Epibranchial placodes and rhombencephalic neural crest provide precursor cells for the geniculate, petrosal, and nodose ganglia. In chick embryos and in Tupaia belangeri, apoptosis in rhombomeres 3 and 5 helps to select premigratory precursor cells and to segregate crest cell streams derived from the even-numbered rhombomeres. Much less is known about the patterns and functions of apoptosis in epibranchial placodes. We found that, in Tupaia belangeri, combined anlagen of the otic placode and epibranchial placode 1 transiently share a primordial low grade thickening with post-otic epibranchial placodes. Three-dimensional reconstructions reveal complementary, spatially, and temporally regulated apoptotic and proliferative events that demarcate the otic placode and epibranchial placode 1, and help to individualize three pairs of epibranchial placodes in a rostrocaudal sequence. Later, rostrocaudal waves of proliferation and apoptosis extend from dorsal to ventral parts of the placodes, paralleled by the dorsoventral progression of precursor cell delamination. These findings suggest a role for apoptosis during the process of neuroblast generation in the epibranchial placodes. Finally, apoptosis eliminates remnants of the placodes in the presence of late invading macrophages.

The expression patterns of erythropoietin (EPO) and its receptor (EPOR) were investigated in the midbrain and in adjacent parts of the synencephalon and hindbrain of embryonic C57Bl mice. On embryonic (E) day 8 (E8), virtually all neuroepithelial cells expressed EPOR. After neural tube closure, subsets of these cells downregulated EPOR. In contrast, radial glial cells were EPOR-immunolabeled from E11 onwards. Simultaneously, subpopulations of early developing neurons upregulated EPO and expressed HIF-1, known to transcriptionally activate EPO. Three-dimensional reconstructions revealed subpopulations of EPO-expressing neurons: (1) in the trigeminal mesencephalic nucleus (TMN), (2) at the rostral transition of the midbrain and synencephalon, (3) in the basal plate of the midbrain, (4) in the trigeminal motor nucleus, and (5) in the trigeminal principal sensory nucleus. In the rostral midbrain and synencephalon, EPO-immunoreactive neurons were attached to EPOR-expressing radial glial cells. The identity of radial glial cells was proven by their immunoreactivity for antibodies against astrocyte-specific glutamate transporter, brain lipid-binding protein, and nestin. From E12.5 onwards EPOR was downregulated in radial glial cells. Viable neurons of the TMN continued to express EPO and upregulated EPOR. Our findings provide new evidence that components of the EPO system are present in distinct locations of the embryonic brain and, by interactions between neurons and radial glial cells as well as among clustered TMN neurons, may contribute to its morphogenesis. Whether the observed expression patterns of EPO and EPOR may reflect EPO-mediated trophic and/or antiapoptotic effects on neurons is discussed.

Development 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).

In all mammals, the mitochondria of the cones of the retina are concentrated in the inner segment. Uniquely in tree-shrews (Tupaia, Scandentia, Mammalia), a "megamitochondrion" exhibiting highly specialized systems of densely packed cristae and a very electron dense matrix, is located apically in the inner segment. The ellipsoid is a solid body containing several megamitochondria and, towards its base, a large number of smaller mitochondria. The refractive index of isolated, but not oriented, inner segments of Tupaia belangeri is higher (XA = 1.405) than in any other mammal studied so far. The consistent geometrical pattern of the multilamellar crista-matrix systems, oriented longitudinally towards the outer segment, suggests an additional optical function of the megamitochondria.

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.