Hans-Jürg Kuhn

The early development of the epicardium in Tupaia belangeri

SummaryDevelopment of the epicardium was studied in embryos of Tupaia belangeri from the 13th to 15th day of ontogeny. The greater part of the epithelium of the epicardium does not differentiate locally from the myoepicardium (cardiac splanchnopleure, splanchnic mesoderm), but rather from the coelomic epithelium of the septum transversum. The myoepicardium of the future atria and ventricles differentiates into myocardial cells only. On ontogenetic day 13, bulbar protrusions (the “villi” of Kurkiewicz 1909) are formed on the surface of the septum transversum and extend into the pericardial cavity, primarily between the sinoatrial and the ventricular regions of the embryonic heart. These protrusions are covered by flattened interdigitating cells, and they are filled with intercellular fluid of the mesenchyme of the septum transversum. Many mitoses are found among the cells. From these protrusions free vesicles are formed which are discharged into the pericardial cavity. The vesicles attach to the surface of the myoepicardium, i.e. to the developing myocardial cells. The vesicles open, and their cells spread out onto the surface of the heart to form the primary epicardium. This process begins on the dorsal surface of the heart, close to the protrusions of the septum transversum, there are, however, further isolated patches of primary epicardium in other regions of the surface of the heart. After the epicardial cells have settled onto the myocardium, mitoses become rare among them. On day 15, most of the myocardium is coated by the primary epicardium and the protrusions on the septum transversum disappear. A “bare” myocardium, as found on ontogenetic days 12 and 13 in Tupaia, might be a primitive (plesiomorphic) condition among chordates. In adult Branchiostoma, the coelomic epithelium which coats the contractile blood vessels had been found to differentiate into muscle cells that remain uncoated on the side facing the coelomic cavity (Franz 1933; Joseph 1914, 1928).

Expression patterns of erythropoietin and its receptor in the developing spinal cord and dorsal root ganglia.

Recombinant human erythropoietin (EPO) is neuroprotective in animal models of adult spinal cord injury, and reduces apoptosis in adult dorsal root ganglia after spinal nerve crush. The present work demonstrates that spinal cord and dorsal root ganglia share dynamic expression patterns of EPO and its receptor (EPOR) during development. C57Bl mice from embryonic days (E) 8 (E8) to E19 were studied. In spinal cord and dorsal root ganglia, EPOR expression in all precursor cells preceded the expression of EPO in subsets of neurons. On E11, EPO-immunoreactive spinal motoneurons and ganglionic sensory neurons resided adjacent to EPOR-expressing radial glial cells and satellite cells, respectively. From E12 onwards, EPOR-immunoreactivity decreased in radial glial cells and, transiently, in satellite cells. Simultaneously, large-scale apoptosis of motoneurons and sensory neurons started, and subsets of neurons were labelled by antibodies against EPOR. Viable neurons expressed EPO and EPOR. Up to E12.5, apoptotic cells were EPOR-immunopositive, but variably EPO-immunonegative or EPO-immunopositive. Thereafter, EPO-immunonegative and EPOR-immunopositive apoptotic cells predominated. Our findings suggest that EPO-mediated neuron-glial and, later, neuron–neuronal interactions promote the differentiation and/or the survival of subsets of neurons and glial cells in central as well as in peripheral parts of the embryonic nervous system. Correspondingly, expression of phospho-Akt-1/protein-kinase B extensively overlapped expression sites of EPO and EPOR, but was absent from apoptotic cells. Identified other sites of EPO and/or EPOR expression include radial glial cells that transform to astrocytes, cells of the floor plate and notochord as well as neural crest-derived boundary cap cells at motor exit points and cells of the primary sympathetic chain.

Pattern of calretinin immunoreactivity in the main olfactory system and the vomeronasal system of the tree shrew, Tupaia belangeri

The distribution of the calcium‐binding protein calretinin was studied in peripheral and central parts of the main olfactory system (MOS) and the vomeronasal system ( VNS ) of adult tree shrew Tupaia belangeri. The calretinin immunoreaction was carried out with a peroxidase‐coupled polyclonal antibody. In the VNS, complete labeling of all receptor cells and vomeronasal nerve fibers was observed, whereas only a subset of the somata and dendrites of receptor cells and of the olfactory nerve fibers of the MOS was immunoreactive. From the immunoreactive dendritic clubs of vomeronasal receptor cells, calretinin‐labeled structures, presumably clumps of microvilli, arose that terminated within immunopositive portions of the mucus. In the main olfactory bulb, the neuropil of some of the glomeruli was immunoreactive. All periglomerular and many mitral cells were labeled. The external plexiform layer was subdivided into a faintly immunoreactive superficial half and a strongly immunoreactive deep half. Immunoreactive basal dendrites of mitral cells could be followed into either the deep half or the superficial half. In the laminated internal granular layer, a subset of immunopositive granule cells extended dendrites into the external plexiform layer. Mitral cells and granule cells with dendrites ascending to different levels of the external plexiform layer may represent functional subclasses. In the accessory olfactory bulb, all vomeronasal nerve fibers, glomeruli, and mitral/tufted cells were labeled, whereas immunoreactive periglomerular cells and internal granule cells were only scattered. In Tupaia, calretinin immunoreactivity is a more general property of the primary projecting neurons of the VNS than of the MOS and possibly indicates the involvement of calretinin in the perception of certain of the olfactory qualities. J. Comp. Neurol. 420:428–436, 2000.

Morphogenesis of megamitochondria in the retinal cone inner segments of Tupaia belangeri (Scandentia)

Abstract.The morphogenesis of the megamitochondria in the retinal cones of prenatal, young postnatal and adult tree shrews (Tupaia belangeri) was studied by transmission electron microscopy and three-dimensional reconstruction techniques. The initial assembly of the supranuclear cone mitochondria and their subsequent migration towards the developing inner segment conform to the morphogenetic pattern known from other mammals. Within the first postnatal week, however, a marked increase in both the number of the cristae and the matrix density occurs in the inner segment mitochondria of Tupaia. These mitochondria then grow, initially exhibiting a basal-to-apical size-gradient. In the 17-day-old Tupaia, this gradient is superseded by a radial size-gradient that, in addition to the single apical megamitochondrion, is characteristically found in the adult Tupaia. The number of megamitochondria remains almost constant from day 12 of postnatal ontogenesis to the adult stage. Each megamitochondrion consists of an apically located body from which several long processes project towards the base of the inner segment. In the older stages, the number of small mitochondria that most probably have budded off from the megamitochondrial processes clearly increases. We consider that megamitochondria in the cone inner segments of Tupaia arise by the growth of a single mitochondrion and not by the fusion of smaller mitochondria.

Pattern of cell death during optic cup formation in the tree shrew Tupaia belangeri.

Developmental cell death during optic cup formation was investigated in the tree shrew Tupaia belangeri. Twenty‐six embryos from days 12 to 16 of prenatal ontogenesis were studied by light microscopy. Prior to the optic vesicle stage, a dorsal area of cell death surrounded the lumen of the V‐shaped optic evagination (phase 1). A ventral band of dead cells, found in the optic vesicle (phase 2), preceded a dorsal focus of cell death (phase 3) previously described as a characteristic avian feature. During further invagination (phase 4), a peak of cell death was represented by a ventrodorsal band extending from the diencephalon over the complete optic anlage. The main areas of cell death found in phases 2 to 4 were, topographically, segments of this band. Also, the distinct areas of cell death reported in the literature for the vertebrate species studied so far fit well into this ventrodorsal band found in Tupaia. Thus, most probably, a common spatio‐temporal sequence of cell death exists in all of them. In Tupaia, dead cells concentrated at the diencephalic insertion of the optic stalk, the suboptic necrotic center (SONC) reported by several authors, were part of the early ventral band of cell death originating from the median floor of the prosencephalon (phase 2). During optic cup formation, the SONC was part of the ventrodorsal band and, thus, was not secondarily formed by the subdivision of a pre‐existing distal ventral area of cell death as reported for several other vertebrates. J. Comp. Neurol. 401:352–366, 1998.

Development of starburst cholinergic amacrine cells in the retina of Tupaia belangeri

“Starburst” cholinergic amacrines specify the response of direction‐selective ganglion cells to image motion. Here, development of cholinergic amacrines was studied in the tree shrew Tupaia belangeri (Scandentia) by immunohistochemistry with antibodies against choline acetyltransferase (ChAT) and neurofilament proteins. Starburst amacrines expressed ChAT much earlier than previously thought. From embryonic day 34 (E34) onward, orthotopic and displaced subpopulations segregated from a single cluster of immunoreactive precursor cells. Orthotopic starburst amacrines rapidly took up positions in the inner nuclear layer. Displaced starburst amacrines were first arranged in a monocellular row in the inner plexiform layer, and, with a delay of 1 week, they descended to the ganglion cell layer. Conversely, dendritic stratification of displaced amacrines slightly preceded that of orthotopic ones. Starburst amacrines expressed the medium‐molecular‐weight neurofilament protein (NF‐M) from E34 to postnatal day 11 (P11) and coexpressed α‐internexin from E36.5 to P11. Consequently, neurofilaments composed of α‐internexin and NF‐M may stabilize developing dendrites of starburst amacrines. During the first 2 postnatal weeks, subpopulations of anti‐NF‐M‐labeled ganglion cells costratified with the preexisting dendritic strata of starburst amacrines in the ON sublamina, OFF sublamina, or both. Hence, anti‐NF‐M‐labeled ganglion cells may include direction‐selective ones. Thereafter, NF‐M and α‐internexin proteins disappeared from starburst amacrines, and NF‐M immunoreactivity was lost in the dendrites of ganglion cells. Our findings suggest that NF‐M and α‐internexin are important for starburst amacrines and ganglion cells to recognize each other and, thus, contribute to the formation of early developing retinal circuits in the inner plexiform layer. J. Comp. Neurol. 502:584–597, 2007.

The earliest invasion of macrophages into the developing brain and eye of the tree shrew Tupaia belangeri.

The earliest occurrence of macrophages was investigated in the brain and optic anlagen of the tree shrew Tupaia belangeri. Nineteen serially sectioned embryos, belonging to five phases of programmed neuroepithelial cell death previously found during optic cup formation, were used. Macrophages were identified by structural criteria and by labelling with the lectin Griffonia simplicifolia I-B4. Macrophages, most probably derived from the yolk sac, are present in the perineural vessels of the phase 1 embryo (V-shaped optic evagination). Within this compartment, their number increases up to phase 4 (advanced invagination) and drops during phase 5. This first wave of macrophages is followed by a second one occurring within the perineural mesenchyme and within the neuroepithelium of the brain and eyes from phase 3 onwards. In the phase 4 embryos, a considerable rise in the number of intraventricular macrophages is noted. During phase 5 (far advanced invagination), marked vascularization of the brain starts, and a peak of macrophages is noted in the neuroepithelium and in the ventricular lumen of the brain. This spatiotemporal pattern suggests that, in Tupaia, the earliest macrophages are simultaneously shifted from perineural vessels into the neuroepithelial walls of the developing brain and, at earlier stages than previously described in other vertebrate species, of the eye anlagen.

The early development of the heart of Tupaia belangeri, with reference to other mammals

SummaryThe development of the heart of Tupaia belangeri from the first endothelial-lined lumina to the cardiac loop is described in 20 embryos with 2 to 14 somites, from ontogenetic days 11 and 12. Bilateral endocardial tubes transporting blood are found in the 8-somite embryo; in the middle cardiac plate, angioblasts and angiocysts are located between them. In the 9-somite embryo, formation of the cardiac loop has started, the endocardial tubes approach each other closely, most of the angiocysts have been incorporated by the expanding endocardial tubes, and fusion of the endocardial lumina has started in the cono-truncal area. Apparently, much of the endocardial cardiac loop found in the 9-somite embryo has been produced by the disproportionate lengthening of a segment of the endocardial tubes, which is very short in the 8-somite embryo. In the 13-somite embryo the endocardial tubes have largely fused, but tube-like strands of endothelia, remnants of the original endothelial walls separating them, form a “palisade” and mark the original boundary between them. Myoepicardial differentiations of the splanchnopleure begin separately on both sides of the embryo and gradually spread craniad until they coalesce in the midline, in front of the anterior intestinal portal. The caudal portions of the endocardial tubes with initial myoepicardial and cardiac jelly differentiations do not contribute to the definitive heart. The anterior intestinal portal is very broad in Tupaia. Contradictions in the literature as to the bilaterality of cardiac primordia of eutherian mammals are discussed. The hypothesis is developed that bilateral endocardial tubes and bilateral myoepicardial differentiations of the splanchnopleure develop in species with a large yolk-sac, relatively late closure of the foregut, and a broad anterior intestinal portal (e.g., Tupaia, ferret, and cat, etc.). This is probably the primitive condition in eutherian mammals. In species with a small yolk-sac and/or reversal of germ layers (man, rodents), the foregut and anterior intestinal portal are formed earlier, the heart primordium reaches its median position ventral to the foregut in the angiocyst-stage, and the first endocardial lumina appear close to the midline. In these species, the primordium of the endocardium seems to be plexiform and without clear evidence for bilaterality.

Ciliogenesis in photoreceptor cells of the tree shrew retina

Abstract Transmission electron microscopy of the retinal cones from several prenatal, young postnatal and adult tree shrews (Tupaia belangeri) reveals that the centrioles, from which the ciliary precursors of the outer segments grow out, are not transported into a pre-existing inner segment, but are positioned under the apical plasma membrane of cone precursor cells all through the inner segment formation. Ciliogenesis starts before or on embryonic day 20 and thus precedes initial formation of the inner segment by 20 days, which is half the gestation period. Thus, the maturation of the outer segment covers a considerably longer period than has been previously described. Published observations from other mammals can be interpreted as conforming with the situation in Tupaia. In other vertebrates, compared to mammals, marked heterochronies do occur. In Tupaia, the centrioles and the cilium are located close to the central longitudinal axis of the photoreceptor precursor cell from the 20-day-old embryo to the 5-day-old juvenile. In this position the microtubule apparatus originating from the centrioles should be most effective in transporting the mitochondria into the inner segment. In the 12-day-old tree shrew, when transport of the mitochondria into the inner segment has been completed, centrioles and cilium have shifted into an eccentric position and the light-collecting megamitochondria have approached the disks of the outer segment. This eccentric position is maintained in all later developmental stages. In certain of the retinal areas of the adult Tupaia, the connecting cilia of neighbouring cones are always positioned on the same side of the inner segments.

Calretinin and FMRFamide immunoreactivity in the nervus terminalis of prenatal tree shrews (Tupaia belangeri)

The distribution and development of FMRFamide- and calretinin-immunoreactive neurons were investigated in the nervus terminalis of prenatal tree shrews from gestation day 19 onwards. The first FMRFamide-immunoreactive cells were observed medially in the olfactory epithelium on gestation day 20. From gestation day 23 onwards, the migrating nervus terminalis ganglion cells showed FMRFamide calretinin immunoreactivity. The distribution pattern of FMRFamide- and calretinin-immunoreactive cells was similar along the migratory route and in the ganglion of the terminal nerve. However, most probably calretinin and FMRFamide were expressed in separate neuronal populations. For the first time in a mammal, FMRFamide and calretinin are reported to occur in the migrating perikarya and neuronal processes of the nervus terminalis during prenatal development. The results suggest (i) an early activation of the rostral FMRFamide-immunoreactive migratory stream comparable to that described for the GnRH-immunoreactive part of the terminal nerve in other mammals and possibly (ii) an involvement of calretinin in mechanisms of cell migration and outgrowth of neuronal processes in the terminal nerve during the studied period.