Search

Extracellular matrix (ECM) provides both physical support and bioactive signals such as growth factors and cytokines to cells at their microenvironment or niche. Engineering the matrix niche becomes an important approach to study or manipulate cellular fate. This work presents an overview on the reconstitution of the ECM niche through a wide range of approaches ranging from coating culture dish with ECM molecules to decellularization of native tissues. In particular, we focused on reconstituting the complex ECM niche through cell-derived matrix (CDM) by reviewing the methodological approaches used in our group to derive ECM from mature cells such as chondrocytes and nucleus pulposus cells (NPCs), undifferentiated stem cells such as mesenchymal stem cells (MSCs), as well as MSCs undergoing chondrogenic and osteogenic differentiation, in 2D or 3D models. Specific attention has also been given to key factors that should be considered in various applications and challenges in relation to the CDM. Last but not the least, a few future perspectives and their significance have been proposed.

Herba eupatorii, one of the most important Chinese medicinal herbs, belongs to the Asteraceae family. In June 2012, a previously unknown disease, tentatively identified as powdery mildew, was observed on H. eupatorii growing in Shangqiu, in eastern Henan Province, China. Symptoms began as white mycelium partially covering upper leaf surfaces; as the disease progressed, it spread to cover entire leaf surfaces. The infected leaves became yellow and necrotic at advanced stages of infection. Specimens consisting of infected leaves were maintained at the Plant-Microbe Interaction Laboratory at Shangqiu Normal University. Microscopic observations of the morphology of the fungus revealed oval primary conidia measuring 18 to 27 × 15 to 22 μm. A long unbranched germ tube that germinated laterally from the ends of conidia was observed in some samples. Conidiophores were cylindrical, simple unbranched, and composed of a basal cell with a swollen base and three to six barrel-shaped conidia formed in chains, measuring 112 to 180 × 9 to 12 μm. Mycelial appressoria were nipple-shaped. Chasmothecia were not observed in the collected samples. To verify the identity of the fungus, the internal transcribed spacer (ITS) rDNA was amplified with ITS1 and ITS4 primers (3) and sequenced. The sequences were deposited as GenBank Accession No. JX546297. Comparison with sequences in the GenBank database revealed that the ITS sequence was 100% homologous with the sequence of Podosphaera fusca on Calendula officinalis (AB525914) (2) and Syneilesis palmata (AB040349) (1). The ITS sequence analysis verified that the causal agent was P. fusca, which is reported to be a cosmopolitan powdery mildew fungus, parasitic on numerous plant species in the Asteraceae family. Koch's postulates were completed by inoculating healthy H. eupatorii plants with a conidial suspension (prepared in distilled water) of 105 conidia/ml collected from infected plants. Five plants were sprayed until the suspension ran off the leaves, while five additional plants were sprayed with distilled water as a control. Plants were maintained in a climate cell under the following conditions: day, 24°C, 16 h; night, 20°C, 8 h; 85% humidity. After 10 days, inoculated plants developed symptoms similar to those observed in the field, whereas control plants remained healthy. Further examination showed that the inoculated plants were infected by P. fusca. To our knowledge, this is the first report of P. fusca affecting H. eupatorii in China. Because there are no fungicides labeled for use on this plant, the appearance of powdery mildew caused by P. fusca could result in substantial production loss of H. eupatorii. References: (1) T. Hirata et al. Can. J. Bot. 78:1521, 2000. (2) S. Takamatsu et al. Persoonia 24:38, 2010. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

In April 2011, there was an outbreak of an infectious disease in southern catfish, Silurus soldatovi meridionalis, (Chen) (15–20 g) in Sichuan Province, China. Two isolates, LW101 and LW102, were isolated from kidney and liver of the sick fish on brain-heart infusion (BHI) agar and were considered to be the cause of this disease based on experimental challenges. The morphological and physiological characteristics as well as the biochemical tests of the two isolates were the same and similar to Edwardsiella ictaluri. Furthermore, the sequencing of the 16S rRNA gene and the gryB gene revealed that the isolates were highly homogeneous with E. ictaluri. On the basis of the phenotypic characteristics and phylogenetic analysis of these genes, both isolates were identified as E. ictaluri. Susceptibility of the isolates to 22 antibiotics was tested using the disc diffusion method. Both isolates showed a similar antibiotic susceptibility, which was characterized by resistance to acetylspiramycin, ampicillin, clarithromycin, penicillin, oxytetracycline, and sinomin (SMZ/TMP); the strains were susceptible to amikacin, chloramphenicol, florfenicol, roxithromycin, ciprofloxacin, norfloxacin, doxycycline, and tenemycin. To our knowledge, this is the first report of E. ictaluri infection in southern catfish.

Controlling cell organization is important in tissue engineering. Guidance by aligned features on scaffolds or stimulation by physical signals can be used to induce cell alignment. We have previously demonstrated a preferred alignment of human MSCs (hMSCs) along the compression loading axis in 3D collagen construct. In this study, we aim to investigate the collagen concentration dependence of the compression-induced hMSC organization. Results demonstrated that the compression-induced alignment and elongation of hMSCs exhibited a biphasic dose-dependent relationship with collagen concentration, and associated well with both collagen ligand density and elastic modulus of the constructs. Moreover, collagen concentration and compression loading significantly affected the expression level of integrin beta 1 and antibody neutralization against this molecule aborted the compression-induced alignment and elongation responses.

Summary Streptococcus agalactiae (Group B streptococcus) has emerged as an important pathogen that affects humans and animals, including aquatic species. S. agalactiae infections are becoming an increasing problem in aquaculture and have been reported worldwide in a variety of fish species, especially those living in warm water. Recently, a very serious infectious disease of unknown aetiology broke out in ya-fish (Schizothorax prenanti) farms in Sichuan Province. A Gram-positive, chain-forming coccus was isolated from moribund cultured ya-fish. The goals of this study were to identify the bacterial strains isolated from diseased fish between 2009 and 2011 in Sichuan Province, China, to evaluate the pathogenicity of the pathogen in ya-fish, crucian carp (Carassius carassius) and the Nile tilapia (Oreochromis niloticus); and to determine the susceptibility of the pathogen strains to many currently available anti-microbial agents. The virulence tests were conducted by intraperitoneal injection of bacterial suspensions. In this study, four strains of a Gram-positive, chain-forming coccus were isolated from moribund cultured ya-fish (S. prenanti). The coccoid microorganism was identified as S. agalactiae using a commercial streptococcal grouping kit and 16S rDNA sequencing analysis. Susceptibility of the isolates to 22 antibiotics was tested using the disc diffusion method. All isolates showed a similar antibiotic susceptibility, which were sensitive to amoxicillin, ciprofloxacin, lomefloxacin, chloramphenicol, rifampin, vancomycin, azithromycin, florfenicol, cefalexin, cefradine and deoxycycline and resistant to gentamicin, sinomin (SMZ/TMP), penicillin, tenemycin, fradiomycin and streptomycin. Furthermore, the virulence tests were conducted by intraperitoneal injection of the isolated strain GY101 in ya-fish, crucian carp and the Nile tilapia. This coccus was lethal to ya-fish, Nile tilapia and crucian carp. The mortality rates of infected ya-fish were 100%, 100%, 60% and 20% at doses of 1.0 × 107, 1.0 × 106, 1.0 × 105 and 1.0 × 104 CFU/fish, respectively. The values were 100%, 80%, 60% and 30% for Nile tilapia and 100%, 60%, 10% and 0% for crucian carp. To our knowledge, this is the first report of S. agalactiae as a pathogen of ya-fish.

It is well established that the heating process during the preparation of dry milk (DMLK) causes structural changes in some milk proteins. However, because such changes are subtle, whether they can be detected by an immunochemical approach remains questionable. The present study attempted to develop a sensitive mAb that might distinguish the DMLK from freshly prepared raw milk. To test this possibility, we immunized mice with commercially prepared DMLK and produced a panel of mAb. From 900 hybridomas screened using an ELISA, 4 clones were found to be specific to DMLK; the other 68 clones recognized both DMLK and raw milk. In contrast to polyclonal antibodies, only the specific mAb could detect the DMLK spiked into the raw milk at as low as 5% in concentration (vol/vol). Western blot analysis shows that these specific mAb were all directed against beta-lactoglobulin (LG) and LG-milk protein conjugates. These mAb reacted with raw milk heated at 95 degrees for 15 min; the reaction with LG-conjugates, however, was abolished when treated with reducing reagent. Thus, results suggests that a new antigenic epitope was exposed in a heating process, and the thio group of LG cross linked with other protein moiety played a provocative role in mAb recognition. A hypothetical model with respect to the interaction between the mAb and DMLK is proposed and discussed.

The radula of the limpet, Cellana toreuma, consists of a continuous series of teeth in various stages of iron biomineralization. The major iron-binding protein of the limpet's iron-containing granule (siderosome) has been purified and identified as ferritin. Limpet ferritin has a M(r) of 575 kDa and can be resolved into two bands by SDS-PAGE analysis, with respective M(r)s of 26 and 21 kDa. The partial N-terminal amino acid sequences of these two subunits were confirmed, and antisera against them were respectively generated. The specifity of these two antisera shows no difference between them. By using transmission electron microscopy and immunogold staining techniques the following two events were revealed: (1) in the superior epithelial cell of the radula, ferritin was disassembled through autophagy or heterophagy before exocytosis; (2) of the limpet ferritin, at least the 26-kDa subunit was found to pass through the microvilli, resulting in the accumulation of iron in the extracellular tooth chamber and the formation of goethite needles. Intracellular ferritin being translocated to the extracellular environment is discussed in the text.

The honeybee is one of the few organisms that can deposit iron minerals intracellularly. Numerous iron granules are formed in the trophocytes, which are located in the abdomen, beginning on the second day after eclosion. The sequential events of iron deposition in honeybees have been determined and the special features of this biomineralization system are (1) that iron deposition vesicles (IDVs) enlarge by fusing with one another; (2) that dense particles (approx. 7.5nm in diameter) are the basic building blocks in the formation of iron granules; and (3) that a cloudy layer just beneath the membrane of IDVs may play an important role in the formation of the dense particles. The iron granules seem to be randomly distributed in the trophocytes of the worker and drone. In the queen, however, they are clustered and peripherally located. This distinct difference in the iron granule distribution between members of the hive suggests that these iron granules may have some biological functions. A detailed analysis of total iron content during the life cycle of honeybees has shown that iron granules in the adult worker contain approximately 1% of the total iron content and also account for approximately 3% of the increase in iron content that occurs between the newly eclosed worker stage and the adult worker stage.

The current study aims to identify the type of cell-matrix adhesions of hMSCs in 3D collagen constructs and to investigate the effects of dynamic compression on the type, morphology and composition of cell-matrix adhesions, particularly to observe whether the compression stimulates the maturation or evolvement of 3D matrix adhesion in hMSC-collagen constructs. Preliminary results demonstrated the colocalization of integrin α 5 β 1 and fibronectin in cell-matrix adhesions in loaded constructs, partially fulfilling the requirements for 3D matrix adhesion to evolve. In addition, fibronectin was shown to be organized into tiny-dotted adhesions in loaded constructs in a loading duration dependent way, suggesting dynamic compression may be able to mature adhesions in the constructs, hopefully into 3D matrix adhesions. It was also demonstrated that hMSCs plated onto their own cell-derived matrices form elongated adhesions which are similar to 3D matrix adhesions formed by fibroblasts. Further characterization on the cell-matrix adhesions of hMSCs in 3D collagen constructs and identification of differences in adhesions between loaded and unloaded constructs are underway.

Previously, we and others have demonstrated the association of a C/T single nucleotide polymorphism (SNP), in the Kozak sequence of CD40, with Graves' disease (GD). Here, using an expanded data set of patients, we confirm the association of the CD40 SNP with GD (n=210, P=0.002, odds ratio (OR)=1.8). Subset analysis of patients with persistently elevated thyroid peroxidase (TPO) and/or thyroglobulin (Tg) antibodies (Abs), (TPO/Tg Abs), after treatment (n=126), revealed a significantly stronger association of the SNP with disease (P=5.2 x 10(-5), OR=2.5) than in GD patients who were thyroid antibody-negative. However, the CD40 SNP was not associated with TPO/Tg Abs in healthy individuals. Next, we tested the CD40 SNP for association with Myasthenia Gravis (MG), which, like GD is an antibody-mediated autoimmune condition. Analysis of 81 MG patients found no association of the SNP with disease. Functional studies revealed significant expression of CD40 mRNA and protein in the thyroid (target tissue in GD) but not in skeletal muscle (target tissue in MG). Combined, our genetic and tissue expression data suggest that the CD40 Kozak SNP is specific for thyroid antibody production involved in the etiology of GD. Increased thyroidal expression of CD40 driven by the SNP may contribute to this disease specificity.

Tall fescue (Festuca arundinacea Schreb), a predominant cool-season perennial grass, is widely used as forage and turf grasses in China. In July 2013, powdery mildew was observed on 10 F. arundinacea lawns (about 0.5 ha in total) in Urumchi, Xinjiang Province, China, with 20 to 30% of the area being infected. Signs of the disease initially appeared as irregular white mycelial colonies on the adaxial surface of infected leaves. As the disease progressed, the colonies covered the whole adaxial surface and white patches appeared on the abaxial surface of infected leaves. Conidiophores were unbranched and cylindrical with swollen bases, measuring 13.3 to 15 × 16.7 to 20 μm, and borne vertically on hyphae. Each conidiophore produced 10 to 18 conidia in a chain. The conidia were oval, one-celled, and colorless, measuring 8.1 to 9.8 × 26 to 29.7 μm. Cleistothecia were black, spherical, and 164.3 to 207.3 μm in diameter, each of which contained 9 to 26 asci. Asci were oblong or ovate, measuring 32.1 to 40 × 85.7 to 96.4 μm. Asci were petiolate, containing eight ascospores. Ascospores were round to oval, colorless, one-celled, measuring 19.1 to 22.5 × 11.7 to 13.6 μm. Based on morphological characteristics of the anamorph and the teleomorph, the fungus was identified as Blumeria graminis (DC.) Speer. Additionally, the internal transcribed spacer (ITS) of 563 bp was amplified from DNA of conidia using ITS1 and ITS4 primers (4). The ITS sequence was deposited in GenBank (Accession No. KF545644). The ITS sequence showed 100% homogeneity with those of B. graminis on Poa pratensis in Swizerland (AB273540) and on P. bulbosa in Iran (AB273551) (1), which further confirmed the identification. Ten 3-week-old healthy plants were inoculated by spraying a spore suspension (1 × 105 conidia ml−1) made from conidia brushed from infected plants, and 10 plants sprayed with sterile distilled water were served as controls. All the plants were placed in the same growth chamber at 20°C, 80% humidity, and 16-h photoperiod. Twenty days after inoculation, typical signs and symptoms of powdery mildew were observed on all the inoculated plants, whereas no symptoms were observed on the controls. Microscopic and ITS analysis showed that the fungus on the inoculated plants is identical to that on diseased field plants. B. graminis on F. arundinacea has been observed in a few European countries (1), Israel (3), and the United States (2). To our knowledge, this is the first report of powdery mildew caused by B. graminis on F. arundinacea in China, which will increase the difficulty to prevent powdery mildew on grasses including cereals. References: (1) U. Braun. The Powdery Mildews (Erysiphales) of Europe. Gustav Fischer Verlag, Jena-Stuttgart-New York, 1995. (2) F. M. Dugan and G. Newcombe. Pacific Northwest Fungi. 2:1-5, 2007. (3) S. O. Voytyuk et al. Biodiversity of the Powdery Mildew Fungi (Erysiphales, Ascomycota) of Israel Vol. 7. Biodiversity of Cyanoprocaryotes, Algae and Fungi of Israel. Koeltz Scientific Books, 2009. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

Swiss chard (Beta vulgaris L. subsp. cicla) is a widely planted vegetable in China. From May to June 2013, an outbreak of powdery mildew on Swiss chard cultivar Fangzheng was observed in the commercial fields in Zhoukou city of Henan Province, located in central China. More than 80% of the plants exhibited symptoms of the disease. At the beginning of infection, circular, white, dust-like colonies of powdery mildew occurred mainly on adaxial surfaces of leaves. As the disease progressed, white mycelia covered the foliar parts of plant. No cleistothecia were found on or in collected samples. Upon microscopic evaluation, conidiophores were unbranched with the length of 63 to 126 and width of 7 to 10 μm (n = 50), produced conidia singly, and composed of a cylindrical foot cell followed by one to three short cells. Conidia were colorless, hyaline, ovoid, measured 29 to 40 × 12 to 18 μm (n = 100), lacked fibrosin bodies, and produced germ tubes on the ends of the conidia. The fungus was identified as Erysiphe betae according to the morphological features (1). To verify the identity, the internal transcribed spacer (ITS) region was amplified with the universal primers ITS1 and ITS4 (2) and sequenced. The ITS sequence obtained was assigned as Accession No. KF268348 in GenBank, which showed 100% homogeneity with two ITS sequences of E. betae isolates from UK (DQ164432 and DQ164436). Koch's postulates were conducted by inoculating 15 healthy 5-week-old plants (cv. Fangzheng) with detached infected leaves, which grew in a growth chamber under 22/16°C (day/night), 50% relative humidity, 120 μmol/m2/s light and a 16-h photoperiod. Fifteen non-inoculated plants grew in another growth chamber with the same conditions as control. Symptoms consistent with the infected field plants were observed on the inoculated plants, while no symptoms were found on the control plants. Microscopic observation revealed that the pathogen growing on the inoculated plants was consistent with the morphology of the original fungus. To our knowledge, this is the first report of E. betae infection on Swiss chard in China (3). References: (1) S. Francis. Mol. Plant Pathol. 3:119, 2002. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990. (3) R. Y. Zheng et al. Page 63 in: Flora Fungorum Sinicorum, Vol. 1, Erysiphales. Science Press, Beijing, 1987.

Sonchus asper is an exotic annual herb belonging to the Asteraceae family. Crude extracts of S. asper leaves have antiphlogistic and antibacterial properties. In May 2011, severe infection of powdery mildew was observed on S. asper in Henan Province, a central area of China. Symptoms began as small, nearly circular white spots that later enlarged and coalesced. As the disease progressed, white mycelia covered the entire plant, including the inflorescence. The voucher specimens are kept at the Key Laboratory of Plant-Microbe Interaction, Shangqiu Normal University. Conidiophores were erect, 108 to 220 × 10 to 12 μm, and composed of a cylindrical foot cell followed by two to three short cells. Nipple-shaped appressoria were formed on ectophytic mycelia. Conidia were hyaline and cylindrical, without distinct fibrosin bodies, and ranging from 29 to 42 × 19 to 24 μm. Long, unbranched germ tubes were formed from the ends of the conidia. These morphological characteristics suggested that the powdery mildew fungus is the anamorph of Golovinomyces cichoracearum (1,2). Chasmothecia were not observed in the collected samples. To confirm the identification, the internal transcribed spacer (ITS) region of the rDNA was amplified with ITS1 and ITS4 primers (4) and sequenced. The ITS sequence obtained was assigned Accession No. JQ010848 in GenBank, and had 99% similarity to two ITS sequences from different G. cichoracearum accessions (HM449077 and AY739111). The ITS sequence analysis further verified that the causal agent of the powdery mildew on S. asper was G. cichoracearum. Pathogenicity was confirmed by inoculating five healthy plants of S. asper with a conidial suspension (105 conidia/ml) from the infected plants, with five noninoculated plants serving as control. Inoculated plants developed the original powdery mildew symptoms after 10 days, whereas control plants remained healthy. G. cichoracearum (synonym Erysiphe cichoracearum) is a cosmopolitan powdery mildew fungus, parasitic on numerous plant species in the Asteraceae family. It was reported to be infecting S. asper in Bulgaria (3). To our knowledge, this is the first report of G. cichoracearum infections on S. asper in China. S. asper is one of the most important Chinese herbal medicines. The sudden appearance of powdery mildew caused by G. cichoracearum on this plant could result in substantial production loss of S. asper unless fungicide applications are employed. References: (1) U. Braun. Beih. Nova Hedwigia 89:1, 1987. (2) R. T. A. Cook and U. Braun. Mycol. Res. 113:616, 2009. (3) G. Negrean and C. M. Denchev. Mycologia Balcanica, 2004, 1:63. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

Euonymus japonicus Thunb. is a popular, woody, ornamental plant in China. From June to September 2010, severe outbreaks of powdery mildew occurred on all investigated E. japonicus plants used for hedges in Zhoukou and Shangqiu cities of Henan province in central China, which is the second largest province in terms of ornamental plant cultivation. A large amount of fungicide was used to prevent the fungal disease, which caused serious environmental pollution and was estimated to result in a 40 to 60% increase in preservation cost of E. japonicus hedges. Gray-white colonies of powdery mildew fungi occurred mainly on the leaves of E. japonicus, on average approximately 50% of the leaves of an individual plant were infected by the fungi. On severely infected leaves, mycelia were amphigenous, thick, forming irregular white patches, and effused to cover the whole surface. Subsequently, these leaves became chlorotic, curled, and withered. The fungus was identified as Erysiphe euonymi-japonici (Vienn.-Bourg.) U. Braun & S. Takam on the basis of the following data of microscopic morphology with scanning electron microscopy and molecular phylogenetic analysis of the internal transcribed spacer (ITS) region. The fungus had unbranched conidiophores with an average length of 57.3 μm and width of 8.8 μm, which was composed of a cylindrical foot cell, 15 to 40 × 6 to 10 μm and one to three shorter cells or cells of about the same length. Conidia were borne singly, cylindrical, or ellipsoid-cylindrical, with an average length of 27.7 μm and width of 10.4 μm. Chasmothecia were not observed in the collected samples during the whole outbreak period. PCR amplification and sequencing of the ITS region was amplified using the universal primers ITS1 and ITS4 (4). The obtained ITS sequence was assigned Accession No. HQ012432 in GenBank, which had a 99 and 98% nt similarity with the ITS sequences (ITS region including the 5.8S rDNA) of two Erysiphe euonymi-japonici isolates from Japan and Argentina in GenBank (Accession Nos. AB250228 and AB250229 (3), respectively). To our knowledge, Erysiphe euonymi-japonici has not been reported previously from central China, although synonymous species, Oidium euonymi-japonici and Microsphaera euonymi-japonici, were reported in southwestern (Sichuan Province) (1) and eastern (Shandong Province) (2) regions. Herbarium specimens are available at the Key Laboratory of Plant Genetics and Molecular Breeding, Zhoukou Normal University, Zhoukou, China. References: (1) H. Feng. J. Sichuan For. Sci. Technol. 13:57, 1992. (2) S. Z. Li et al. J. Shandong For. Sci. Technol. 46:40, 1995. (3) S. Limkaisang et al. Mycoscience 47:327, 2006. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

The siliceous cell wall of diatoms is formed in a silica deposition vesicle that is delimited by a membrane, the silicalemma. Once the siliceous wall matures, it is expelled and a new plasmalemma is formed underneath. Underlying wall formation is a multitude of events and processes, some of which are now known. A comparative study on wall morphogenesis in seven centric diatoms leads to the following conclusions: (1) the silica deposition vesicle is formed by the coalescence of small vesicles; (2) the silicalemma becomes part of the organic casing of the mature siliceous wall; (3) at least four morphological forms of deposited silica can be seen during the development of wall components; (4) microtubules, serving as cytoskeletons, are associated with the formation of certain wall components initiated from a cytoplasmic protrusion. These events are discussed in detail.

The present study on a centric diatom,Ditylum brightwellii, includes two parts: detection of sugars in the silica deposition vesicle (SDV) with lectins and labeling the developing siliceous cell wall in the SDV with rhodamine 123. Cells with developing valves are treated with SDS to remove all the cytoplasmic contents, then either stained with fluorescein labeled lectins or thin-sectioned and stained with colloidal gold labeled lectins. The results show that mannose is part of the organic matrix in the SDV. Rhodamine 123, a non-toxic fluorescent laser dye, enters the cell immediately and is trapped in the SDV probably by the high reducing potential of the SDV. Silica is co-deposited with rhodamine 123 in the SDV, and the resulting valves and girdle bands become fluorescent. Implications of this study for the mechanism of silicification are discussed.

The growth and shape of the membraneous labyrinth appears to be organized and restricted by the formation of the cartilaginous capsule. Coiling of the cochlear duct appears to be the result of the physical barrier and support provided by the cartilage cells differentiating from mesenchyme surrounding the otocyst. In addition, tissue interactions between this mesenchyme and the developing cochlear duct may be the source of a genetic message for the coiling mechanism.

A dichotomy of hair-cell types has been found in the bullfrog sacculus, and considerable evidence supports the view that one type (“peripheral”) is transformed during macular growth to the other type (“central”). Between the periphery and the center of the macula, one finds a gradation of form from “peripheral” to “central” type. Occasionally in adults and more often in stage-26 tadpoles one finds the presumably younger peripheral type of hair cell occurring well beyond the limits of the macula proper. The apparent morphogenic sequence for saccular hair cells is (1) development of a kinocillum on an endolymphatic epithelial cell, (2) gradual transformation of microvilli into stereocilia, (3) growth of the stereocilia and development of kinociliary bulb, (4) achievement of final size and form.