Convergent Acquisition of Nonembryonic Development in Styelid Ascidians.

Asexual propagation and whole body regeneration are forms of nonembryonic development (NED) widespread across animal phyla and central in life history and evolutionary diversification of metazoans. Whereas it is challenging to reconstruct the gains or losses of NED at large phylogenetic scale, comparative studies could benefit from being conducted at more restricted taxonomic scale, in groups for which phylogenetic relationships are well established. The ascidian family of Styelidae encompasses strictly sexually reproducing solitary forms as well as colonial species that combine sexual reproduction with different forms of NED. To date, the phylogenetic relationships between colonial and solitary styelids remain controversial and so is the pattern of NED evolution. In this study, we built an original pipeline to combine eight genomes with 18 de novo assembled transcriptomes and constructed data sets of unambiguously orthologous genes. Using a phylogenomic super-matrix of 4,908 genes from these 26 tunicates we provided a robust phylogeny of this family of chordates, which supports two convergent acquisitions of NED. This result prompted us to further describe the budding process in the species Polyandrocarpa zorritensis, leading to the discovery of a novel mechanism of asexual development. Whereas the pipeline and the data sets produced can be used for further phylogenetic reconstructions in tunicates, the phylogeny provided here sets an evolutionary framework for future experimental studies on the emergence and disappearance of complex characters such as asexual propagation and whole body regeneration.

Japan's initiative on rare and undiagnosed diseases (IRUD): towards an end to the diagnostic odyssey.

Japan has been facing challenges relating to specifically defined rare diseases, called Nan-Byo in Japanese (literally 'difficult'+'illness'), and has already taken measures for them since 1972. This governmental support has surely benefited Nan-Byo patients; however, those suffering from medically unidentified conditions do not fall into this scheme and thus still confront difficulty in obtaining an examination, a diagnosis, and a treatment. To identify such rare and often undiagnosed diseases, we must integrate systematic diagnosis by medical experts with phenotypic and genetic data matching. Thus, in collaboration with Nan-Byo researchers and the Japanese universal healthcare system, the Japan Agency for Medical Research and Development launched the Initiative on Rare and Undiagnosed Diseases (IRUD) in 2015. IRUD is an ambitious challenge to construct a comprehensive medical network and an internationally compatible data-sharing framework. Synergizing with existing next-generation sequencing capabilities and other infrastructure, the nationwide medical research consortium has successfully grown to accept more than 2000 undiagnosed registrants by December 2016. We also aim at expanding the concept of microattribution throughout the initiative; that is, proper credit as collaborators shall be given to local primary care physicians, nurses and paramedics, patients, their family members, and those supporting the affected individuals whenever appropriate. As it shares many challenges among similar global efforts, IRUD's future successes and lessons learned will significantly contribute to ongoing international endeavors, involving players in basic research, applied research, and societal implementation.

Looking for putative phenoloxidases of compound ascidians: haemocyanin-like proteins in Polyandrocarpa misakiensis and Botryllus schlosseri.

Phenoloxidases (POs) and haemocyanins constitute a family of copper-containing proteins widely distributed among invertebrates. Both of them are able, under appropriate conditions, to convert polyphenols to quinones and induce cytotoxicity through the production of reactive oxygen species, a fundamental event in many immune responses. In ascidians, PO activity has been described and studied in both solitary and colonial species and the enzyme is involved in inflammatory and cytotoxic reactions against foreign cells or molecules, and in the formation of the cytotoxic foci which characterise the nonfusion reaction of botryllids. Expressed genes for two putative POs (CiPO1 and CiPO2) have been recently identified in C. intestinalis. In the present study, we determined the cDNA sequences of two haemocyanin-like proteins from two colonial ascidians: Botryllus schlosseri from the Mediterranean Sea and Polyandrocarpa misakiensis from Japan. Multiple sequence alignments evidenced the similarity between the above sequences and crustacean proPOs whereas the analysis of the three-dimensional structure reveals high similarity with arthropod haemocyanins which share common precursors with arthropod proPOs. Botryllus HLP grouped in the same cluster with Ciona POs, whereas Polyandrocarpa HLP clustered with arthropod haemocyanins; all of them share the full conservation of the six histidines at the two copper-binding sites as well as of other motifs, also found in arthropod haemocyanin subunits, involved in the regulation of enzyme activity. In situ hybridisation indicated that the genes are transcribed inside morula cells, a characteristic haemocyte type in ascidians where PO activity is located, at the beginning of their differentiation. These results represent a first attempt to identify candidate molecules responsible of the PO activity in compound ascidians.

Expression and function of myc during asexual reproduction of the budding ascidian Polyandrocarpa misakiensis.

The budding ascidian Polyandrocarpa misakiensis proliferates asexually by budding. The atrial epithelium is a multipotent but differentiated tissue, which transdifferentiates into various tissues and organs after the bud separates from the parental body. We isolated cDNA clones homologous to the myc proto-oncogene from P. misakiensis. The cDNA, named Pm-myc, encoded a polypeptide of 639 amino acid residues, containing Myc-specific functional motifs, Myc box I and Myc box II, and the basic helix-loop-helix domain. Expression of Pm-myc was observed in the atrial epithelium in the organ-forming region of the developing bud, where the epithelial cells dedifferentiate and re-enter the cell cycle. The expression was also observed in fibroblast-like cells, which are known to participate in the organogenesis together with the epithelial cells. Unexpectedly, the atrial epithelium expressed Pm-myc more than one day before the dedifferentiation. The organogenesis was disturbed by Pm-myc-specific double-stranded RNA. In situ hybridization revealed that Pm-myc-positive fibroblast-like cells disappeared around the organ primordium of the dsRNA-treated bud. The results suggest that the mesenchymal-epithelial transition of fibroblast-like cells is important for the organogenesis in this budding ascidian species.

Piwi-expressing hemoblasts serve as germline stem cells during postembryonic germ cell specification in colonial ascidian, Botryllus primigenus.

Animals that propagate asexually are exciting models to investigate the cellular system, which produces germline cells constitutively throughout life. The present research investigated whether piwi was a germline-specific marker in the colonial ascidian Botryllus primigenus. An approximately 2.8 kb long cDNA fragment was cloned and termed BpPiwi, since the obtained amino acid sequence (874 aa) contained PAZ and PIWI domains. BpPiwi was expressed specifically by germline cells such as the loose cell mass (germline precursor cells), oocytes, spermatogonia, and spermatocytes. In addition, BpPiwi transcripts were also detected in some coelomic cells in the hemocoel and tunic vessels. BpPiwi(+) coelomic cells possessed similar morphological features to hemoblasts (stem cells). The concentration of BpPiwi(+) cells was found to be significantly lower than that obtained for hemoblasts suggesting that BpPiwi(+) cells comprise a fraction of hemoblasts. Further, the ability of BpPiwi(+) cells to serve as somatic stem cells was examined. No BpPiwi signals were detected from somatic hemoblasts forming vascular buds. The genetic knockdown of BpPiwi induced by siRNA injection resulted in the formation of a defective germline precursor. These results suggest that BpPiwi(+) hemoblasts reside in the hemocoel and tunic vessels and function as germline stem cells in the postembryonic colony. Based on the findings of the characterization of three effective germline genes piwi, vasa, and nanos, we propose that germline stem cells reside as BpPiwi(+)/BpVas(-)/BpNos(+) hemoblasts in B. primigenus.

Regeneration of the gut requires retinoic acid in the budding ascidian Polyandrocarpa misakiensis.

The protochordate ascidian Polyandrocarpa misakiensis has a striking ability to regenerate. When the posterior half of the adult body is amputated, the anterior half completely loses the esophagus, stomach and intestine. These organs are reconstituted in a week. Histological observation revealed that the regeneration involves transdifferentiation of the atrial epithelium near the cut surface. The morphological features of the gut primordium were similar to those observed in the developing bud of this species. Inhibitors of the synthesis of retinoic acid (RA) suppressed the formation of the gut. 13-cis RA rescued the regenerates from the inhibitor-induced hypoplasia. These results suggest that RA is required for the regeneration of the gut. A gene encoding the RA receptor (Pm-RAR) and its target gene, TRAMP, were expressed in and around the regenerating gut. Pm-RAR-specific and TRAMP-specific double-stranded RNA molecules inhibited the regeneration of the gut, indicating that the RA signal is mediated at least in part by Pm-RAR and TRAMP. These results suggested that RA triggers the transdifferentiation of the atrial epithelium into the gut in regenerating animals, as it does during asexual reproduction.

The role of Nanos homologue in gametogenesis and blastogenesis with special reference to male germ cell formation in the colonial ascidian, Botryllus primigenus.

We examined the structure, expression, and possible functions of a nanos homologue during gametogenesis and blastogenesis in the colonial ascidian Botryllus primigenus. An approximately 1.3-kb-long cDNA was cloned; it was termed BpNos since the deduced amino acid sequence (288 aa) contained 2 Nanos-like CCHC zinc finger motifs. Immature and mature male germ cells expressed BpNos most strongly, while loose aggregates of hemoblasts, multipotent epithelial cells (in developing buds) and a few coelomic cells in the hemocoel and tunic vessels weakly expressed BpNos. No signals were detected from female germ cells. To determine possible functions of BpNos, B. primigenus colonies were injected with BpNos short interfering (si)RNA. Buds developed normally, showing that BpNos plays a limited role in B. primigenus blastogenesis. However, the developing buds possessed no spermatogonia and spermatocytes in the testes, although oocytes developed normally. In the knockdown colonies, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay (TUNEL)-positive male germ cells were observed, suggesting that BpNos siRNA treatment might induce apoptosis. In conclusion, BpNos is a weak marker of germline precursor cells and multipotent somatic epithelial cells but a strong marker of spermatogonia and spermatocytes. A major function of BpNos may be the maintenance of male germline cells.

Cell proliferation dynamics of somatic and germline tissues during zooidal life span in the colonial tunicate Botryllus primigenus.

Botryllus primigenus is a colonial tunicate in which three successive generations develop synchronously. To identify proliferation centers and possible adult stem cells during asexual reproduction, somatic and germline cells were labeled with 5-bromo-2'-deoxyuridine (BrdU). In the youngest generation, multipotent epithelial cells exhibited an average labeling index (LI) of 30% 24 hr after BrdU injection. In the middle generation, the LI of organ rudiments decreased gradually and reached zero by the beginning of the eldest generation. Exceptionally, cells of specialized tissues such as the pharyngeal inner longitudinal vessel and the posterior end of the endostyle continued DNA synthesis and mitosis even in the eldest generation. Proliferating somatic and germline cells of younger generations expressed a Botryllus myc homolog (BpMyc), but adult tissues did not. This result strongly suggests that in B. primigenus undifferentiated progenitor cells are discernible from possible adult stem cells by the presence or absence of BpMyc.

Multipotent epithelial cells in the process of regeneration and asexual reproduction in colonial tunicates.

The cellular and molecular features of multipotent epithelial cells during regeneration and asexual reproduction in colonial tunicates are described in the present study. The epicardium has been regarded as the endodermal tissue-forming epithelium in the order Enterogona, because only body fragments having the epicardium exhibit the regenerative potential. Epicardial cells in Polycitor proliferus have two peculiar features; they always accompany coelomic undifferentiated cells, and they contain various kinds of organelles in the cytoplasm. During strobilation a large amount of organelles are discarded in the lumen, and then, each tissue-forming cell takes an undifferentiated configuration. Septum cells in the stolon are also multipotent in Enterogona. Free cells with a similar configuration to the septum inhabit the hemocoel. They may provide a pool for epithelial septum cells. At the distal tip of the stolon, septum cells are columnar in shape and apparently undifferentiated. They are the precursor of the stolonial bud. In Pleurogona, the atrial epithelium of endodermal origin is multipotent. In Polyandrocarpa misakiensis, it consists of pigmented squamous cells. The cells have ultrastructurally fine granules in the cytoplasm. During budding, coelomic cells with similar morphology become associated with the atrial epithelium. Then, cells of organ placodes undergo dedifferentiation, enter a cell division cycle, and commence morphogenesis. Retinoic acid-related molecules are involved in this dedifferentiation process of multipotent cells. We conclude that in colonial tunicates two systems support the flexibility of tissue remodeling during regeneration and asexual reproduction; dedifferentiation of epithelial cells and epithelial transformation of coelomic free cells.

Strain analysis of arsenic-doped silicon using CBED rocking curves of low-order reflections.

Local lattice strains of semiconductor devices have been so far examined using higher order Laue zone (HOLZ) line patterns of convergent-beam electron diffraction (CBED). Recently, strain analyses in highly strained regions near interfaces have been reported using split HOLZ line patterns. In the present paper, it is demonstrated for arsenic-doped silicon that the use of CBED rocking curves of low-order reflections provides a promising new tool for the determination of strain distributions of highly strained specimen areas. That is, the anomalous intensity increase in the CBED rocking curves of low-order reflections is explained using a model structure with a strain gradient in the electron beam direction, which is similar to the models used for the split HOLZ line patterns.

Body muscle-cell differentiation from coelomic stem cells in colonial tunicates.

Body muscle-cell differentiation was ultrastructurally examined in palleal buds of the colonial tunicate Symplegma reptans. Undifferentiated coelomic cells accumulate near the primordial oral siphon and associate with the basal lamina beneath the epidermis. They initially display the characteristics of hemoblast cells that have a large nucleus with a prominent nucleolus and narrow cytoplasm filled with polysomes. However, they soon become unique due to the development of an indented contour of the nucleus. When the basal lamina of the epidermis develops into the fibrous extracellular matrix (ECM), the muscle precursor cell has the deeply-notched nucleus, and thick and thin filaments in the cytoplasm facing the ECM. Collagen fibril-like structures appear in the ECM. Myofilaments are arranged with the ratio of thick to thin filaments being 1:2.5. Dense bodies and plaques become evident before the oral siphon is perforated. These results show that in S. reptans, the sphincter muscle cells arise from undifferentiated hemoblasts, and that their differentiation begins with a morphological change in their nuclei. Epidermal cells and/or the ECM may have an inductive effect on muscle cell differentiation.

Involvement of vasa homolog in germline recruitment from coelomic stem cells in budding tunicates.

We investigated the mechanism by which germline cells are recruited in every asexual reproductive cycle of the budding tunicate Polyandrocarpa misakiensis using a vasa homolog (PmVas) as the germline-specific probe. A presumptive gonad of Polyandrocarpa arose as a loose cell aggregate in the ventral hemocoel of a 1-week-old developing zooid. It developed into a compact clump of cells and then separated into two lobes, each differentiating into the ovary and the testis. The ovarian tube that was formed at the bottom of the ovary embedded the oogonia and juvenile oocytes, forming the germinal epithelium. PmVas was expressed strongly by loose cell aggregates, compact clumps, and peripheral germ cells in the testis and germinal epithelium. No signals were detected in growing buds and less than 1-week-old zooids, indicating that germ cells arise de novo in developing zooids of P. misakiensis. Cells of the loose cell aggregates were 5-6 mum in diameter. They looked like undifferentiated hemoblasts in the hemocoel. To examine the involvement of PmVas in the germline recruitment at postembryonic stages, both growing buds and 1-week-old developing zooids were soaked with double-stranded PmVas RNA. The growing buds developed into fertile zooids expressing PmVas, whereas the 1-week-old zooids developed into sterile zooids that did not express PmVas. In controls (1-week-old zooids) soaked with double-stranded lacZ RNA, the gonad developed normally. These results strongly suggest that in P. misakiensis, PmVas plays a decisive role in switching from coelomic stem cells to germ cells.

Molecular collaborations between serpins and trefoil factor promote endodermal cell growth and gastrointestinal differentiation in budding tunicates.

We present evidence supporting novel collaborations between the serine protease inhibitor (serpin) and the trefoil factor during the budding stage of the tunicate Polyandrocarpa misakiensis. Using a maltose-binding protein/P-serpin fusion protein, two polypeptides of 40 kDa and 45 kDa were pulled down from Polyandrocarpa homogenates. Based on their partial amino acid sequence data, a single cDNA (928 bp) was cloned. It encodes a polypeptide that has five tandem repeats of a trefoil consensus motif. Thus, we termed the cDNA P-trefoil. Both P-trefoil and P-serpin were expressed exclusively by coelomic cells during budding. P-Trefoil was expressed mainly by coelomic cells throughout the asexual life cycle of Polyandrocarpa, while P-Serpin was localized particularly in coelomic cells and in the extracellular matrix in developing buds. The native P-Trefoil protein showed aminopeptidase activity. It induced cell growth in cultured Polyandrocarpa cells at a concentration of 8 microg/mL. P-Serpin reinforced this activity of P-Trefoil. Further, a mixture of P-Trefoil and P-Serpin exhibited the in vitro induction of a gut-specific alkaline phosphatase. These results show for the first time that a serpin can interact with a trefoil factor to play a role in the cellular growth and differentiation of the gastric epithelium.

In vitro culture of mesenchymal lineage cells established from the colonial tunicate Botryllus primigenus.

Body trunks were isolated from juvenile zooids of the Japanese colonial tunicate Botryllus primigenus and cultured in vitro to establish tissue-specific cell lines. Epidermal cells from some explants spread and formed a flat sheet consisting of vacuolated cells. They then dissociated into single cells, and their growth stopped within two weeks. Continuously proliferating cells were established from four explants. After the 20th implantation, nuclear and mitochondrial DNAs were extracted from these cells. The nucleotide sequences of proliferating cell nuclear antigen (PCNA) and mitochondrial large ribosomal RNA (mtlrRNA) completely matched the PCNA and mtlrRNA taken from living colonies of B. primigenus; this shows that the four independently proliferating cells were indeed of the Botryllus origin. One cell line (Bp0306E10) comprised round-shaped cells with a diameter of 8-10 microm. These cells have been cultured in vitro with a doubling time of approximately 24 hours since June, 2003. The BrdU labeling index was approximately 2%. Monoclonal antibodies raised against the cultured cells recognized a 28 kDa polypeptide and stained free mesenchymal cells in vivo. G418-resistant subclonal cells could be established by introducing a tunicate retrotransposon loaded with the neomycin resistance gene into the cells by electroporation. This study is the first to succeed in producing a sustainable cell culture of Botryllus.

Postembryonic epigenesis of Vasa-positive germ cells from aggregated hemoblasts in the colonial ascidian, Botryllus primigenus.

We investigated whether Vasa was a germline-specific marker in the colonial ascidian Botryllus primigenus, and whether it was inducible epigenetically in the adult life span. We cloned a Botryllus Vasa homologue (BpVas). The deduced open reading frame encoded 687 amino acid residues. It was expressed specifically by germline cells such as the loose cell mass, oogonia and juvenile oocytes in the ovary, and the primordial testis (compact cell mass), spermatogonia and juvenile spermatocytes in the testis. The loose cell mass, the most primitive germline cells, showed an ultrastructure of undifferentiated cells known as hemoblasts. The hemoblasts did not contain electron-dense materials or a mitochondrial assembly in the cytoplasm. These organelles appeared later in the oogonia and oocytes. When the loose cell mass and developing germ cells were eliminated by extirpating all zooids and buds from the colonies, BpVas transcripts disappeared completely from the vascularized colonies. After 14 days, when the colonies regenerated by vascular budding, BpVas-positive cells reappeared in some cases, and in 30 day colonies, BpVas-positive germ cells were observed in all the regenerated colonies. These results show that in B. primigenus, germ cells are inducible de novo from the Vasa-negative cells even at postembryonic stages.

Structure of a cDNA for Ciona Cytochrome b(5) and the ubiquitous expression of mRNA in embryonic tissues.

A cDNA clone for cytochrome b(5) was isolated from a cDNA library of an ascidian, Ciona savignyi, by a plaque hybridization method using a digoxigenin-labeled cDNA for the soluble form of human cytochrome b(5). The cDNA is composed of 5'- and 3'-noncoding sequences, and a 396-base pair coding sequence. The 3'-noncoding sequence contains polyadenylation signal sequences. The amino acid sequence of 132 residues deduced from the nucleotide sequence of the cDNA showed 61% identity and 82% similarity to the cytochrome b(5) of another ascidian species, Polyandrocarpa misakiensis, which we previously cloned. The amino-terminal hydrophilic domain of 98 residues contains well-conserved structures around two histidine residues for heme binding. A cDNA expression system was constructed to prepare a putative soluble form of Ciona cytochrome b(5). The recombinant soluble cytochrome b(5) showed an asymmetrical absorption spectrum at 560 nm as is shown by mammalian cytochromes b(5) upon reduction with NADH and NADH-cytochrome b(5) reductase. The recombinant Ciona cytochrome b(5) is reduced by NADH-cytochrome b(5) reductase with an apparent K(m) value of 3.3 microM. This value is similar to that of the cytochrome b(5) of Polyandrocarpa misakiensis. The expression of Ciona cytochrome b(5) mRNA during development was examined by an in situ hybridization method and ubiquitous expression in embryonic tissues was observed. The results indicate that cytochrome b(5) plays important roles in various metabolic processes during development.

Phospholipids and their derivatives as mitogen and motogen of budding tunicates.

In order to discover novel invertebrate cytokines from the budding tunicate, Polyandrocarpa misakiensis, we treated the water-insoluble fraction of tunicate homogenates with trypsin. The extracts showed remarkable activities to promote the growth and motility of tunicate cells. The activities were heat-stable and proteinase K-resistant. After anion exchange chromatography, the activities were eluted with detergents such as 0.1% deoxycholic acid. The Fourier transform infrared spectrum indicated large amounts of fatty acids and phospholipids instead of polypeptides in the extracts. Consistently, the activities were extractable with organic solvents such as chloroform. Long chains of n-3 polyunsaturated free fatty acids (FFA), phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) were the major components in the lipid-soluble fraction. A cDNA for FFA-releasing enzyme phospholipase A(2) (PLA(2)) was cloned. The expression of this gene could be seen in epidermal cells during budding. The recombinant protein, as in the case of the authentic PLA2, preferred PC and PE as substrates, followed by PS and PI. The resultant FFAs only promoted cell growth, while the remaining lysophospholipids stimulated cell motility. The former contained unsaturated fatty acids (C18:1, C20:5, and C22:6) while the latter did not, suggesting that unsaturated fatty acids are responsible for mitogenic activity in tunicate cells. These results show for the first time that phospholipids and their derivatives are bio-mediators promoting cell growth and cell motility in invertebrates.

Molecular cloning and characterization of a novel glutathione S-transferase gene induced by light stimulation in the protozoan Blepharisma japonicum.

A cDNA clone that is inducible by light stimulation was cloned by a differential screening method from a cDNA library of the protozoan Blepharisma japonicum, and the light-dependent expression was checked by semi-quantitative reverse transcription polymerase chain reaction analysis. Sequence analysis showed that the cDNA encodes a glutathione S-transferase (GST) that has not been characterized in the protozoa. Multiple alignment of B. japonicum GST (BjGST1), known protozoan, and mammalian alpha-, micro-, pi-, sigma-, theta-, zeta-, kappa-, and omega-class GSTs suggested that the BjGST1 may be a novel class GST. Furthermore, highly conserved amino acid residues among the GSTs and the substrate specificity of recombinant BjGST1 showed that BjGST1 is related to alpha-, micro-, pi-, and sigma-class GSTs rather than the other class of GSTs.

Acquisition of retinoic acid signaling pathway and innovation of the chordate body plan.

Retinoic acid (RA) regulates many of the chordate-specific and vertebrate-specific characters. These include the anteroposterior pattern of the dorsally located central nervous system, pharynx with gill slits, neural crest cells, limb morphogenesis and anteroposteriorly organized vertebrae. The necessity of endogenous RA and the RA receptor (RAR) has been demonstrated by mutant analyses, vitamin A-deficient animals and various other methods. Since RAR has been identified only in chordates, the acquisition of the RAR-mediated RA signaling pathway is thought to be an important event for the innovation of the chordate body plan. RA-synthesizing aldehyde dehydrogenases and RA-degrading enzymes also seem to be chordate-specific. The expression pattern of these genes in ascidian embryos is similar to that in vertebrate embryos. These results suggest that the RA signaling cascade, with various regulators and modifiers, had been already well established in the common chordate ancestor. RA also regulates morphogenesis during the asexual reproduction of ascidians, suggesting that RA may also have played a part in producing diversity within the chordate groups.

Detection of characteristic signals from as-doped (<1 at.%) regions of silicon by transmission electron microscopy and convergent-beam electron diffraction.

Characteristic signals were detected from As-doped (< 1 at.%) regions of silicon by dark-field transmission electron microscopy and convergent-beam electron diffraction. A slight intensity increase was observed in 220 dark-field images, which may be explained by an increase of scattering amplitude due to the As doping. The doped region showed a much higher intensity in 004 dark-field images. The characteristic high intensity was observed for specimens with As concentrations of about 0.09-0.8 at.%. Convergent-beam electron diffraction patterns obtained from the As-doped region showed a characteristic rocking curve for 004 reflection. These characteristics should originate from incoherent elastically scattered electrons due to a static lattice distortion around the doped As atoms. The observed characteristics in dark-field images and rocking curves of the 004 reflection should be a good probe not only for investigating the concentration of doped atoms in Si lattice, but also for the amount of impurity and/or point defects in other crystalline materials.