CircRNAs in the tree shrew (Tupaia belangeri) brain during postnatal development and aging.

Circular RNAs (circRNAs) are a novel type of non-coding RNA expressed across different species and tissues. At present, little is known about the expression and function of circRNAs in the tree shrew brain. In this study, we used RNA-seq to identify 35,007 circRNAs in hippocampus and cerebellum samples from infant (aged 47-52 days), young (aged 15-18 months), and old (aged 78-86 months) tree shrews. We observed no significant changes in the total circRNA expression profiles in different brain regions over time. However, circRNA tended to be downregulated in the cerebellum over time. Real-time RT-PCR analysis verified the presence of circRNAs. KEGG analysis indicated the occurrence of ubiquitin-mediated proteolysis, the MAPK signaling pathway, phosphatidylinositol signaling system, long-term depression, the rap1 signaling pathway, and long-term potentiation in both brain regions. We also observed that 29,087 (83.1%) tree shrew circRNAs shared homology with human circRNAs. The competing endogenous RNA networks suggested novel_circRNA_007362 potential functions as a 24-miRNAs sponge to regulate UBE4B expression. Thus, we obtained comprehensive circRNA expression profiles in the tree shrew brain during postnatal development and aging, which might help to elucidate the functions of circRNAs during brain aging and in age-related diseases.

Scleral gene expression during recovery from myopia compared with expression during myopia development in tree shrew.

PURPOSE: During postnatal refractive development, the sclera receives retinally generated signals that regulate its biochemical properties. Hyperopic refractive error causes the retina to produce "GO" signals that, through the direct emmetropization pathway, cause scleral remodeling that increases the axial elongation rate of the eye, reducing the hyperopia. Myopia causes the retina to generate "STOP" signals that produce scleral remodeling, slowing the axial elongation rate and reducing the myopia. Our aim was to compare the pattern of gene expression produced in the sclera by the STOP signals with the GO gene expression signature we described previously. METHODS: The GO gene expression signature was produced by monocular -5 diopter (D) lens wear for 2 days (ML-2) or 4 days (ML-4); an additional "STAY" condition was examined after eyes had fully compensated for a -5 D lens after 11 days of lens wear (ML-11). After 11 days of -5 D lens wear had produced full refractive compensation, gene expression in the STOP condition was examined during recovery (without the lens) for 2 days (REC-2) or 4 days (REC-4). The untreated contralateral eyes served as a control in all groups. Two age-matched normal groups provided a comparison with the treated groups. Quantitative real-time PCR was used to measure mRNA levels for 55 candidate genes. RESULTS: The STAY group compensated fully for the lens (treated eye versus control eye, -5.1±0.2 D). Wearing the lens, the hyperopic signal for elongation had dissipated (-0.3±0.3 D). In the STOP groups, the refraction in the recovering eyes became less myopic relative to the control eyes (REC-2, +1.3±0.3 D; REC-4, +2.6±0.4 D). In the STAY group, three genes showed significant downregulation. However, many genes that were significantly altered in GO showed smaller, nonsignificant, expression differences in the same direction in STAY, suggesting the gene expression signature in STAY is a greatly weakened form of the GO signature. In the STOP groups, a different gene expression pattern was observed, characterized by mostly upregulation with larger fold differences after 4 days than after 2 days of recovery. Eleven of the 55 genes examined showed significant bidirectional GO/STOP regulation in the ML-2 and REC-2 groups, and 13 genes showed bidirectional regulation in the ML-4 and REC-4 groups. Eight of these genes (NPR3, CAPNS1, NGEF, TGFB1, CTGF, NOV, TIMP1, and HS6ST1) were bidirectionally regulated at both time points in the GO and STOP conditions. An additional 15 genes showed significant regulation in either GO or STOP conditions but not in both. CONCLUSIONS: Many genes are involved in scleral remodeling and the control of axial length. The STOP (recovery) gene expression signature in the sclera involves some of the same genes, bidirectionally regulated, as the GO signature. However, other genes, regulated in GO, are not differentially regulated in STOP, and others show differential regulation only in STOP.

[Molecular evidence on the phylogenetic position of tree shrews].

The tree shrew is currently located in the Order Scandentia and is widely distributed in Southeast Asia, South Asia, and South China. Due to its unique characteristics, such as small body size, high brain-to-body mass ratio, short reproductive cycle and life span, and low-cost of maintenance, the tree shrew has been proposed as an alternative experimental animal to primates in biomedical research. However, there is unresolved debate regarding the phylogenetic affinity of tree shrews to primates and their phylogenetic position in Euarchontoglires. To help settle this debate, we summarized the available molecular evidence on the phylogenetic position of the tree shrew. Most nuclear DNA data, including recent genome data, suggested that the tree shrew belongs to the Euarchonta clade harboring primates and flying lemurs (colugos). However, analyses of mitochondrial DNA (mtDNA) data suggested a close relationship to lagomorphs and rodents. These different clustering patterns could be explained by nuclear gene data and mtDNA data discrepancies, as well as the different phylogenetic approaches used in previous studies. Taking all available conclusions together, the robust data from whole genome of this species supports tree shrews being genetically closely related to primates.

[Measurement and analysis of anatomical parameter values in tree shrews].

Anatomical parameter values in tree shrews are major biological characteristic indicators in laboratory animals. Body size, bones and mammilla, organ weights, coefficient intestinal canal and other anatomical data were measured and analyzed in laboratory domesticated tree shrews (7 to 9 months of age). Measurement of 31 anatomical parameters showed that body height, width of the right ear, ileum and colon had significant differences between males and females (P<0.05). Highly significant differences were also found in body slanting length, chest depth, torso length, left and right forelimb length, right hind limb length, left and right ear length, left ear width, keel bone length, left and right tibia length, duodenum and jejunum (P<0.01). With body length as the dependent variable, and tail length, torso length, right and left forelimb length, and left and right hind limb length as independent variables for stepwise regression analysis, the regression equation for body length = 13.90 + tail length × 0.16. The results of 37 organs weights between female and male tree shrews showed very significant differences (P<0.01) for weight of heart, lungs, spleen, left and right kidney, bladder, left and right hippocampus, left submandibular gland, and left and right thyroid gland, as well as significant (P<0.05) differences in the small intestine, right submandibular gland, and left adrenal gland. The coefficient of heart, lung, stomach, bladder, small and large intestine, brain, right hippocampus, and left adrenal gland showed highly significant differences (P<0.01), while differences in the right kidney, left hippocampus, left submandibular gland, right adrenal gland, and left and right thyroid gland were significant (P<0.05). With animal weight as the dependent variable and indicators of heart, lung, liver, spleen, left and right kidney and brain as independent variables for stepwise regression analysis, the regression equation showed that weight = 62.73 + left kidney × 79.21 + heart × 24.09. Female and male laboratory domesticated tree shrews showed certain influences in body size, organ weight and coefficient, and intestinal canal regarding anatomical parameters. This experiment provides basic data for studies on laboratory tree shrews and animal models.

Ontogenetic development of locomotion in small mammals--a kinematic study.

Comparative studies of locomotion indicate that limb design and performance are very similar in adult mammals of small to medium size. The present study was undertaken to test whether basic therian limb pattern is present during postnatal development. Kinematic data were collected from juveniles of two eutherian species in a cross-sectional study, using cinevideography. The tree shrew Tupaia glis and the cui Galea musteloides were selected because of their different reproductive strategies, which could result in differences in the development of locomotor abilities. The aims of this study were to describe the process by which young animals develop the adult pattern of locomotion and the extent to which this process varies in two species with very different postnatal ontogenies. Despite their different life histories, the development of kinematic parameters in the altricial tree shrew and the precocial cui are surprisingly similar. General limb design, performance, and timing of segment and joint movements in the young animals were similar to adults in both species, even from the first steps. Touch-down of the forelimb occurred at the position below the eye in all individuals and limb position was highly standardized at touch-down; no major changes in segment and joint angles were observed. Significant changes occurred at lift-off. With increasing body mass, limb segments rotated more caudally, which resulted in larger limb excursions and relatively longer steps. Developmental changes in locomotor abilities were similar in both species; only the time necessary to reach the adult performance was different. Despite the widely assumed maturity of locomotor abilities in precocial young, the first steps of the cui juveniles were not similar to the movements of adults. The adult locomotor pattern was reached within the first postnatal week in the cui and by the time they leave the nest in the tree shrew (39 days after birth; individual P39). These results suggest that during the evolution of precocial development only processes independent of exercise or gravity can be shifted into the intrauterine period. However, development of locomotor ability dependents on exercise, and adjustments and training occur during growth. Therefore, only the time necessary to reach maturity was clearly shortened in the precocial juvenile relative to the ancestral altricial condition.

Development of the lateral geniculate nucleus: interactions between retinal afferent, cytoarchitectonic, and glial cell process lamination in ferrets and tree shrews.

We have studied the relationship of retinal afferents, glial cell processes, and neuronal cytoarchitectonics in the lateral geniculate nucleus (LGN) of two species: tree shrews (Tupaia belangeri) and ferrets (Mustela putoris). Both species are relatively immature at birth, allowing the development of these features to be studied in the perinatal period. Retinal afferents, visualized by intraocular injection of a wheat germ agglutinin/horseradish peroxidase conjugate (WGA-HRP), are apparently the first elements of the developing LGN to exhibit a characteristic layered pattern in tree shrews and ferrets. Some radial glia still remain in the LGN of both species as the retinal afferents are in the process of segregating. Glial cell processes were visualized immunohistochemically with antibodies to glial fibrillary acidic protein (GFAP) or vimentin. In both the ferret and tree shrew, layering of glial cell processes is first seen as the overlap of retinal terminal fields diminishes. In the tree shrew LGN, these bands of dense glial cell staining are seen in apparent future cellular layers, whereas in the ferret, glial cell banding appears in interlaminar zones. If one or both eyes are removed at birth in tree shrews (before LGN cell layers are formed), the glial cell pattern seen 1 week later is in accord with the distribution of surviving nerve cells. The glial processes do not appear to invade regions left by degenerating retinal terminals or dying LGN cells. Several days after the appearance of layered glial cell processes (in the tree shrew) or at about the same time as glial layering (in the ferret), the first interlaminar spaces develop between neuronal cells, marking the beginning of cytoarchitectonic lamination, with its distinctive alternating cell-rich and cell-poor zones. Over the next several weeks, LGN neurons in both species continue to segregate into characteristic layers until the final, adult pattern of neuronal lamination is evident; as this process is completed, glial cell lamination disappears. These observations suggest that glial cells may be involved in establishing the neuronal layers that characterize the mature LGN of many species.

Vimentin: changes in distribution during brain development.

This paper examines both the anatomical changes in the distribution of vimentin intermediate filament protein and the biochemical changes in vimentin and its degradative enzyme during postnatal brain development in the tree shrew (Tupaia belangeri). A pattern of multiple immunoreactive bands at birth (postnatal day 0, or P0) was revealed in nitrocellulose blots of polyacrylamide gels ("Western blots"). These multiple bands gradually disappear during development, and in the adult a single band at the published molecular weight for vimentin (57 kD) is seen. This pattern of bands probably reflects shifts in the activity of a calcium-activated vimentin protease. The changes in the anatomical distribution of vimentin-immunoreactive (vimentin+) cells and their fine processes parallel the biochemical shifts seen in immunoblots. We have examined the neocortex, lateral geniculate nucleus (LGN), and hippocampus in detail. During the first postnatal week, vimentin+ glia, especially radial glia, are prominent in both neocortex and hippocampus. In contrast, only a few vimentin+ radial glia remain in the thalamus at this age. Vimentin+ glia appear to coincide with bundles of axons and often seem to outline subdivisions of thalamic nuclei. Additionally, cellular layers of the lateral geniculate nucleus (LGN) appear to stain with antibodies to vimentin several days before the characteristic neuronal cell layers appear in this area. During the second postnatal week, vimentin+ cells appear in "patches" throughout the cortex. Some subdivisions of the thalamus and hippocampus (as defined by cytoarchitectonic differences in the adult) are distinct when the tissue is stained with an antibody to vimentin, even though a conventional Nissl stain at this age shows no apparent delineation in these same regions. Finally, in the adult, only a few vimentin+ cells remain, primarily in the white matter. Taken together, these results suggest that the remodeling of vimentin+ intermediate filaments in immature glial cells (including radial glia) is paralleled by the action of the enzyme which breaks down these filaments. The apparent activity of this enzyme is high early in development as radial and other glia are rapidly dividing and undergoing morphological changes, with a decrease in activity in the juvenile and adult brain, as immature glial cells are supplanted by mature forms.

Development of acetylcholinesterase activity in the lateral geniculate nucleus.

The pattern of acetylcholinesterase activity in the tree shrew (Tupaia belangeri) lateral geniculate nucleus (LGN) undergoes a number of striking changes during postnatal development. The adult tree shrew LGN is made up of six cellular layers divided by relatively cell-free interlaminar zones. At birth, however, the nucleus appears unlaminated when processed with conventional Nissl-staining techniques. The cellular lamination appears during the first postnatal week. The eyes open much later, typically at the end of the third week after birth. In the adult tree shrew, acetylcholinesterase (AChE) activity is found throughout the nucleus (both within and between the six cellular layers). In most sections examined, reaction product is slightly more intense in the lateral cell layers (4, 5, and 6). This is in sharp contrast to the pattern at birth (postnatal day zero, or P0). The detectable AChE activity at this age is apparently found in inchoate layers 1-2 and 4-5. Within these pairs, areas innervated by the ipsilateral eye (i.e., incipient layers 1 and 5) appear to contain more reaction product. From P0 to P4, the density of AChE activity increases in layers 1-2 and 4-5 and becomes detectable in the barely evident layers 3 and (usually) 6 at this age. By the middle of the second postnatal week, after laminae are clearly apparent with a Nissl stain, AChE activity has increased and is mainly associated with each cellular layer in the nucleus. During the third week after birth this pattern undergoes a radical shift. The most intense AChE activity is now in the interlaminar zones. Finally, as the adult pattern emerges, AChE activity increases in the cellular layers and all areas of the nucleus exhibit relatively high levels of AChE activity. Superimposed on this changing laminar pattern of AChE activity are changes related to the retinotopic map within the nucleus. Portions of the LGN representing central vision develop their characteristic pattern of activity several days ahead of the regions representing more peripheral visual field locations. AChE activity is also found transiently in the optic tract near the LGN during the first 3 postnatal weeks. Two (possibly three) groups of AChE-carrying fibers can be traced from the optic chiasm to their apparent sites of termination (or origin) in the parabigeminal nucleus, ventral lateral geniculate nucleus, and dorsal LGN. The activity present in the optic tract disappears shortly after eye opening.(ABSTRACT TRUNCATED AT 400 WORDS)

Postnatal differentiation of the gametogenic and endocrine functions of the testis in the tree-shrew (Tupaia belangeri).

Testicular development was studied in Tupaia belangeri (tree-shrew) from birth to sexual maturity. At birth the seminiferous cords contained peripheral supporting cells and centrally located gonocytes. Large foetal Leydig cells were prominent in the interstitium. The mitotic index of the gonocytes was low at birth and rose to peak levels at Day 20, following the regression of the foetal generation of Leydig cells, and during the nadir in circulating testosterone concentrations. Mitotic activity returned to low levels at Day 30 in association with the reappearance of differentiated Leydig cells and the first signs of increased androgenesis. The negative temporal relationship between mitogenesis and androgenic function suggests that the proliferation of the gonocytes does not require, and may be inhibited by, high titres of androgens. Post-mitotic development of the gonocytes occurred during a period of rising testosterone levels, and the first appearance of spermatogonia coincided with peak testosterone levels. This indicates that androgens may be specifically involved in the initiation of spermatogenesis. Spermatogenesis progressed to completion during a phase of declining testosterone levels. The precise temporal correlations established during post-natal development suggest that the tree-shrew is a suitable animal model for studies on the endocrine control of the initiation of spermatogenesis in primates.

Growth and reproductive development in the male tree shrew (Tupaia belangeri) from birth to sexual maturity.

The growth and reproductive development of the male tree shrew were studied from birth to sexual maturity. An infantile phase from birth to Day 30 was characterized by the rapid involution of the testis and adrenal gland from a fetal condition followed by a nadir in testosterone levels and slow growth and differentiation of the testis and accessory sex organs. The initiation of puberty occurred collaterally with the emergence of the young from the nest and was marked by a sharp rise in testosterone levels from Days 30 to 35 to maximum levels at Days 40-55. Peak testosterone levels were temporally correlated with the onset of maximum growth and differentiation of the testis and accessory sex organs, descent of the testis, development of the scrotum, and a pronounced peak in the weight-velocity curve. The rapid growth of males at puberty contributed to a moderate degree of sexual dimorphism in this species. Puberty was attained at about Day 90 with the completion of spermatogenesis and the functional differentiation of the accessory sex organs. The postnatal development of the tree shrew conforms with the general primate pattern. The precise endocrine correlates established during puberty make Tupaia belangeri a useful small animal model for the study of puberty in primates.

Ultrastructure of the developing tree shrew lateral geniculate nucleus.

We examined the ultrastructural development of the lateral geniculate nucleus (LGN) in postnatal tree shrews to distinguish which features, if any, show a correlation with the development of cell layers. Our data indicate that synaptogenesis has begun at birth (PO) which is prior to the development of cell layers. At postnatal day 8 (P8), when laminar borders can be distinguished, the majority of synaptic profiles are still immature. Although some mature synaptic profiles can be identified at P8 and especially at P15, complex synaptic arrangements characteristic of adults are absent at that time. Growth cones are present at all 3 ages but are less prevalent at P8 and P15 than at birth and appear to be present in slightly higher concentrations in the interlaminar spaces. These results suggest that LGN cell layer formation does not correlate with the beginning of synaptogenesis but that it may correlate with an increase in growth cones in the interlaminar spaces.

Presence of retinogeniculate fibers is essential for initiating the formation of each interlaminar space in the lateral geniculate nucleus.

We demonstrated in a previous study that, following neonatal bilateral enucleation in the tree shrew, interlaminar spaces (ILSs) in the dorsal lateral geniculate nucleus fail to form. In the present study we sought to determine which aspects of ILS formation are dependent upon retinal input. Accordingly, we studied the degree of ILS formation in tree shrews which were bilaterally enucleated either during ILS formation on postnatal day 3 (P3) or just after all ILS were apparent but before they had reached a mature width (P15). Our results indicate that retinal input is necessary for the initial formation of each ILS, but that it is not required for the maturation or maintenance of ILSs which have already begun to form.

The role of retinogeniculate afferents in the development of connections between visual cortex and the dorsal lateral geniculate nucleus.

The role of retinogeniculate afferents in the development of patterns of connections between visual cortex and the lateral geniculate nucleus (LGN) was addressed by studying the effect of bilateral enucleation at birth on those patterns of connections in tree shrew. In normal adult tree shrews there are six LGN cell layers separated by cell-sparse interlaminar spaces. The reciprocal connections between the LGN and visual cortex are restricted to a column running across all six LGN layers; the geniculocortical projection arises from the cell layers while the corticogeniculate projection terminates primarily in the interlaminar spaces. At birth, when the experimental animals were bilaterally enucleated, the retinogeniculate fibers have begun to segregate by eye but neither the cytological characteristics of individual layers nor the interlaminar spaces have yet formed, and the corticogeniculate fibers have not entered the nucleus. Bilateral enucleation does not prevent the development of the cytological characteristics of individual layers but the interlaminar spaces do not develop. The results of [3H]proline/HRP injections into visual cortex in animals bilaterally enucleated at birth indicate that in the absence of retinogeniculate fibers, and thus interlaminar spaces, the corticogeniculate fibers do not concentrate at the laminar borders but instead spread across all six LGN cell layers. Despite the failure of this projection to concentrate at laminar borders, the corticogeniculate fibers do terminate within a restricted projection column.

Early postnatal development of laminar characteristics in the dorsal lateral geniculate nucleus of the tree shrew.

Three characteristics distinguish the six layers of the adult tree shrew dorsal lateral geniculate nucleus (LGN). First, interlaminar spaces divide the nucleus into cell layers. Second, input from the two eyes projects to the nucleus such that two layers (1 and 5) receive input from the ipsilateral retinal and four layers (2, 3, 4, and 6) receive input from the contralateral retina. Finally, distinct cytological features characterize individual layers. In this report, we describe the postnatal development of LGN layers in the tree shrew in terms of the development of these three characteristics. At birth, the nucleus appears homogenous in Nissl-stained sections. Thus, no interlaminar spaces are present and all cells look similar in shape, size, and staining intensity. However, autoradiographic data show that, at birth, retinal afferents are segregated in an adult-like pattern. Interlaminar spaces begin to be evident between layers innervated by opposite eyes on postnatal day 2. Several days later, interlaminar spaces between layers innervated by the same eye (i.e., the borders of layer 3) appear, while the others continue to widen. Although some cytological maturation begins before interlaminar space formation, it is not until interlaminar spaces are apparent that features such as differential staining intensity and cell size can be used to distinguish individual layers. The results suggest that the three characteristics that define LGN layers in the tree shrew may be temporally separate events in the developing nucleus. Thus, retinal afferents are segregated prior to interlaminar space formation which, in turn, is initiated prior to final maturation of the cytological features that characterize the cell layers. This may indicate a degree of developmental independence among these maturational events.

[Qualitative and quantitative study on ontogenesis of nucleus nervi hypoglossi in male Tupaia belangeri]. / Qualitative und quantitative Untersuchung des Nucl. n. hypoglossi an einer ontogenetischen Reihe von männlichen Tupaia belangeri

The change of the number of neurons, of the freshvolume, of the density of neurons, of the relative volume (freshvolume of hypoglossal nucleus/freshvolume of the whole brain) and of the length of the hypoglossal nucleus of 30 male Tupaia belangeri aged 36 to 388 days of ontogenesis has been studied using the logistic function (see article) for increasing values and the exponential function (see article) for decreasing values. The number of neurons is decreasing at 36 days of ontogenesis from 5700 to 5150 on the adult level, the density of neurons from 30000 N/mm3 to 6000 N/mm3 and the relative volume from 0,8% to 0,26%. The freshvolume is increasing from 0,31 mm3 on birth to 0,92 mm3 on the adult level and the length from 1,74 mm to 2,41 mm. The development of hypoglossal nucleus of Tupaia belangeri during ontogenesis starts very early in comparison with the whole brain. The half-value time of the growth of freshvolume is 53 days of ontogenesis and that of the growth of length is 34 days of ontogenesis. The maximum of spontaneous cell loss, stated here too for the hypoglossal nucleus, probably lies before the 36th day of ontogenesis. The decrease of the relative volume during ontogenesis is a further characteristic fact of the very early start of the development of hypoglossal nucleus of Tupaia belangeri during ontogenesis.