrices (ECMs) that is induced by matrix metalloproteinases (MMPs) and is thought to be one of the triggers of the regeneration. We studied this remodeling in limbs of Japanese newt, Cynops pyrrhogaster, by using five genes of newt MMPs (nMMPs) as probes: nMMP9, nMMP3/10-a, nMMP3/10-b, and nMMP13 that had been characterized previously, and nMMPe that was newly cloned in the present study. nMMPe was 502 amino acid residues long and showed a low homology to other known vertebrate MMPs. Reverse transcriptase-polymerase chain reactions analysis localized the transcript of nMMPe in the apical epidermal cap (AEC) and the non-blastemal wound epidermis but not in the blastemal mesenchyme or the normal epidermis. Northern blot analysis localized the transcripts of nMMP9, nMMP3/10-a, and nMMP13 in the bone of regenerating limbs, whereas those of nMMP3/10-b in AEC. mRNA in situ hybridization experiments identified the nMMP-expressing cells. nMMP9 gene was strongly expressed in chondrocytes of the cartilage of epiphysis. Of interest, basal cells of AEC, but not those of the normal skin, expressed nMMP3/10-b intensely. Immunohistochemical analysis showed that the nMMP9 proteins synthesized by chondrocytes were secreted and distributed widely in the basement membrane of bone and ECMs of the amputation plane. These nMMPs characterized in the present study might cooperatively work to remodel ECMs of regenerating limbs.

Metal ion-responsive transgenic Xenopus laevis as an environmental monitoring animal.

We generated germ line-transgenic Xenopus laevis that monitors environmental heavy metal ions. Sperm nuclei were transduced with cDNA of enhanced green fluorescent protein (EGFP) driven by murine metallothionein-1 gene promoters and were microinjected into unfertilized eggs. The eggs developed to sexually matured adults. The transgenic tadpoles at the premetamorphic stage were reared in water containing Zn(2+) and Cd(2+) separately at the concentrations of 0.38-1.52 and 0.09-0.44 µM, respectively. These animals responded to Zn(2+) at as low as 0.38 µM and Cd(2+) at as low as 0.44 µM. The level of EGFP fluorescence emitted by tadpoles increased as the concentration increased up to 1.52 µM and the exposure time prolonged up to 120 h. The fluorescent response was much more sensitive to Cd(2+) than to Zn(2+). We concluded that these transgenic tadpoles are useful as an animal indicator of environmental heavy metal ions.

Molecular identification of the skin transformation center of anuran larval skin using genes of Rana adult keratin (RAK) and SPARC as probes.

Anuran larval skin undergoes a process of metamorphosis into pre-adult and adult skin. Basal skein, larval basal and adult basal cells are basement membrane-attaching cells in the larval, pre-adult and adult epidermis, respectively, and are identified as cells expressing genes of RLK (Rana larval keratin), both RLK and RAK (Rana adult keratin), and RAK. Larval to pre-adult skin conversion takes place in the histological entity called the skin transformation center (STC). The present study performed a cDNA subtractive gene screening on cDNA of the larval and the pre-adult skin, and cloned the secreted protein acidic and rich in cysteine (SPARC) gene as an upregulated gene in the larva to pre-adult skin conversion. RAK gene-positive basal skein cells and fibroblasts in and around the STC were weakly and strongly sparc-positive, respectively. Using sparc and rak, we redefined the STC and visualized it on a histological section as an approximately 150 microm-long region that contained about 20 rak-negative and weakly sparc-positive basal cells. Intense sparc expression was observed in basal skein cells, but not in larval basal cells, suggesting that SPARC acts as a suppressor of rak during epidermal differentiation. This suggestion was tested by investigating the effect of SPARC on cultured larval basal cells. We observed that SPARC suppressed the expression of rak, but not rlk.

Metamorphosis-dependent transcriptional regulation of xak-c, a novel Xenopus type I keratin gene.

Anuran larvae transform their epidermis to the adult counterpart during metamorphosis. The major event of this process is the proliferation of larval epidermal basal cells and their differentiation into adult ones. The present study isolated novel type I keratin cDNA dubbed xak-c (Xenopus adult keratin-c) that was exclusively expressed in adult epidermal basal cells. The gene started its expression in the larval epidermis at the onset of metamorphosis. Thyroid hormone (TH) induced the precocious expression of the gene in the epidermis of premetamorphic tadpoles. To study the transcriptional regulation of this gene in relation to epidermal metamorphosis, a 2.8 kb 5'-flanking region of xak-c was cloned and its promoter activity was investigated. Gene constructs were made so as to contain the xak-c promoter region and gene of EGFP or luciferase as a reporter gene and were transfected into various types of cells, which revealed that the 5'-flanking region had an epidermal cell-specific transcriptional activity in both anurans and mammals. Larval skin tissues of Xenopus were transfected with the constructs and cultured in the presence and absence of TH, which showed that the promoter region is responsive to TH, although the region did not contain the consensus TH response element-like sequence. In sharp contrast, the promoter region did not respond to TH in the adult skin, clearly indicating that the cloned region contains specific sequences that respond to metamorphosis-dependent transcription factor(s).

Characterization of human stellate cell activation-associated protein and its expression in human liver.

This study cloned cDNA of human homologue (hSTAP) of rat stellate cell activation-associated protein (rSTAP). hSTAP gene is on chromosome 17q and is composed of four exons. Various types of cells including hepatic stellate cells expressed hSTAP mRNA. Recombinant hSTAP was a heme protein with the activity of peroxidase. hSTAP can be used as a marker of quiescent stellate cells in human liver.

Lineage of anuran epidermal basal cells and their differentiation potential in relation to metamorphic skin remodeling.

The anuran remodels the larval epidermis into the adult one during metamorphosis. Larval and adult epidermal cells of the bullfrog were characterized by determining the presence of huge cytoplasmic keratin bundles and the expression profiles of specific marker genes, namely colalpha1 (collagen alpha1 (I)), rlk (larval keratin) and rak (adult keratin). We identified four types of epidermal basal cells: (i) basal skein cells that have keratin bundles and express colalpha1 and rlk; (ii) rak+-basal skein cells that have keratin bundles and express colalpha1, rlk, and rak; (iii) larval basal cells that express rlk and rak; and (iv) adult basal cells that express rak. These traits suggested that these basal cells are on the same lineage in which basal skein cells are the original progenitor cells that consecutively differentiate into rak+-basal skein cells into larval basal cells, and finally into adult basal cells. To directly verify the differentiation potential of larval basal cells into adult ones, the mono-layered epidermis composed of larval basal cells was cultured in the presence of aldosterone and thyroid hormone. In this culture, larval basal cells differentiated into adult basal cells that reconstituted the adult epidermis. Thus, it was concluded that larval basal cells are the direct progenitor cells of the adult epidermal stem cells.

Characterization of gene of anuran cathepsin D as a metamorphosis-associated enzyme.

Cathepsin D is a member of the lysosomal aspartic proteinases and has been claimed to play a crucial role in the breakdown of larval tissues during anuran metamorphosis. The present study aimed to characterize the gene of anuran cathepsin D as an approach for elucidating the molecular mechanism of involution of larval tissues. Three overlapping cDNA of Xenopus cathepsin D were amplified by the polymerase chain reaction and were cloned, which covered 590 bp, 355 bp and 790 bp sequences of the region between two enzyme active sites, a 5'-end and 3'-end regions of the gene, respectively. Altogether they revealed 1.6 kbp encoding 422 amino acids of the enzyme. Xenopus cathepsin D showed about 60% homology to mammalian enzymes at the amino acid sequence level. Northern blot analyses revealed the gradual increase of cathepsin D transcripts in the tail during prometamorphosis, reaching a peak at stage 61 and 62. Thyroid hormone notably enhanced the signals of northern blotting of the tail. The in situ hybridization histochemistry showed that both epidermal cells in the basal layer and subepidermal mesenchymal cells embedded in the thick layer of collagen fibers produce high levels of mRNA of cathepsin D in response to thyroid hormone, suggesting active contribution of the cells closely located at the basement membrane to the breakdown of tail tissues.