Transcriptome analysis reveals a complex response to the RGNNV/SJNNV reassortant Nervous Necrosis Virus strain in sea bream larvae.

The gilthead sea bream (Sparus aurata) is a marine fish of great importance for Mediterranean aquaculture. This species has long been considered resistant to Nervous Necrosis Virus (NNV), an RNA virus that causes massive mortalities in several farmed fish animals. However, the recent appearance of RGNNV/SJNNV reassortant strains started to pose a serious threat to sea bream hatcheries, as it is able to infect larvae and juveniles of this species. While host response to NNV has been extensively studied in adult fish, little attention has been devoted to early life history stages, which are generally the most sensitive ones. Here we report for the first time a time-course RNA-seq analysis on 21-day old fish gilthead sea bream larvae experimentally infected with a RGNNV/SJNNV strain. NNV-infected and mock-infected samples were collected at four time points (6 h, 12 h, 24 h, and 48 h post infection). Four biological replicates, each consisting of five pooled larvae, were analysed for each time point and group. A large set of genes were found to be significantly regulated, especially at early time points (6 h and 12 h), with several heat shock protein encoding transcripts being up-regulated (e.g. hspa5, dnaj4, hspa9, hsc70), while many immune genes were down-regulated (e.g. myd88 and irf5 at T06, pik3r1, stat3, jak1, il12b and il6st at T12). A gene set enrichment analysis (GSEA) identified several altered pathways/processes. For instance, the formation of peroxisomes, which are important anti-viral components as well as essential for nervous system homeostasis, and the autophagy pathway were down-regulated at 6 h and 24 h post infection (hpi). Finally, two custom "reactomes" (i.e. significant gene sets observed in other studies) were defined and used. The first reactome integrated the transcriptomic response to NNV in different fish species, while the second one included all genes found to be stimulated either by interferon (IFN) or by IFN and Chikungunya virus in zebrafish. Genes in both reactomes showed predominant up-regulation at 6hpi and 12hpi and a general down-regulation at 24hpi. Such evidence suggest a certain degree of similarity between the response of sea bream and that of other fish species to NNV, while the observed down-regulation of IFN- and viral-stimulated pathways argues for a possible interference of NNV against the host response.

Development and testing of a combined species SNP array for the European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata).

SNP arrays are powerful tools for high-resolution studies of the genetic basis of complex traits, facilitating both selective breeding and population genomic research. The European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata) are the two most important fish species for Mediterranean aquaculture. While selective breeding programmes increasingly underpin stock supply for this industry, genomic selection is not yet widespread. Genomic selection has major potential to expedite genetic gain, particularly for traits practically impossible to measure on selection candidates, such as disease resistance and fillet characteristics. The aim of our study was to design a combined-species 60 K SNP array for European seabass and gilthead seabream, and to test its performance on farmed and wild populations from numerous locations throughout the species range. To achieve this, high coverage Illumina whole-genome sequencing of pooled samples was performed for 24 populations of European seabass and 27 populations of gilthead seabream. This resulted in a database of ~20 million SNPs per species, which were then filtered to identify high-quality variants and create the final set for the development of the 'MedFish' SNP array. The array was then tested by genotyping a subset of the discovery populations, highlighting a high conversion rate to functioning polymorphic assays on the array (92% in seabass; 89% in seabream) and repeatability (99.4-99.7%). The platform interrogates ~30 K markers in each species, includes features such as SNPs previously shown to be associated with performance traits, and is enriched for SNPs predicted to have high functional effects on proteins. The array was demonstrated to be effective at detecting population structure across a wide range of fish populations from diverse geographical origins, and to examine the extent of haplotype sharing among Mediterranean farmed fish populations. In conclusion, the new MedFish array enables efficient and accurate high-throughput genotyping for genome-wide distributed SNPs for each fish species, and will facilitate stock management, population genomics approaches, and acceleration of selective breeding through genomic selection.

Performance and precision of double digestion RAD (ddRAD) genotyping in large multiplexed datasets of marine fish species.

The development of Genotyping-By-Sequencing (GBS) technologies enables cost-effective analysis of large numbers of Single Nucleotide Polymorphisms (SNPs), especially in "non-model" species. Nevertheless, as such technologies enter a mature phase, biases and errors inherent to GBS are becoming evident. Here, we evaluated the performance of double digest Restriction enzyme Associated DNA (ddRAD) sequencing in SNP genotyping studies including high number of samples. Datasets of sequence data were generated from three marine teleost species (>5500 samples, >2.5 × 1012 bases in total), using a standardized protocol. A common bioinformatics pipeline based on STACKS was established, with and without the use of a reference genome. We performed analyses throughout the production and analysis of ddRAD data in order to explore (i) the loss of information due to heterogeneous raw read number across samples; (ii) the discrepancy between expected and observed tag length and coverage; (iii) the performances of reference based vs. de novo approaches; (iv) the sources of potential genotyping errors of the library preparation/bioinformatics protocol, by comparing technical replicates. Our results showed use of a reference genome and a posteriori genotype correction improved genotyping precision. Individual read coverage was a key variable for reproducibility; variance in sequencing depth between loci in the same individual was also identified as an important factor and found to correlate to tag length. A comparison of downstream analysis carried out with ddRAD vs single SNP allele specific assay genotypes provided information about the levels of genotyping imprecision that can have a significant impact on allele frequency estimations and population assignment. The results and insights presented here will help to select and improve approaches to the analysis of large datasets based on RAD-like methodologies.

Development of a real-time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues.

The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single-step, high-sensitivity real-time PCR assay for simultaneous detection and quantification of P. damselae was designed targeting partial of the sequence of the bamB gene and tested for specificity and sensitivity on laboratory-generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non-target bacterial species proved the assay was able to discriminate Phdd-Phdp subspecies from diverse hosts/geographical origins and between non-target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between Phdp-Phdd. This is the first time that a molecular method for P. damselae diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease.

Gene expression profiling of thymus in beef cattle treated with prednisolone.

Glucocorticoids (GCs) are extensively used in livestock production, not only for their anti-inflammatory properties but also to improve the quality and quantity of meat in veal and beef production. In Italy, an increase in GC-positive cases has been observed in cattle since 2008, particularly prednisolone (PDN). Recent studies clearly demonstrate that both histopathological analysis and high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) were unable to detect PDN treatments. The aim of this study was to identify transcriptomic signatures of PDN administration in the thymus of experimentally treated animals by comparison with untreated controls, in order to identify gene expression changes or pathways alteration induced by the corticosteroid treatment. Microarray data analysis showed substantial modifications in thymus gene expression profiles after PDN treatment. Several of the 388 differentially expressed genes encoded pro-inflammatory and anti-inflammatory mediators or immune regulators which showed that PDN might have a role in the regulation of immunologic homeostasis, act on both innate and acquired components of the immunity and mainly induce the activation of immune tolerance and anti-inflammatory pathways. Thus, this study allowed to deepen the effects of PDN on the immune system and showed the potentiality of gene expression profiling by DNA-microarray as a powerful tool to complement the existing methods against the illegal use of growth promoting hormones, especially when working on samples collected after slaughtering.

Quantitative trait loci for resistance to fish pasteurellosis in gilthead sea bream (Sparus aurata).

Fish pasteurellosis is a bacterial disease causing important losses in farmed fish, including gilthead sea bream, a teleost fish of great relevance in marine aquaculture. We report in this study a QTL analysis for resistance to fish pasteurellosis in this species. An experimental population of 500 offspring originating from eight sires and six dams in a single mass-spawning event was subjected to a disease challenge with Photobacterium damselae subsp. piscicida (Phdp), the causative agent of fish pasteurellosis. A total of 151 microsatellite loci were genotyped in the experimental population, and half-sib regression QTL analysis was carried out on two continuous traits, body length at time of death and survival, and for two binary traits, survival at day 7 and survival at day 15, when the highest peaks of mortality were observed. Two significant QTLs were detected for disease resistance. The first one was located on linkage group LG3 affecting late survival (survival at day 15). The second one, for overall survival, was located on LG21, which allowed us to highlight a potential marker (Id13) linked to disease resistance. A significant QTL was also found for body length at death on LG6 explaining 5-8% of the phenotypic variation.

High uptake of colonoscopy in first-degree relatives of patients with colorectal cancer in a healthcare region: a population-based, prospective study.

BACKGROUND AND STUDY AIMS: A screening program in first-degree relatives (FDRs) of colorectal cancer (CRC) patients (index patients) was started in Trentino, Italy, to analyze factors that influence uptake of CRC screening among invited FDRs (first objective) and to describe colorectal findings among those undergoing colonoscopy (secondary objective). PATIENTS AND METHODS: FDRs aged between 45 and 75 years were invited; exclusion criteria were: colonoscopy or barium enema in the preceding 5 years, a history of familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, inflammatory bowel diseases, and severe comorbidities. FDRs who were eligible but were not invited for screening because consent was not obtained from the index patients were considered as the control group. FDRs were invited by the education campaign targeted at the population at risk (both study and control groups); in the study group, interventions targeting individuals at risk (letters, phone calls, face-to-face counseling) were implemented. RESULTS: Starting from 626 new index cases of diagnosed CRC, 725 FDRs were invited to counseling; 77.6 % of these attended for colonoscopy in the study group vs. 8 % in the control group ( P < 0.0001). Predictors of colonoscopy uptake were FDR age above 60 years [odds ratio (OR) 2.50, 95 %CI 1.72 - 3.62], complex family history (simple family history: one CRC at age above 60 years; complex family history: one CRC at age below 60 or two or more CRC; OR 1.54; 95 %CI 1.04 - 2.33) and living in a rural area (OR 1.64, 95 %CI 1.12 - 2.44). Of the 560 FDRs in the study group, 186 (33.8 %) had adenomas, and 48 (8.8 %) had advanced adenomas or cancer. CONCLUSIONS: Interventions that target FDRs of patients with CRC, especially those younger than 60 years, with a complex family history of CRC and who live in a rural area, may improve uptake of CRC screening via colonoscopy.

Direct identification of Photobacterium damselae subspecies piscicida by PCR-RFLP analysis.

Fish pasteurellosis is an infectious disease that affects several teleost species living in temperate marine waters. The pathogen responsible, Photobacterium damselae subspecies piscicida, shows high genetic similarity with P. damselae subsp. damselae, making subspecies discrimination extremely laborious. Here we report for the first time a PCR-RFLP method for the identification of P. damselae subsp. piscicida without prior isolation in pure culture. Genomic sequence information was obtained through cloning and sequencing of RAPD products. Two P. damselae-specific primer pairs were developed and tested on 17 strains of P. damselae subsp. piscicida, 10 strains of P. damselae subsp. damselae, and 6 closely related control species. High sensitivity was achieved in PCR amplification on serially diluted samples (<180 fg of pure bacterial DNA or <10 fg, depending on the amplified fragment). Restriction analysis of PCR products showed a unique digestion profile for all P. damselae subsp. piscicida strains. The same PCR-RFLP method was implemented on total DNA samples extracted from experimentally infected sea bream and sea bass. Positive results were obtained on fish with clear signs of the disease as well as on challenged, but asymptomatic, fish. The method presented here might provide a useful tool for both prevention and rapid diagnosis of fish pasteurellosis.

Contribution of adventitial fibroblasts to neointima formation and vascular remodeling: from innocent bystander to active participant.

The adventitial layer surrounding the blood vessels has long been exclusively considered a supporting tissue the main function of which is to provide adequate nourishment to the muscle layers of tunica media. Although functionally interconnected, the adventitial and medial layers are structurally interfaced at the external elastic lamina level, clearly distinguishable at the maturational phase of vascular morphogenesis. Over the last few years the "passive" role that the adventitia seemed to play in experimental and spontaneous vascular pathologies involving proliferation, migration, differentiation, and apoptosis of vascular smooth muscle cells (VSMCs) has been questioned. It has been demonstrated that fibroblasts from the adventitia display an important partnership with the resident medial VSMCs in terms of phenotypic conversion, proliferation, apoptotic, and migratory properties the result of which is neointima formation and vascular remodeling. This article is an attempt at reviewing the major themes and more recent findings dealing with the phenotypic conversion process that leads adventitial "passive" (static) fibroblasts to become "activated" (mobile) myofibroblasts. This event shows some facets in common with vascular morphogenesis, ie, the process of recruitment, incorporation, and phenotypic conversion of cells surrounding the primitive endothelial tube in the definitive vessel wall. We hypothesize that during the response to vascular injuries in the adult, "activation" of adventitial fibroblasts is, at least in part, reminiscent of a developmental program that also invests, although with distinct spatiotemporal features, medial VSMCs.

Evaluation of the precision of magnetic resonance phase velocity mapping for blood flow measurements.

Evaluating the in vivo accuracy of magnetic resonance phase velocity mapping (PVM) is not straightforward because of the absence of a validated clinical flow quantification technique. The aim of this study was to evaluate PVM by investigating its precision, both in vitro and in vivo, in a 1.5 Tesla scanner. In the former case, steady and pulsatile flow experiments were conducted using an aortic model under a variety of flow conditions (steady: 0.1-5.5 L/min; pulsatile: 10-75 mL/cycle). In the latter case, PVM measurements were taken in the ascending aorta of ten subjects, seven of which had aortic regurgitation. Each velocity measurement was taken twice, with the slice perpendicular to the long axis of the aorta. Comparison between the measured and true flow rates and volumes confirmed the high accuracy of PVM in measuring flow in vitro (p > 0.85). The in vitro precision of PVM was found to be very high(steady: y = 1.00x + 0.02, r = 0.999; pulsatile: y = 0.98x + 0.72, r = 0.997; x: measurement #1, y: measurement #2) and this was confirmed by Bland-Altman analysis. Of great clinical significance was the high level of the in vivo precision (y = 1.01x - 0.04, r = 0.993), confirmed statistically (p = 1.00). In conclusion, PVM provides repeatable blood flow measurements. The high in vitro accuracy and precision, combined with the high in vivo precision, are key factors for the establishment of PVM as the "gold-standard" to quantify blood flow.

Fish oil supplementation prevents neointima formation in nonhypercholesterolemic balloon-injured rabbit carotid artery by reducing medial and adventitial cell activation.

We asked whether balloon-injured neointima formation in the presence of high/low serum cholesterol (CT) levels might be affected by dietary supplementation with fish oil (FO). To test this hypothesis, we examined the differentiation, proliferation, or apoptosis profile of smooth muscle cell (SMC) and adventitial cell response to a mild injury induced via a Fogarty catheter in the carotid artery of adult rabbits that had been fed a standard chow or 0.5% CT-enriched diet starting 4 weeks before the lesion. One week before surgery, animals received FO supplementation. This regimen was continued for the following 3 weeks. The effect of FO on the early proliferative/migratory response of carotid SMCs was also examined in 2- and 7-day-injured normocholesterolemic rabbits. As controls, animals subjected to 3-week endothelial injury and animals kept on a 7-week CT diet were used. Carotid cryosections from the various animal groups were evaluated for morphometry (image analysis), differentiation (immunofluorescence with monoclonal antibodies specific for smooth muscle markers, ie, myosin isoforms, SM22, and fibronectin), proliferation (bromodeoxyuridine incorporation), and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling). FO treatment significantly reduced the development of intimal thickening in normocholesterolemic rabbits but had no efficacy in the presence of relatively higher serum CT levels. At day 2 (adventitia) and day 7 (neointima, media, and adventitia), the proliferation index of SMCs in FO-treated injured rabbits was markedly lower than in untreated injured controls. Concomitantly with the antiproliferative effect, FO was able to decrease the size of 2 cell types involved in the cell growth response to endothelial injury, namely, the "fetal-type" medial SMC subpopulation and the fibroblast-derived adventitial myofibroblasts. Thus, in our experimental conditions, a low CT level is a permissive condition for FO to prevent neointima formation to a considerable extent. This event is attributable to the early postinjury effect of FO on SMC/adventitial cell proliferation/differentiation patterns.

Differential expression of SM22 isoforms in myofibroblasts and smooth muscle cells from rabbit bladder.

The E-11 and 1-B8 monoclonal antibodies raised to the smooth muscle (SM)-specific SM22 protein from pig stomach were used to study the in vivo and in vitro phenotypic characteristics of myofibroblasts (MF) and SM cells (SMC) from the bladder detrusor muscle and serosal thickening of male rabbit. The 22-kDa SM22 band found in the SM extract appeared to be composed of distinct isoforms when examined in non-equilibrium two-dimensional gel electrophoresis (2D-EF): alpha (the most basic), beta, gamma, and delta (the most acidic) in the ratio of 34(alpha):23(beta):36(gamma):8(delta). Western blots of 2D-electrophoresed bladder extracts treated with E-11 and 1-B8 showed that alpha, beta, and delta, but not gamma isoforms were labeled with E-11, whereas alpha, beta, and gamma isoforms were stained with 1-B8. This differential immunoreactivity was not influenced by phosphorylation. The tissue distribution of SM22 immunostaining was heterogeneous in the bladder SM and serosal thickening developed as a consequence of partial outflow obstruction of the urinary bladder. At the cellular level, the 1-B8 and E-11 antibodies stained the SMC in a "diffuse" (the whole cytoplasm) and "honeycomb" (the peripheral cytoplasm) manner, whereas MF immunostaining was quite homogeneous. The two antibodies also reacted with cultured primary bladder SMC and MF grown in low serum conditions showing a heterogeneous SM22 cell distribution but an identical subcellular localization, i.e., the actin-containing filamentous network, distinguishable in part from that found in vivo. The immunocytochemical, Western blotting and 2D-EF patterns of MF from thickened serosa indicated that the gamma isoform alone is expressed in this tissue. This SM22 variant appeared before the completion of the cellular transition from MF to fully differentiated SMC. This pattern is reminiscent of bladder ontogenesis where SM22 expression in the developing bladder wall precedes that of SM myosin. Taken together these data suggest that: (i) SM22 isoforms are differently assorted in MF vs. SMC; (ii) SM22 is a SMC-lineage marker inasmuch as its expression occurs in an experimental condition characterized by a time-related cell phenotypic transition from MF to SMC, and (iii) cell conversion ability of serosal cells in the adult might take place via the reactivation of a specific "foetal" gene programme.

Smooth muscle-specific SM22 protein is expressed in the adventitial cells of balloon-injured rabbit carotid artery.

During the "response-to-injury" process after a mechanical insult to the porcine coronary arteries, the adventitial cells acquire the structural characteristics of myofibroblasts before being incorporated into smooth muscle (SM) layer. We assessed whether the SM-specific SM22 protein can be used as a tracer of adventitial cell-myofibroblast differentiation in the mild balloon injury of rabbit carotid artery. To achieve this goal, we used 2 monoclonal anti-SM22 antibodies (E-11 and 1-B8) and a molecular probe for the SM22alpha mRNA isoform in immunocytochemical and in situ hybridization experiments. The differentiation profile and the migratory and proliferative ability of activated adventitial cells were evaluated by a panel of antibodies to some SM and nonmuscle antigens and pulse- and end-labeling with bromo-deoxyuridine, respectively. In adventitial cells, SM22 antigenicity and SM22alpha mRNA were detectable at days 2 and 4 and, to a lesser extent, at days 7 and 21 after injury, particularly near the adventitia-media interface and mostly colocalizing with bromo-deoxyuridine-positive cells. The pulse-labeling experiments showed that the large majority of these cells penetrated the outermost layer of the tunica media without migrating to the subendothelial region. The phenotypic features of activated migrating and nonmigrating adventitial cells resembled those of vimentin-actin myofibroblast subtype and fetal-type SM cells. These findings indicate that a direct exposure of adventitia to the lumen is not required for phenotypic changes and proliferation/migration of these cells. After comparison of the SM22 expression in arterial vessels during early stages of development, we hypothesize that in the injured carotid artery the mural incorporation of adventitial cells and the spatiotemporal activation of SM22 expression are reminiscent of the vascular morphogenetic process and suggest the existence of a stem cell-like reservoir in adventitia. The early adventitial upregulation of SM22 expression in the injured vessel might be related to a multistep transition process in which nonmuscle cells are converted to myofibroblasts and, possibly, to SM cells.

Use of navigator-echo-gated MRI to diagnose a coronary shunt involving an anomalous origin of the right coronary artery from the pulmonary artery.

Origin of the right coronary artery from the main pulmonary artery is an anomaly that can cause formation of a left-to-right coronary shunt, leading to myocardial ischemia and early onset of congestive heart failure. We describe a case in which magnetic resonance imaging was able to show the anomalous origin of the right coronary artery, and magnetic resonance phase velocity mapping was able to demonstrate the presence of a left-to-right shunt through the coronary artery by showing retrograde flow in the right coronary artery.

Quantification of mitral regurgitation with MR phase-velocity mapping using a control volume method.

Reliable diagnosis and quantification of mitral regurgitation are important for patient management and for optimizing the time for surgery. Previous methods have often provided suboptimal results. The aim of this in vitro study was to evaluate MR phase-velocity mapping in quantifying the mitral regurgitant volume (MRV) using a control volume (CV) method. A number of contiguous slices were acquired with all three velocity components measured. A CV was then selected, encompassing the regurgitant orifice. Mass conservation dictates that the net inflow into the CV should be equal to the regurgitant flow. Results showed that a CV, the boundary voxels of which excluded the region of flow acceleration and aliasing at the orifice, provided accurate measurements of the regurgitant flow. A smaller CV provided erroneous results because of flow acceleration and velocity aliasing close to the orifice. A large CV generally provided inaccurate results because of reduced velocity sensitivity far from the orifice. Aortic outflow, orifice shape, and valve geometry did not affect the accuracy of the CV measurements. The CV method is a promising approach to the problem of quantification of the MRV.

Quantification of the aortic regurgitant volume with magnetic resonance phase velocity mapping: a clinical investigation of the importance of imaging slice location.

BACKGROUND AND AIMS OF THE STUDY: Current techniques for assessment of aortic regurgitation (AR) are mainly qualitative. Magnetic resonance phase velocity mapping (PVM) provides accurate measurements of arterial blood blow. In AR, the aortic regurgitant volume (ARV) can be quantified with a single imaging slice measurement in the ascending aorta. The aim was to use PVM to: (i) quantify the regurgitant volume in patients with AR using an in vitro validated technique; and (ii) confirm in vivo our previous in vitro findings of the importance of measurement location. METHODS: Four healthy volunteers and 19 patients with AR, varying from mild to severe, were examined in a 1.5 Tesla MRI scanner. In 13 patients, the slice was placed: (i) between the aortic valve and the coronary ostia; (ii) at the sinotubular junction (SJ); and (iii) 2 cm above the SJ. In six patients, one measurement was taken as close as technically possible to the aortic valve. PVM measurements of the ARV were compared with angiographic/echocardiographic AR grading. RESULTS: No ARV was measured in healthy subjects. In patients, PVM results correlated well with angiographic/echocardiographic data. Repeatability of the PVM results was excellent and interobserver variability very small. The measured ARV decreased as the slice distance from the aortic valve increased, due to aortic compliance, in agreement to previous in vitro results. Close to the valve, acceleration did not affect the accuracy of velocity measurements. CONCLUSIONS: PVM has great potential to measure AR in a purely quantitative manner. Measurement location is important and results suggest that the closer the measurement to the valve the more accurate the ARV quantification.

Phenotypic changes in the regenerating rabbit bladder muscle. Role of interstitial cells and innervation on smooth muscle cell differentiation.

We have studied the phenotypic changes in regenerating smooth muscle (SM) tissue of detrusor muscle after local application of a necrotizing, freeze-thaw injury to the serosal surface of rabbit bladder. Bromo-deoxyuridine (BrdU) incorporation and immunofluorescence studies were performed on bladder cryosections from day 2 up to day 15 after surgery with monoclonal antibodies specific for some cytoskeletal markers [desmin, vimentin, non-muscle (NM) myosin] and for SM-specific proteins (alpha-actin, myosin, and SM22). Four days after lesion, some clls incorporated in regenerating SM bundles are BrdU positive, but all display a phenotypic pattern identical to that of the interstitial, highly proliferating cells, i.e., expression of vimentin. By days 7-15 the differentiation profile of regenerating SM returns to that of uninjured SM tissue (appearance of desmin, SM-type alpha-actin, and SM myosin). A chemical denervation achieved by 6-hydroxydopamine treatment for 2 weeks induces the formation of vimentin/SM alpha-actin/NM myosin/SM22-containing myofibroblasts in the interstitial, fibroblast-like cells of uninjured bladder. In the bladder wall, alteration of reinnervation during the regenerating SM process produces: (1) in the outer region, the activation of vimentin/SM alpha-actin/desmin myofibroblasts in the de novo SM cell bundles; and (2) in the inner region of bladder, including the muscularis mucosae, the formation of proliferating, fully differentiated SM cells peripherally to newly formed SM cell bundles. These findings suggest that: (1) the de novo SM tissue formation in the bladder can occur via incorporation of interstitial cells into growing SM bundles; and (2) the alteration of reinnervation during the regenerating process induces a spatial-specific differentiation of interstitial myofibroblasts in SM cells before SM cell bundling.