Jaco Hagoort

Reconstruction of the Patterns of Gene Expression in the Developing Mouse Heart Reveals an Architectural Arrangement That Facilitates the Understanding of Atrial Malformations and Arrhythmias

Firm knowledge about the formation of the atrial components and of the variations seen in congenital cardiac malformations and abnormal atrial rhythms is fundamental to our understanding of the normal structure of the definitive atrial chambers. The atrial region is relatively inaccessible and has continued to be the source of disagreement. Seeking to resolve these controversies, we made three-dimensional reconstructions of the myocardial components of the developing atrium, identifying domains on the basis of differential expression of myocardial markers, connexin40, and natriuretic precursor peptide A. These reconstructions, made from serial sections of mouse embryos, show that from the outset of atrial development, the systemic and pulmonary veins are directly connected to the atrium. Relative to the systemic junctions, however, the pulmonary venous junction appears later. Our experience shows that three-dimensional reconstructions have three advantages. First, they provide clear access to the combined morphological and molecular data, allowing clarification and verification of morphogenetic concepts for nonmorphological experts and setting the scene for further discussion. Second, they demonstrate that, from the outset, the myocardium surrounding the pulmonary veins is distinct from that clothing the systemic venoatrial junctions. Third, they reveal an anatomical and molecular continuity between the entrance of the systemic venous tributaries, the internodal atrial myocardium, and the atrioventricular region. All these regions are derived from primary myocardium, providing a molecular basis for the observed nonrandom distribution of focal right atrial tachycardias.

The interactive presentation of 3D information obtained from reconstructed datasets and 3D placement of single histological sections with the 3D portable document format

Interpretation of the results of anatomical and embryological studies relies heavily on proper visualization of complex morphogenetic processes and patterns of gene expression in a three-dimensional (3D) context. However, reconstruction of complete 3D datasets is time consuming and often researchers study only a few sections. To help in understanding the resulting 2D data we developed a program (TRACTS) that places such arbitrary histological sections into a high-resolution 3D model of the developing heart. The program places sections correctly, robustly and as precisely as the best of the fits achieved by five morphology experts. Dissemination of 3D data is severely hampered by the 2D medium of print publication. Many insights gained from studying the 3D object are very hard to convey using 2D images and are consequently lost or cannot be verified independently. It is possible to embed 3D objects into a pdf document, which is a format widely used for the distribution of scientific papers. Using the freeware program Adobe Reader to interact with these 3D objects is reasonably straightforward; creating such objects is not. We have developed a protocol that describes, step by step, how 3D objects can be embedded into a pdf document. Both the use of TRACTS and the inclusion of 3D objects in pdf documents can help in the interpretation of 2D and 3D data, and will thus optimize communication on morphological issues in developmental biology.

Development of the building plan of the heart.

Abstract: In this communication we discuss the formation of the synchronously contracting chambered heart from a peristaltically contracting linear heart tube. It is proposed that members of the T‐box family of transcription factors play a crucial role in the formation of the building plan of the formed heart. Tbx5 may confer venoarterial polarity to the heart tube, whereas Tbx2 initially and Tbx3 in later developmental stages prevent the cardiac inflow tract, atrioventricular region, outflow tract, as well as the cardiac inner curvatures from chamber differentiation. With the exception of the outflow tract that becomes incorporated into the ventricles, these regions contribute to the cardiac conduction system.

An interactive three-dimensional digital atlas and quantitative database of human development

Digital reconstruction of human development The detailed morphology of human development has intrigued scientists and the medical field alike. However, the scarcity of specimens hampers detailed mapping of tissue architecture. Furthermore, inaccuracies in the description of human development have crept into textbooks from observations of animal models that are extrapolated to humans. By mapping normal developmental processes and patterns, such as the growth and relative placement of organs, congenital anomalies can be better understood. de Bakker et al. generated interactive three-dimensional digital reconstructions based on the Carnegie collection of histologically sectioned human embryos spanning the first 2 months of gestation. These interactive models will serve as educational and scientific resources for normal and abnormal human development. Science, this issue p. 10.1126/science.aag0053 Interactive three-dimensional models unveil early human development. INTRODUCTION The basic human body plan, the arrangement of organs in the body, is laid down during embryonic development. Insight into the formation of this plan informs researchers and clinicians about normal development versus the development of congenital malformations, the latter of which have an incidence of 3% in the human population and cause up to one-quarter of all neonatal deaths. Despite modern technologies such as three-dimensional imaging, the intricate morphogenesis of the developing human body is difficult to understand. Textbooks on human development are often based on the works of early embryologists, some published more than 100 years ago. Because of the limited availability of human embryonic specimens, it is difficult or impossible to independently verify the information carried in these textbooks, or even to assess whether this information is derived from studies on human or animal models. RATIONALE Current imaging and computer technology make it possible to reconstruct human development with sufficient resolution to visualize organ development. Stained histological sections (mainly from the Carnegie Collection of human embryos) were digitized, tissues and organs were identified, and knowledge-driven modeling was applied to correct imperfections in the three-dimensional reconstructions. RESULTS We created a digital atlas with 14 interactive three-dimensional models of human embryology and a database encompassing 34 embryos spanning the first 2 months of human development. Approximately 15,000 histological sections from the Carnegie Collection were analyzed by trained biomedical students under expert supervision, and up to 150 organs and structures were identified and digitally labeled in each section. The labeled structures were then spatially reconstructed in such a way that the relation between the reconstruction and the original images was preserved. We tested the reproducibility of the manual tracing of the different organs and found that the variability in volumes of segmented structures ranged from 0.3% to 2% between students for simple and complex structures, respectively. The 3D models, supplemented by an object tree with structures named in accordance with the international standard of embryonic terminology, the Terminologia Embryologica, are presented as interactive 3D-PDFs, which facilitates exploration of the complex relations between the different organs and allows researchers to develop an independent view of their spatial relations. The 3D reconstructions enable the measurement of the growth of the individual organs and structures, the assessment of the changing position of organs relative to vertebral segments during development, and the verification of remaining ambiguities in the descriptions of the development of organs. CONCLUSION The morphology presented in this atlas is directly connected to the original sections of the embryos in the Carnegie Collection—a connection that was in danger of being lost, with present-day textbook morphology becoming increasingly schematic and deviating from the original substrate. A number of detailed analyses of the development of the kidney, pharyngeal arch cartilages, and notochord show that the current descriptions of the development of these organs are based on comparative animal models rather than on factual observations in human specimens. These examples demonstrate the scientific value of the atlas. This atlas will therefore serve as an educational and reference resource for students, clinicians, and scientists interested in human development and development-related congenital diseases. The 3D-PDFs of the reconstructions, as well as original and labeled images, are freely available (http://3datlasofhumanembryology.com). Lateral views of a model of a 7.5-week-old human embryo (16 mm). Left: Skeletal system. Center: Cardiovascular system with transparent heart muscle. Venous system is shown in blue, arterial system in purple, liver vessels in red, and umbilical vein in pink. Right: Reconstructed organs, except skin. Note, for example, the neural tube in green and the nerves in yellow. Scale bar, 2.5 mm. Current knowledge about human development is based on the description of a limited number of embryonic specimens published in original articles and textbooks, often more than 100 years ago. It is exceedingly difficult to verify this knowledge, given the restricted availability of human embryos. We created a three-dimensional digital atlas and database spanning the first 2 months of human development, based on analysis of nearly 15,000 histological sections of the renowned Carnegie Collection of human embryonic specimens. We identified and labeled up to 150 organs and structures per specimen and made three-dimensional models to quantify growth, establish changes in the position of organs, and clarify current ambiguities. The atlas provides an educational and reference resource for studies on early human development, growth, and congenital malformations.

Cardiomyocyte-specific miRNA-30c over-expression causes dilated cardiomyopathy.

MicroRNAs (miRNAs) regulate many aspects of cellular function and their deregulation has been implicated in heart disease. MiRNA-30c is differentially expressed in the heart during the progression towards heart failure and in vitro studies hint to its importance in cellular physiology. As little is known about the in vivo function of miRNA-30c in the heart, we generated transgenic mice that specifically overexpress miRNA-30c in cardiomyocytes. We show that these mice display no abnormalities until about 6 weeks of age, but subsequently develop a severely dilated cardiomyopathy. Gene expression analysis of the miRNA-30c transgenic hearts before onset of the phenotype indicated disturbed mitochondrial function. This was further evident by the downregulation of mitochondrial oxidative phosphorylation (OXPHOS) complexes III and IV at the protein level. Taken together these data indicate impaired mitochondrial function due to OXPHOS protein depletion as a potential cause for the observed dilated cardiomyopathic phenotype in miRNA-30c transgenic mice. We thus establish an in vivo role for miRNA-30c in cardiac physiology, particularly in mitochondrial function.

Towards a 3-dimensional atlas of the developing human embryo: The Amsterdam experience

Knowledge of complex morphogenetic processes that occur during embryonic development is essential for understanding anatomy and to get insight in the pathogenesis of congenital malformations. Understanding these processes can be facilitated by using a three-dimensional (3D) developmental series of human embryos, which we aim to create in this project. Digital images of serial sections of 34 human embryos of the Carnegie Collection between Carnegie stages 7 (15-17 days) and 23 (56-60 days) are used to create 3D reconstructions of different organ systems. The software package Amira is used to align the sections and to create the 3D reconstructions. In this midway evaluation we show the first results of the atlas, containing 34 embryos with more than 13.500 manually annotated sections. The 3D models can be interactively viewed within a 3D-pdf. This will be the first complete digital 3D human embryology atlas of this size, containing all developing organ systems.

Measurement and 3D-Visualization of Cell-Cycle Length Using Double Labelling with Two Thymidine Analogues Applied in Early Heart Development

Organ development is a complex spatial process in which local differences in cell proliferation rate play a key role. Understanding this role requires the measurement of the length of the cell cycle at every position of the three-dimensional (3D) structure. This measurement can be accomplished by exposing the developing embryo to two different thymidine analogues for two different durations immediately followed by tissue fixation. This paper presents a method and a dedicated computer program to measure the resulting labelling indices and subsequently calculate and visualize local cell cycle lengths within the 3D morphological context of a developing organ. By applying this method to the developing heart, we show a large difference in cell cycle lengths between the early heart tube and the adjacent mesenchyme of the pericardial wall. Later in development, a local increase in cell size was found to be associated with a decrease in cell cycle length in the region where the chamber myocardium starts to develop. The combined application of halogenated-thymidine double exposure and image processing enables the automated study of local cell cycle parameters in single specimens in a full 3D context. It can be applied in a wide range of research fields ranging from embryonic development to tissue regeneration and cancer research.

3D Topography of the Young Adult Anal Sphincter Complex Reconstructed from Undeformed Serial Anatomical Sections

Background Pelvic-floor anatomy is usually studied by artifact-prone dissection or imaging, which requires prior anatomical knowledge. We used the serial-section approach to settle contentious issues and an interactive 3D-pdf to make the results widely accessible. Method 3D reconstructions of undeformed thin serial anatomical sections of 4 females and 2 males (21–35y) of the Chinese Visible Human database. Findings Based on tendinous septa and muscle-fiber orientation as segmentation guides, the anal-sphincter complex (ASC) comprised the subcutaneous external anal sphincter (EAS) and the U-shaped puborectal muscle, a part of the levator ani muscle (LAM). The anococcygeal ligament fixed the EAS to the coccygeal bone. The puborectal-muscle loops, which define the levator hiatus, passed around the anorectal junction and inserted anteriorly on the perineal body and pubic bone. The LAM had a common anterior attachment to the pubic bone, but separated posteriorly into puborectal and “pubovisceral” muscles. This pubovisceral muscle was bilayered: its internal layer attached to the conjoint longitudinal muscle of the rectum and the rectococcygeal fascia, while its outer, patchy layer reinforced the inner layer. ASC contraction makes the ano-rectal bend more acute and lifts the pelvic floor. Extensions of the rectal longitudinal smooth muscle to the coccygeal bone (rectococcygeal muscle), perineal body (rectoperineal muscle), and endopelvic fascia (conjoint longitudinal and pubovisceral muscles) formed a “diaphragm” at the inferior boundary of the mesorectum that suspended the anorectal junction. Its contraction should straighten the anorectal bend. Conclusion The serial-section approach settled contentious topographic issues of the pelvic floor. We propose that the ASC is involved in continence and the rectal diaphragm in defecation.

Architectural differences in the anterior and middle compartments of the pelvic floor of young‐adult and postmenopausal females

The pelvic floor guards the passage of the pelvic organs to the exterior. The near‐epidemic prevalence of incontinence in women continues to generate interest in the functional anatomy of the pelvic floor. However, due to its complex architecture and poor accessibility, the classical ‘dissectional’ approach has been unable to come up with a satisfactory description, so that many aspects of its anatomy continue to raise debate. For this reason, we opted for a ‘sectional’ approach, using the Chinese Visible Human project (four females, 21–35 years) and the Visible Human Project (USA; one female, 59 years) datasets to investigate age‐related changes in the architecture of the anterior and middle compartments of the pelvic floor. The puborectal component of the levator ani muscle defined the levator hiatus boundary. The urethral sphincter complex consisted of a circular proximal portion (urethral sphincter proper), a sling that passed on the vaginal wall laterally to attach to the puborectal muscle (urethral compressor), and a circular portion that surrounded the distal urethra and vagina (urethrovaginal sphincter). The exclusive attachment of the urethral sphincter to soft tissues implies dependence on pelvic‐floor integrity for optimal function. The vagina was circular at the introitus and gradually flattened between bladder and rectum. Well‐developed fibrous tissue connected the inferior vaginal wall with urethra, rectum and pelvic floor. With eight‐muscle insertions, the perineal body was a strong, irregular fibrous node that guarded the levator hiatus. Only loose areolar tissue comprising a remarkably well developed venous plexus connecting the middle and superior parts of the vagina with the lateral pelvic wall. The posterolateral boundary of the putative cardinal and sacrouterine ligaments coincided with the adventitia surrounding the mesorectum. The major difference between the young‐adult and postmenopausal pelvic floor was the expansion of fat in between the components of the pelvic floor. We hypothesize that accumulation of pelvic fat compromises pelvic‐floor cohesion, because the pre‐pubertal pelvis contains very little fibrous and adipose tissue, and fat is an excellent lubricant.

Architecture of structures in the urogenital triangle of young adult males; comparison with females

The fibro‐muscular architecture of the urogenital triangle remains contentious. Reasons are small size of the constituting structures and poor visibility with most imaging methods. We reinvestigated the area in serial sections of three males (21–38 years old) of the American and Chinese Visible Human Projects and two 26‐week‐old male fetuses, and compared the findings with earlier observations in females. The mass of the levator ani muscle was approximately twofold smaller and its funnel shape steeper in males than females. In the levator hiatus, a strand of the smooth longitudinal muscle layer of the rectum, the ‘rectourethral (RU) muscle’, extended anteriorly from the anorectal bend to the penile bulb. Fibrous tissue that formed in the inferior reach of the fetal RU muscle identified the location of the developing perineal body (PB) and divided the muscle into posterior ‘rectoperineal’ and anterior ‘deep perineal’ portions. In males, the PB remained small and bipartite, so that the RU muscle presented as an undivided midline structure. The well‐developed female PB, instead, intertwined with the deep perineal muscle and both structures passed the vagina bilaterally to form the perineal membrane in the posterior portion of the urogenital triangle. The urethral rhabdosphincter extended in the anterior portion of the urogenital triangle between the penile bulb inferiorly and the bladder neck superiorly, and consisted of a well‐developed circular ‘membranous’ portion with bilateral posteroinferior ‘wings’ and a thinner ‘prostatic’ portion on the prostate anterior side. In men, muscles occupy the urogenital triangle, but additional tightening of the locally fibrous adipose tissue by the superficial transverse perineal muscle appears necessary to generate functional support in women. An interactive 3D pdf file with these anatomical details (available online) should allow more accurate interpretation of ultrasound, computed tomography and magnetic resonance images.