Early embryonic development and transplantation in tree shrews.

As a novel experimental animal model, tree shrews have received increasing attention in recent years. Despite this, little is known in regards to the time phases of their embryonic development. In this study, surveillance systems were used to record the behavior and timing of copulations; embryos at different post-copulation stages were collected and cultured in vitro; and the developmental characteristics of both early-stage and in vitro cultured embryos were determined. A total of 163 females were collected following effective copulation, and 150 were used in either unilateral or bilateral oviduct embryo collections, with 307 embryos from 111 females obtained (conception rate=74%). Among them, 237 embryos were collected from 78 females, bilaterally, i.e., the average embryo number per female was 3.04; 172 fertilized eggs collected from 55 females, bilaterally, were cultured for 24-108 h in vitro for developmental observations; finally, 65 embryos from 23 bilateral cases and 70 embryos from 33 unilateral cases were used in embryo transplantation.

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.

The morphogenesis of the arteries of the pelvic extremity. A comparative study of mammals with special reference to the tree shrew Tupaia belangeri (Tupaiidae, Scandentia, Mammalia).

The ontogeny of the arteries of the pelvic extremity of Tupaia belangeri was investigated by light microscopy on the basis of serial sections of 30 embryos, dating from day 17 to day 42 post-copulation. In Tupaia, the gestational period takes approximately 43 days. Additionally, a 3-D reconstruction of the pelvic region and the right leg of a 22-day embryo was prepared. The arteries of an adult Tupaia were studied on the basis of a corrosion cast. The results were compared with the ontogeny of the arterial system of other mammals. In the 17-day embryo, the anlage of the pelvic extremity is penetrated by a capillary plexus. In the 18-day embryo, the a. ischiadica reaches the pelvic limb bud, representing the primary axial artery. On day 19, its r. perforans tarsi extends from the plantar to the dorsal aspect of the foot plate. The a. ischiadica is the main artery of the leg until the stage of the 22-day embryo. Afterwards, the peripheral arteries supplied by it are taken over by the a. iliaca externa and its extension, the a. femoralis. The a. iliaca externa springs from the a. iliaca communis in the 19-day embryo. From day 21 to day 22, the capillary plexus, which is nourished by the a. femoralis, closely approaches the a. ischiadica, and finally, a connecting branch joins the a. ischiadica. The a. ischiadica is then reduced to the a. glutea caudalis, and the aa. femoralis, poplitea profunda (at the cranial aspect of the m. popliteus), and interossea become the main arteries of the pelvic extremity. The a. poplitea superficialis, lying at the caudal aspect of the m. popliteus, and its continuation in the crural region, the a. peronea, develop until the 25-day embryo. The a. peronea gives rise to an r. perforans which penetrates the membrana interossea towards the dorsum of the foot. As a result of a shift of the origin of the a. iliaca externa in the proximal direction, the length of the a. iliaca communis gradually decreases until, on day 24, the a. iliaca externa springs directly from the lateral wall of the aorta. In the 20-day embryo, the a. iliaca externa gives rise to an a. circumflexa ilium profunda towards the lateral pelvic wall, and in 23-day embryos, to the a. profunda femoris. The main branches of the a. profunda femoris develop until day 24. At the same time, the aa. circumflexa femoris lateralis and nutricia ossis femoris arise from the a. femoralis. The a. saphena, which is already recognizable in the 23-day embryo, gives rise to the a. genus descendens, and as an a. plantaris medialis, to four aa. digitales plantares communes (I-IV) at the planta pedis. The development of the a. tibialis cranialis on day 25 takes place independently and without any topographic relation to the a. saphena, which functionally replaces the a. tibialis cranialis in some other mammals. In the 26-day embryo, the aa. peronea and tibialis cranialis extend to the dorsum of the foot where they continue as the aa. dorsales pedis profunda and superficialis. The fourth main artery of the lower leg, the a. caudalis femoris, which is first observed in the 20-day embryo, reaches the lateral aspect of the foot on day 24. Its r. calcaneus runs to the planta pedis. In 30-day embryos, the aa. digitales plantares propriae have differentiated. The corresponding dorsal arteries and the superficial plantar vascular are develop until day 35, so that all important arteries of the pelvic extremity, which are seen in the corrosion cast of the adult, are recognizable. Among the embryos and the adult Tupaia studied, individual variation is minimal. The developmental stage at which the arteries of the leg acquired a secondary vascular wall was ascertained. Only a vessel with a primary vascular wall can dissolve into a capillary plexus later on (e.g., a. interossea). In contrast, the course of an artery which has acquired a secondary vascular wall is determined, because modifications of the course of a vessel often need a capillary plexus as an intermediate st

FMRFamide immunoreactivity and the invasion of adenohypophyseal cells into the neural lobe in the developing pituitary of the tree shrew Tupaia belangeri.

Ontogenetic development of FMRFamide immunoreactivity in the cells and nerve fibers of the pituitary was studied in the tree shrew Tupaia belangeri. Up to the 26th day of gestation (E26), no FMRFamide immunoreactivity was visible. From E27 onwards it increased continuously until prenatally, on E41, the adult pattern was reached in the adenohypophysis, although at a lower intensity. In the adult Tupaia, as in the other mammals studied so far, a finely stained FMRFamide-immunoreactive fiber network was visible in the neural lobe and the infundibular stalk. As in several other adult mammals including man, endocrine cells in the pars intermedia and numerous scattered cells in the pars distalis were labeled, in contrast to several reports on rats and our studies on Galago, showing no FMRFamide-immunoreactive cells in these locations of the pituitary. With reference to the 'basophil invasion', we found FMRFamide-immunoreactive endocrine cells invading the neural lobe from the pars intermedia during the pituitary development. The distribution pattern of FMRFamide immunoreactivity in Tupaia indicates that the mammalian counterparts of FMRFamide may function as neuromodulators, neurotransmitters or as hormones already in defined prenatal stages.

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

The 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.

The early development of the epicardium in Tupaia belangeri.

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).

[The ontogenesis of the manubrium sterni in Tupaia belangeri]. / Die Ontogenese des Manubrium sterni bei Tupaia belangeri.

The morphogenesis of the manubrium sterni was studied in a series of dated embryos of Tupaia belangeri. In addition to the sternal bands, the "paired suprasternal Anlage" takes part in the shaping of the manubrium sterni as reported by Klima (1968) for other mammals. It forms skeletal elements that mediate between the clavicle and the manubrium: the sternocalvicular ligament and the paired prominence on the dorsal surface of the manubrium, which underlies the clavicles. The paired prominence corresponds to the praeclavium present in some therians. Very probably, the discus articularis of the sternoclavicular articulation of some primates can be attributed to the suprasternal Anlage. There was, however, no indication that the ossa suprasternalia of primates develop from the suprasternal Anlagen: In Tupaia these Anlagen do not form the cranial part of the manubrium. Klima's "unpaired Anlage" develops differently in Tupaia than in other therians. It consists of connective tissue and is not integrated into the manubrium. It presents an insertion surface for the M. pectoralis major, which shifts its origin onto the manubrium, after the sternal bands have fused. The homology of the "unpaired Anlage" and the "pars chondralis interclaviculae" is doubtful.

[Ontogeny and morphology of the round window and aqueduct of cochlea in Tupaia and other mammals]. / Die Ontogenese und Morphologie der Fenestra rotunda und des Aquaeductus cochleae von Tupaia und anderen Säugern.

Studied the morphogenesis of the Fenestra rotunda and of the Aquaeductus cochleae in a series of 23 dated embryos and postnatal stages of Tupaia belangeri. The ontogeny of the Fenestra rotunda is the result of the caudal growth of the Processus recessus (DE BEER 1937). The Processus arises from the caudal ridge of the floor of the cochlear part of the otic capsule. On the 28th d of ontogeny (the gestation period of Tupaia belangeri is 43 d), it is fused with the lateral edge of the parachordal plate. On the 40th d, the Processus recessus joins the ventral surface of the canalicular part of the otic capsule, which develops a small cartilaginous process to meet it. In Tupaia, the Processus recessus is a large cartilaginous plate in a nearly horizontal position. It does not reach the plane of the Foramen perilymphaticum. The Processus recessus can be regarded as a part of the parachordal plate that was shifted laterally together with the Recessus scalae tympani by the enlargement of the cochlear part of the otic capsule in the ancestors of living mammals. The Processus forms the floor of the Aquaeductus cochleae, by which the laterally shifted Recessus scalae tympani of mammals remains connected with the cranial cavity. The Aquaeductus cochleae contains the Ductus perilymphaticus connecting the Cavum perilymphaticum of the inner ear with the Cavum leptomeningeum. The Fenestra rotunda of mammals is homologous with the lateral aperture of the Recessus scalae tympani of reptiles. In some mammals (e.g. Micropotamogale), the Membrana tympani secundaria spans the lateral aperture of the Recessus scalae tympani, as in many reptiles. Both the Membrana tympani secundaria of reptiles and that of mammals are homologous. Secondarily, in a large number of therian mammals (e.g. Myotis [Frick 1952]), the tympanic cavity extends into the Recessus scalae tympani displacing the Membrana tympani secundaria medially from the lateral aperture of the Recessus scalae tympani (= Fenestra rotunda of mammals) and even into the plane of the Foramen perilymphaticum. Thereby the Fossula fenestrae rotundae is formed, which in bounded medially by the Membrana tympani secundaria.

Ontogeny and cranial morphology of the tympanic region of the Tupaiidae, with special reference to Ptilocercus.

The structure of the tympanic region of the skull of Ptilocercus lowii was studied in an embryo of 30 mm crown-rump length and in 5 osteocrania. As in Tupaia, the anterior wall of the bulla of Ptilocercus is not completed by a tympanic process of the alisphenoid, contrary to earlier reports. Ptilocercus resembles Tupaia in the following derived characters. The ventral wall of the tympanic cavity is formed by a rostral entotympanic and by a caudal tympanic process of the petrosal. The entotympanic develops in primary connection with the tubal cartilage. The tympanic aperture of the auditory tube is bordered by the entotympanic. The ring-shaped tympanicum is covered by the entotympanicum and is aphaneric. The musculus tensor tympani is lacking. Among mammals, these characters can be regarded as synapomorphic for the Tupaiidae, that is, to have been present in the common ancestor of the two subfamilies. From the evidence of the tympanic region, the Tupaiidae, therefore, form a monophyletic group. Besides these synapomorphies, there are remarkable differences between Ptilocercus and Tupaia in the structure of the bulla. In Ptilocercus the bulla is smaller and less pneumatized than in Tupaia. An anterior intrabullar septum, present in Tupaia, is lacking in Ptilocercus. The epitympanic wing of the alisphenoid is smaller in Ptilocercus than in Tupaia. A lateral prefacial commissure of the tegmen tympani is present in Ptilocercus, but absent in Tupaia. The caudal tympanic process of the petrosal is larger in Ptilocercus than in Tupaia. These characters are autapomorphic for the Ptilocercinae and for the Tupaiinae, respectively. They demonstrate that the auditory bulla of Ptilocercus and that of Tupaia have evolved independently to a considerable extent. An early phylogenetic separation of their respective ancestors seems likely. The tympanic region of the skull provides no evidence for close relationships of the tree shrews to the primates or to any other eutherians. The classification of the Tupaiidae in a separate order, Scandentia, is supported.