The embryonic expression patterns of zebrafish genes encoding LysM-domains.

The function and structure of LysM-domain containing proteins are very diverse. Although some LysM domains are able to bind peptidoglycan or chitin type carbohydrates in bacteria, in fungi and in plants, the function(s) of vertebrate LysM domains and proteins remains largely unknown. In this study we have identified and annotated the six zebrafish genes of this family, which encode at least ten conceptual LysM-domain containing proteins. Two distinct sub-families called LysMD and OXR were identified and shown to be highly conserved across vertebrates. The detailed characterization of LysMD and OXR gene expression in zebrafish embryos showed that all the members of these sub-families are strongly expressed maternally and zygotically from the earliest stages of a vertebrate embryonic development. Moreover, the analysis of the spatio-temporal expression patterns, by whole mount and fluorescent in situ hybridizations, demonstrates pronounced LysMD and OXR gene expression in the zebrafish brain and nervous system during stages of larval development. None of the zebrafish LysMD or OXR genes was responsive to challenge with bacterial pathogens in embryo models of Salmonella and Mycobacterium infections. In addition, the expression patterns of the OXR genes were mapped in a zebrafish brain atlas.

Knockdown of the glucocorticoid receptor alters functional integration of newborn neurons in the adult hippocampus and impairs fear-motivated behavior.

Glucocorticoids (GCs) secreted after stress reduce adult hippocampal neurogenesis, a process that has been implicated in cognitive aspects of psychopathology, amongst others. Yet, the exact role of the GC receptor (GR), a key mediator of GC action, in regulating adult neurogenesis is largely unknown. Here, we show that GR knockdown, selectively in newborn cells of the hippocampal neurogenic niche, accelerates their neuronal differentiation and migration. Strikingly, GR knockdown induced ectopic positioning of a subset of the new granule cells, altered their dendritic complexity and increased their number of mature dendritic spines and mossy fiber boutons. Consistent with the increase in synaptic contacts, cells with GR knockdown exhibit increased basal excitability parallel to impaired contextual freezing during fear conditioning. Together, our data demonstrate a key role for the GR in newborn hippocampal cells in mediating their synaptic connectivity and structural as well as functional integration into mature hippocampal circuits involved in fear memory consolidation.

p140Cap suppresses the invasive properties of highly metastatic MTLn3-EGFR cells via impaired cortactin phosphorylation.

We have recently shown that the adaptor protein p140Cap regulates tumor properties in terms of cell motility and growth. Here, by using the highly metastatic rat adenocarcinoma cell line MTLn3-epidermal growth factor receptor (EGFR), we assess the role of p140Cap in metastasis formation. Orthotopic transplantation of MTLn3-EGFR cells over-expressing p140Cap in Rag2(-/-)γ(c)(-/-) mice resulted in normal primary tumor growth compared with the controls. Strikingly, p140Cap over-expression causes an 80% inhibition in the number of lung metastases. p140Cap over-expressing cells display a 50% reduction in directional cell migration, an increased number and size of focal adhesions, and a strong impairment in the ability to invade in a 3D matrix. p140Cap over-expression affects EGFR signaling and tyrosine phosphorylation of cortactin in response to EGF stimulation. Intriguingly, p140Cap associates with cortactin via interaction with its second proline-rich domain to the cortactin SH3 domain. The phosphomimetic cortactin tyrosine 421 mutant rescues migration and invasive properties in p140Cap over-expressing cells. Taken together, these data demonstrate that p140Cap suppresses the invasive properties of highly metastatic breast carcinoma cells by inhibiting cortactin-dependent cell motility.

Analysis of cardiac development in the turtle Emys orbicularis (Testudines: Emidydae) using 3-D computer modeling from histological sections.

In this article we present a 3-D modeling study of cardiac development in the European pond turtle, Emys orbicularis (of the reptilian order Testudines). The study is aimed at elucidating the embryonic development of the horizontal septum in the ventricle and underscoring the importance of 3-D reconstructions in studying morphogenesis. Turtles possess one common ventricle, partly divided into three cava by a vertical and a horizontal septum, of which the embryonic origins have so far not been described. We used serial sectioning and computerized high-resolution 3-D reconstructions of different developmental stages to create a chronological overview of cardiogenesis, in order to study this process. This has yielded a new understanding of the development of the horizontal septum and (directly related) the looping of the heart tube. This looping is found to be markedly different from that in the human heart, with the turtle having two clear bends in the part of the heart tube leaving the primitive ventricle, as opposed to one in humans. It is this particular looping that is responsible for the formation of the horizontal septum. In addition to our findings on the ventricular septation this study has also yielded new insights into the developmental origins of the pulmonary vein. The 3-D reconstructions were built using our platform TDR-3-D base and enabled us to study the developmental processes in specific parts of the turtle heart separately and in three dimensions, over time. The complete 3-D reconstructions have been made available to the reader via internet using our 3-D model browser application, which allows interactive viewing of the models. The browser application can be found on bio-imaging.liacs.nl/galleries/emysorbicularis/TurtleGallery.html, along with additional images of both models and histological sections and animation sequences of the models. By allowing the reader to view the material in such an interactive way, we hope to make optimal use of the new 3-D reconstruction techniques and to engage the reader in a more direct manner.

Mining and analysing spatio-temporal patterns of gene expression in an integrative database framework.

Mining patterns of gene expression provides a crucial approach in discovering knowledge such as finding genetic networks that underpin the embryonic development. Analysis of mining results and evaluation of their relevance in the domain remains a major concern. In this paper we describe our explorative studies in support of solutions to facilitate the analysis and interpretation of mining results. In our particular case we describe a solution that is found in the extension of the Gene Expression Management System (GEMS), i.e. an integrative framework for spatio-temporal organization of gene expression patterns of zebrafish to a framework supporting data mining, data analysis and patterns interpretation As a proof of principle, the GEMS has been equipped with data mining functionality suitable for spatio-temporal tracking, thereby generating added value to the submission of data for data mining and analysis. The analysis of the genetic networks is based on the availability of domain ontologies which dynamically provides meaning to the discovered patterns of gene expression data. Combination of data mining with the already presently available capabilities of GEMS will significantly augment current data processing and functional analysis strategies.

Genomic annotation and transcriptome analysis of the zebrafish (Danio rerio) hox complex with description of a novel member, hox b 13a.

The zebrafish (Danio rerio) is an important model in evolutionary developmental biology, and its study is being revolutionized by the zebrafish genome project. Sequencing is at an advanced stage, but annotation is largely the result of in silico analyses. We have performed genomic annotation, comparative genomics, and transcriptional analysis using microarrays of the hox homeobox-containing transcription factors. These genes have important roles in specifying the body plan. Candidate sequences were located in version Z v 4 of the Ensembl genome database by TBLASTN searching with Danio and other vertebrate published Hox protein sequences. Homologies were confirmed by alignment with reference sequences, and by the relative position of genes along each cluster. RT-PCR using adult Tübingen cDNA was used to confirm annotations, to check the genomic sequence and to confirm expression in vivo. Our RT-PCR and microarray data show that all 49 hox genes are expressed in adult zebrafish. Significant expression for all known hox genes could be detected in our microarray analysis. We also find significant expression of hox 8 paralogs and hox b 7 a in the anti-sense direction. A novel gene, D. rerio hox b 13 a, was identified, and a preliminary characterization by in situ hybridization showed expression at 24 hpf at the tip of the developing tail. We are currently characterizing this gene at the functional level. We argue that the oligo design for microarrays can be greatly enhanced by the availability of genomic sequences.

Automated topographical cell proliferation analysis.

BACKGROUND: Cell proliferation is often studied using the incorporation of bromodeoxyuridine (BrdU). Immunohistochemical staining is then used to detect BrdU in the nucleus. To circumvent the observer bias and labor-intensive nature of manually counting BrdU-labeled nuclei, an automated topographical cell proliferation analysis method is developed. METHODS: Sections stained with fluorescein-labeled anti-BrdU and counterstained with To-Pro-3 are scanned using confocal laser scanning microscopy (CLSM). For every point in the image, the nucleus density of BrdU-labeled nuclei and the total nucleus density of the neighborhood of that point are calculated from the BrdU and the To-Pro-3 signal, respectively. The ratio of these densities gives an indication of the amount of cell proliferation at that point. The automated measure is validated by comparing it with the ratio of BrdU-stained nuclei to the total number of nuclei obtained from a manual count. RESULTS: A positive correlation is found between the automated measure and the ratios calculated from the manual counting (r = 0.86, P < 0.001). Calculating the topographical cell proliferation using the automated method is faster and does not suffer from interobserver variability. CONCLUSIONS: Automated topographical cell proliferation analysis is a fast method to objectively find differences in cell proliferation within a tissue. This can be visualized by a topographical map that corresponds to the tissue under study.

Septation and valvar formation in the outflow tract of the embryonic chick heart.

There is no agreement, in the chick, about the number of the endocardial cushions within the outflow tract or their pattern of fusion. Also, little is known of their relative contributions to the formation of the arterial valves, the subpulmonary infundibulum, and the arterial valvar sinuses. As the chick heart is an important model for studying septation of the outflow tract, our objective was to clarify these issues. Normal septation of the outflow tract was studied in a series of 60 staged chick hearts, by using stained whole-mount preparations, serial sections, and scanning electron microscopy. A further six hearts were examined subsequent to hatching. At stage 21, two pairs of endocardial cushions were seen within the developing outflow tract. One pair was positioned proximally, with the other pair located distally. By stage 25, a third distal cushion had developed. This finding was before the appearance of two further, intercalated, endocardial cushions, also distally positioned, which were first seen at stage 29. In the arterial segment, the aortic and pulmonary channels were separated by the structure known as the aortopulmonary septum. The dorsal limb of this septum penetrated the distal dorsal cushion, whereas the ventral limb grew between the remaining two distal cushions, both of which were positioned ventrally. The three distal endocardial cushions, and the two intercalated endocardial cushions, contributed to the formation of the leaflets and sinuses of the arterial roots. The two proximal cushions gave rise to a transient septum, which later became transformed into the muscular component of the subpulmonary infundibulum. Concomitant with these changes, an extracardiac tissue plane was formed which separated this newly formed structure from the sinuses of the aortic root. Our study confirms that three endocardial cushions are positioned distally, and two proximally, within the developing outflow tract of the chick. The pattern of the distal cushions, and the position of the ventral limb of the aortopulmonary septum, differs significantly from that seen in mammals.

Misexpression of noggin leads to septal defects in the outflow tract of the chick heart.

BMP-2 and BMP-4 are known to be involved in the early events which specify the cardiac lineage. Their later patterns of expression in the developing mouse and chick heart, in the myocardium overlying the atrioventricular canal (AV) and outflow tract (OFT) cushions, also suggest that they may play a role in valvoseptal development. In this study, we have used a recombinant retrovirus expressing noggin to inhibit the function of BMP-2/4 in the developing chick heart. This procedure resulted in abnormal development of the OFT and the ventricular septum. A spectrum of abnormalities was seen ranging from common arterial trunk to double outlet right ventricle. In hearts infected with noggin virus, where the neural crest cells have been labelled, the results show that BMP-2/4 function is required for the migration of neural crest cells into the developing OFT to form the aortopulmonary septum. Prior to septation, misexpression of noggin also leads to a decrease in the number of proliferating mesenchymal cells within the proximal cushions of the outflow tract. These results suggest that BMP-2/4 function may mediate several key events during cardiac development.

Anatomy of ureterovesical junction and distal ureter studied by endoluminal ultrasonography in vitro.

PURPOSE: The emerging technique of endoluminal ultrasonography (ELUS) provides a new modality for endoscopic visualization of the urinary tract which needs to be further evaluated. We studied the normal anatomy of distal ureter and ureterovesical junction using ELUS. MATERIALS AND METHODS: An assessment of in vitro ELUS ureteric images undertaken at 1 mm. intervals from 8 fresh human cadaver pelvis blocs of bladder and distal ureter were compared with findings of serial histological sections of the same specimens (stained for cholinesterase isoenzymes) to assess the degree of correlation. Computer-assisted 3D reconstructions were made. RESULTS: The different components (ureteric, detrusor and periureteric tissue) of the UVJ could be identified on the basis of echogenicity and form, but differentiation between the respective muscle layers in the wall of the ureter or of the detrusor was not possible. Nevertheless, ureteric volume measurements and an assessment of transmural ureteric length and the angle of passage through the bladder wall were possible. CONCLUSIONS: ELUS is able to differentiate between the ureteric and detrusor muscle and the UVJ gross anatomy can be reconstructed. ELUS technology, however, fails to differentiate between individual muscular layers of the ureter or the detrusor. Further improvement in ELUS is mandatory.

Developmental bioinformatics: linking genetic data to virtual embryos.

This paper discusses current efforts to produce databases of gene expression for the major model embryos used in developmental biology. The efforts to build these resources were motivated by the need for immediate internet access to all types of research data, and the production of these databases is a major and new challenge for bioinformatics. Thus far bioinformatics has mainly been concerned with textually oriented resources and data, much of it concerned with gene and protein sequences. Because the genetic basis of developmental biology is integrated with developmental anatomy, these databases require the use of images to link molecular data with spatial information. In order to standardise database formats, digital atlases of some model systems are being produced that include integrated anatomical descriptions and these are being linked to appropriate genetic data. Integrating such image-based, searchable data into databases makes new demands on the field of bioinformatics and we consider here the imaging modalities that are used to obtain information and we discuss in particular the production of 3D images from serial sections. Next, we consider how to integrate textual and spatial descriptions of gene expression and the key tool needed to make this possible, i.e. anatomical nomenclature. A short review of internet resources on developmental biology is also given and future prospects for the development of these databases are discussed.

Normal and abnormal embryonic development of the anorectum in human embryos.

In the literature, some controversy still exists about the normal and abnormal development of the human anorectum. Therefore, a three-dimensional and histological study was performed on human embryos. In early anorectal development (< or = 49 days postfertilization), the cloaca plays a crucial role, separated from the amniotic cavity by its cloacal membrane. In the cloaca, the yolk sac/primitive hindgut and allantois/primitive urogenital sinus enter. During the embryonic caudal folding process, incorporation of these structures occurs, including their surrounding extraembryonic mesoderm, which fuses to form the urorectal septum. Consequently, this septum does not grow in the direction of the cloacal membrane, and fusion of these structures is likewise never observed. The cloaca remains as such until the cloacal membrane ruptures by apoptotic cell death. The dorsal part of the cloaca then becomes part of the amniotic cavity, and is by no means involved in the development of the anorectum. The tip of the urorectal septum will become the perineal area. Soon after rupture of the cloacal membrane, during late anorectal development (> or = 49 days postfertilization), a secondary occlusion of the anorectal canal occurs, first due to adhesion, followed by formation of an epithelial "plug" at the level of the anal orifice. Recanalization, by apoptotic cell death, of this secondary occluded anal orifice occurs later during development. Based on these embryological observations, congenital anorectal malformations with an abnormal communication to the exterior are best explained as early embryonic defects. The abnormal communications, usually called fistulae, should be regarded as ectopic anal orifices. Anorectal malformations with the anus in normal position are best explained as late embryonic defects.

Quantitative graphical description of portocentral gradients in hepatic gene expression by image analysis.

The liver consists of numerous repeating, randomly oriented, more or less cylindrical units, the lobules. Although enzyme-histochemical or microbiochemical assays accurately reflect zonal differences in lobular enzyme content, their results cannot be directly compared to biochemical assays. This is because section-based assays typically sample along a linear portocentral column of cells, even though periportal regions contribute substantially more to hepatic volume than pericentral regions. We have developed a time-efficient approach that depends on image analysis to determine the prevalence of hepatocytes (pixels) with a defined cellular concentration of a particular gene product (absorbance), and that generates a graph with the average absorbance per hepatocyte on the ordinate and the percentage of hepatocytes with absorbances in each of a predetermined range of absorbances incrementally summed on the abscissa. The direction of the gradient is read directly from the section. The gradient is a graphical representation of the two-dimensional distribution pattern of the gene product between the portal tracts and the central veins. The total surface area underneath the resulting graph represents the integrated absorbance and is equivalent to the outcome of a biochemical assay. The typical linear portocentral gradient can be derived from that representing the two-dimensional distribution if we assume that liver lobules are uniformly cylindrical or prismatic. The analysis, therefore, yields a quantitative description of the relation between the enzymatic phenotype of hepatocytes and their position on a normalized portocentral radius. We have used the procedure to compare portocentral gradients of different enzymes in the same liver and of the same enzyme in different livers. In addition, bipolar portocentral gradients of the same enzyme in the same liver were analyzed.

Three-dimensional reconstruction of colon carcinoma metastases in liver.

Resection of liver metastases in patients with colon cancer increases survival but success depends on removal of all tumour tissue. For this purpose, understanding of spatial relationships between metastases and liver architecture is essential. Because metastatic cancer growth is essentially a three-dimensional (3D) event, we decided to apply 3D reconstruction techniques to study these spatial relationships between metastases and liver structures such as blood vessels, stroma and the liver capsule (Glisson's capsule). Colon carcinoma metastases were experimentally induced in rat liver by injection of colon cancer cells (CC531) into the portal vein. Three weeks later, livers from these animals and control livers were removed and immediately frozen in liquid nitrogen. Thirty-seven to 110 consecutive sections were used for each 3D reconstruction of 26 metastases in eight livers. Contours of different structures were stained by (immuno)histochemical means, traced in each section and stored in a database. From the contour model, a volume model was generated. Among the 26 metastases, seven were found to grow distantly from the liver capsule. They were small and consisted of well-differentiated cancer cells that were totally surrounded by a basement membrane and stroma which was always connected with adjacent blood vessels of a portal tract. The remaining 19 metastases showed a more advanced pattern of development. Infiltration of poorly differentiated colon cancer cells progressed through the stroma at various sites and areas of direct contact between cancer cells and hepatocytes were frequently found. This type of outgrowth of cancer cells was only found when metastases had made contact with the liver capsule. However, some areas in sections of these advanced stages still resembled small metastases. On the basis of these findings, we conclude that stroma-affects the differentiation pattern of cancer cells and has at least a dual role in tumour growth. On the one hand it limits invasion of cancer cells in the surrounding host tissue. On the other hand, stroma formation at the capsule, which consists mainly of granulation tissue, facilitates outgrowth of the tumours. Furthermore, our 3D reconstructions demonstrate the spatial heterogeneity of larger metastases and the importance of a 3D approach to understand growth and development of metastases in general and colon cancer metastases in the liver in particular.

Endoluminal ultrasound of the urethra: a new modality for cross-sectional imaging of the urethra?

Endoluminal ultrasound (ELUS) with high-frequency transducers is a new technique for imaging tubular structures. In combination with a rotating mirror, 360 degrees cross-sectional images of the wall can be obtained. Because of the high frequency, the axial resolution is much higher and thus more detail can be seen. In the study reported in this article, ELUS was performed to image the wall of the female pig urethra to see whether cross-sectional images obtained by ELUS could be correlated with anatomic cross sections of the urethra. Commercially available transducers with a frequency of 30 and 20 MHz were used, the latter having the best suitable frequency for this procedure. The images were of high quality and the different anatomic layers could be clearly visualized. The mucosa/submucosa, the external sphincter, and the surrounding serosa were all identifiable. The correct interpretation was also confirmed by histological cross-section study. We believe that endoluminal ultrasound is a very promising technique for imaging the urethra. Further studies need to be conducted to improve the catheters for urological use and to develop the clinical usefulness of this technique.

Functional anatomy of the human ureterovesical junction.

BACKGROUND: The valve function of the ureterovesical-junction (UVJ) is responsible for protection of the low pressure upper urinary tract from the refluxing of urine from the bladder. Controversy about the microanatomy of the human ureterovesical-junction persists. METHODS: Ten (3 male and 7 female) fresh cadaveric bladders (mean age 70 years old) were studied. The bladders were fixed within 24 hours postmortem, frozen, and serially sectioned. Acetyl- and butyryl- (nonspecific) cholinesterase activity were visualised as described by Karnovsky and Roots. The three-dimensional distribution of the different muscle groups participating in the formation of the UVJ was reconstructed. RESULTS: Three different muscle groups were identified: (1) the detrusor muscle and the deep trigone were mainly acetylcholinesterase-positive, (2) the inner and outer layer of the ureteric muscle were butyrylcholinesterase-positive and merged into a single longitudinal layer at the level of the UVJ and form the superficial trigone distally to the ureteric orifices, and (3) the muscularis mucosae is a discontinuous butyrylcholinesterase-positive layer in the bladder that is absent from the trigone. No evidence of any muscular connection was found between the ureter and bladder musculature. CONCLUSIONS: The anatomy of the UVJ as observed by us suggests the following model of the ureteric peristalsis. The urine bolus arrives in the ureteric lumen at the UVJ level. The ureter can only shorten its length, slides freely in its tunnel, and discharges the urine bolus in the bladder cavity. Ureteric constriction due to the peristalsis and thickening of the contracted portion of the ureter prevents the upstream leakage. Distal spreading of the ureteric "peristalsis" in the superficial trigone increases the submucosal ureteric length and prevents reflux.

Design and implementation of a database and program for 3D reconstruction from serial sections: a data-driven approach.

This paper describes a structural approach for a standard setup of a computer program for 3D reconstruction from serial sections. Three-dimensional reconstruction as a technique increases in importance as, along with modern immunohistochemical techniques, it is a tool in the understanding of three-dimensional development patterns. In order to apply 3D reconstruction technique in a standard laboratory setup, an attempt was made to streamline the input and the manipulation of the data such that results are obtained easily. One will find a combination of two approaches in this paper: the first is a strict ordering of the complex data, and the second is an ordering of the processes that one wishes to apply on the data (together, these two approaches constitute an information analysis); because it was observed that developmental biologists tend to work from simple lines to describe their observations, the contour model was chosen as the vehicle to build a reconstruction model from. Consequently, the data is ordered in a database that has to be manipulated to get the data out in the desired format. The most important output format is a display of the reconstructed contour stack on a graphical computer screen. Together with the other data manipulation processes, such as the input, the inspection, the revision (correction), and the reconstruction, all processes are described using the reconstruction of an 11 embryonic days (ED) rat embryo as an example. Finally, the merits of the program are illustrated with an example from the development of the human embryonic heart.

Vascular branching pattern and zonation of gene expression in the mammalian liver. A comparative study in rat, mouse, cynomolgus monkey, and pig.

BACKGROUND: A significant part of the liver volume consists of regions in which hepatocytes are in close contact with large branches of the afferent (portal vein) or efferent (hepatic vein) vessels. As most studies have addressed zonation of gene expression around the parenchymal branches of the portal and hepatic vein only, the patterns of gene expression in hepatocytes surrounding larger vessels are largely unknown. METHODS: For that reason, we studied the patterns of expression of the mRNAs and proteins of the pericentral marker enzymes glutamine synthase, ornithine aminotransferase, and glutamate dehydrogenase and the periportal marker enzymes phosphoenolpyruvate carboxykinase and carbamoylphosphate synthase in the rat liver, in relation to the branching pattern of the afferent and efferent hepatic veins with immuno and hybridocytochemical techniques. These patterns of expression were compared with those seen in mouse, monkey, and pig liver. RESULTS: The distribution patterns of the genes studied appear to reflect the "intensity" of the pericentral and periportal environment, glutamine synthase and phosphoenolypyruvate carboxykinase requiring the most pronounced environment, respectively. The patterns of gene expression around the large branches of the portal and hepatic vein were found to be related to the parenchymal branches in the neighbourhood of these large blood vessels. Only the cells of the limiting plate retain their periportal and pericentral phenotype for those marker enzymes that do not require a pronounced periportal or pericentral environment to be expressed. GS-negative areas in the pericentral limiting plate appear to correlate with a local absence of draining central veins, and become more frequent and extensive around the larger branches of the hepatic vein. CONCLUSIONS: The similarity of the observed patterns of gene expression of the genes studied in mouse, rat, monkey, pig, and man suggests that they reflect a general feature of gene expression in the mammalian liver. A comparison of mouse, rat, pig, and human liver suggests that the presence of glutamine synthase-negative areas reflects the branching order of the efferent hepatic blood vessel.

Atrioventricular conduction system in hearts with muscular ventricular septal defects in the setting of complete transposition.

The detailed structure of a ventricular septal defect was compared in 90 hearts with complete transposition (concordant atrioventricular and discordant ventriculoarterial connections) and in 102 hearts with concordant connections at both junctions; the latter group was selected to include only cases with the septums aligned in the normal way. The interventricular communications observed in 13% of the group with complete transposition, which, in our material, had no counterpart in the hearts with concordant segmental connections, were of special interest. These defects, completely surrounded by muscle, were positioned around the midline on the right side of the septum but always lay under or partially under the septal leaflet of the tricuspid valve. The medial papillary muscle group was always to the "left hand margin" of the defect as seen by the surgeon. Because these defects lay within the boundaries set by the septal leaflet of the tricuspid valve, they would conform to the criteria for classification as inlet muscular defects but could equally be described as central or subtricuspid. It is significant that, in all those cases with histologic sectioning, the axis of atrioventricular conduction tissue ran to the surgeon's right hand margin. This position is markedly different from the pattern found in typical defects of the inlet septum, which are completely surrounded by muscle and extend to the posterior wall of the heart. In this more common situation, the conduction axis runs above the left hand margin of the defect. This finding has obvious implications for surgical treatment.

The functional anatomy of the ureterovesical junction.

OBJECTIVE: To obtain a new insight into the anti-reflux mechanism of the ureterovesical junction by studying the topographical anatomy of the juxta- and intravesical ureter and its relationship to the surrounding bladder musculature. MATERIALS AND METHODS: Fresh pig bladders were fixed, frozen and serially sectioned. Enzyme histochemistry was performed to demonstrate tissue acetyl- and butyryl- (non-specific) cholinesterase. Smooth muscle cells were identified by immunohistochemistry using a monoclonal anti-alpha actin smooth muscle antibody. Three-dimensional computer reconstructions of the different muscle groups of the bladder and ureterovesical junction were generated. RESULTS: On the basis of expression patterns of the cholinesterase isozymes, five different groups of muscles were identified: the detrusor, the muscularis mucosae of the bladder, the muscle layer of the intravesical ureter and the separate inner and outer muscular layers of the pelvic ureter. CONCLUSION: No separate ureteric sheath was identified. There appeared to be few or no (muscular) connections between the ureter and the bladder musculature. The muscle layer of the ureter ended beneath the mucosa of the bladder, without extension into the trigone. The submucosal section of the ureter was very short, although its length was thought to be of importance in the anti-reflux mechanism. Examination using enzyme histochemistry demonstrated a muscularis mucosae in the bladder which was absent from the trigone.