Development of visual projections in the marsupial, Setonix brachyurus

Summary Retinal projections to the primary visual centres were studied following injection of tritiated proline into one eye in the Marsupial, Setonix brachyurus between 10 and 100 days postnatal and in adults. Initially, projections from the two eyes overlapped extensively, particularly between 20 and 50 days. There was a gradual refinement thereafter, including a segregation of inputs from the two eyes within both the lateral geniculate nucleus (LGN) and superior colliculus (SC) by 70 days. Such refinement in visual centres is discussed in relation to the concurrent emergence of retinal ganglion cell density gradients, a decrease in ganglion cell numbers, cell death in the ganglion cell layer and loss of optic axonal profiles.     

The effects of a transient increase in temperature on cell generation and cell death in the hippocampus and amygdala of the wallaby, Setonix brachyurus (quokka)

We examined the effects of a single exposure to a temperature elevation of 2° C for a 2 h period on the developmental processes of cell division and cell death in the hippocampus and the amygdala of the quokka. Animals aged postnatal day (P) 40–45 were injected with tritiated (³H-) thymidine and then exposed to either 37° C (normal) or 39° C (±0.2° C) in an incubator for a duration of 2 h. The young were then returned to the nipple and, after a period of 24 h, were sacrificed. Brains were sectioned and selected sections processed for autoradiography, and some were counterstained. Cell division taking place at the time of heating was estimated by counting ³H-thymidine-labelled cells and at the time of sacrifice by counting mitotic figures. Dying cells were visualised as pyknotic profiles in cresyl-violet-stained sections. In both the amygdala and the hippocampus, the number of ³H-thymidine-labelled cells (cells dividing in situ during the heating period) was significantly lower in the experimental than the control group. Such cells were glia in the amygdala and granule cells and glia in the hippocampus. However, the number of dying cells or mitotic figures (cells dividing at the time of sacrifice) did not differ significantly between the two groups. By comparison, the number of ³H-thymidine-labelled cells, dying cells or mitotic figures did not significantly differ in the diencephalon. Therefore, a brief exposure to a slight elevation in temperature results in an immediate alteration in cell division in the hippocampus and amygdala. These findings have implications for the role played by raised temperature, such as during virus infection, in producing developmental anomalies of the brain. Received: 17 November 1997 / Accepted: 5 May 1998     

Functional respiratory morphology in the newborn quokka wallaby (Setonix brachyurus)

A morphological and morphometric study of the lung of the newborn quokka wallaby (Setonix brachyurus) was undertaken to assess its morphofunctional status at birth. Additionally, skin structure and morphometry were investigated to assess the possibility of cutaneous gas exchange. The lung was at canalicular stage and comprised a few conducting airways and a parenchyma of thick-walled tubules lined by stretches of cuboidal pneumocytes alternating with squamous epithelium, with occasional portions of thin blood-gas barrier. The tubules were separated by abundant intertubular mesenchyme, aggregations of developing capillaries and mesenchymal cells. Conversion of the cuboidal pneumocytes to type I cells occurred through cell broadening and lamellar body extrusion. Superfluous cuboidal cells were lost through apoptosis and subsequent clearance by alveolar macrophages. The establishment of the thin blood-gas barrier was established through apposition of the incipient capillaries to the formative thin squamous epithelium. The absolute volume of the lung was 0.02 +/- 0.001 cm(3) with an air space surface area of 4.85 +/- 0.43 cm(2). Differentiated type I pneumocytes covered 78% of the tubular surface, the rest 22% going to long stretches of type II cells, their precursors or low cuboidal transitory cells with sparse lamellar bodies. The body weight-related diffusion capacity was 2.52 +/- 0.56 mL O(2) min(-1) kg(-1). The epidermis was poorly developed, and measured 29.97 +/- 4.88 microm in thickness, 13% of which was taken by a thin layer of stratum corneum, measuring 4.87 +/- 0.98 microm thick. Superficial capillaries were closely associated with the epidermis, showing the possibility that the skin also participated in some gaseous exchange. Qualitatively, the neonate quokka lung had the basic constituents for gas exchange but was quantitatively inadequate, implying the significance of percutaneous gas exchange.     

Generation of retinal cells in the wallaby, Setonix brachyurus (quokka)

We have examined the generation of retinal cells in the wallaby, Setonix brachyurus (quokka). Animals received a single injection of tritiated thymidine between postnatal days 1-85 and retinae were examined at postnatal day 100. Retinae were sectioned, processed for autoradiography and stained with Cresyl Violet. Ganglion cells were labelled by injection of horseradish peroxidase into the optic tracts and primary visual centres. Other cells were classified according to their morphology and location. Retinal cell generation takes place in two phases. During the first phase, which concludes by postnatal day 30, cells destined to lie in all three cellular layers of the retina are produced. In the second phase, which starts by postnatal day 50, cell generation is almost entirely restricted to the inner and outer nuclear layers. Cells produced in the first phase are orthotopic and displaced ganglion cells, displaced and orthotopic amacrine cells, horizontal cells and cones. Glia in the ganglion cell layer, orthotopic amacrine cells, bipolar and horizontal cells. Muller glia, and rods are generated in the second phase. Cells became heavily labelled with tritiated thymidine in the central retina before postnatal day 7, over the entire retina (panretinal) by postnatal day 7 and from postnatal day 18, only in the periphery. The second phase of cell generation is initiated at P50, in a region extending from the optic nerve head to mid-temporal retina. Subsequently, cells are generated in annuli, centred on mid-temporal retina, which are seen at progressively more peripheral locations. Therefore, cell addition to the inner and outer nuclear layers continues for longer in peripheral than in mid-temporal retina. We suggest that such later differential cell addition to the inner and outer nuclear layers contributes to an asymmetric increase in retinal area. This non-uniform growth presumably results in more expansion of the ganglion cell layer peripherally than in mid-temporal retina and may play a role in establishing density gradients of ganglion cells.     

Histological and electron microscopic milestones in the development of the retina of a marsupial wallaby, Macropus eugenii

Summary Retinal differentiation in the pouch young of the wallaby Macropus eugenii was characterized microscopically and morphometrically. Mitosis occurs until early in the second month in the central retina, and until early in the fourth month, peripherally. Separation of the neuroblast layer by the outer plexiform layer did not immediately halt cell division. The retinal surface continued to expand well past the time of cessation of proliferation. Cell death in the ganglion cell layer continued through the fourth month centrally and to nearly five months in the periphery. The major period of cell death was coincident with the segregation of retinal afferents and the refinement of topography in the superior colliculus and dorsal lateral geniculate necleus. Beginning in the third month retinal thickness, measured between the outer limiting membrane and nerve fiber layer declined equally in peripheral and central regions. At all stages the combined thicknesses of the outer and inner nuclear layer in the retinal periphery was greater than that in the center. Together with a late thickening of the inner plexiform layer, the data are consistent with the suggestion that expansion of peripheral non-ganglion cell elements may play a role in development of center to periphery differences in ganglion cell distributions.Retinal differentiation of the wallaby follows the pattern of most mammals. The onset of development of key milestones for the acquisition of retinal function occurred in the sequence: conventional synapse formation prior to ribbon synapse formation in the inner plexiform layer, and photoreceptor outer segment differentiation prior to terminal triad synapse formation.     

Horizontal cells in the retina of the brush-tailed possum

Most species of eutherian (placental) mammals examined have two types of horizontal cell, one is axonless and the other has a short axon. We have recently shown that a marsupial, the quokka wallaby, also has two types of horizontal cell and that the axonless cell in this species has unusual stubby processes that pass through the inner nuclear layer to reach the inner plexiform layer. In order to discover whether these descending processes are a feature of marsupials in general, I examined the morphology of retinal horizontal cells in the brush-tailed possum, using horseradish peroxidase labelling. There are two types of horizontal cell in the possum. One type is axonless and has long, fine dendrites somewhat similar to that in the quokka; however, there are several marked differences between the axonless cells seen in the two species. The axonless cell in the possum has on average ten secondary dendrites, twice as many as seen in the quokka. These dendrites are arranged in a radial distribution, unlike those in the quokka, which are polarised in a direction often orthogonal to the overlying ganglion cell axons. Axonless horizontal cells in the possum do not have descending processes that reach the inner plexiform layer as has been seen in the quokka. The second horizontal cell type, the shortaxon cell, has an axon and an axonal arbor and is similar to the short-axon cell seen in the retina of the quokka. Therefore, the morphology of the axonless horizontal cell appears to be variable, while that of the short-axon cell is conserved in marsupials as in eutherians.     

The immunocytochemical localization of neuronal nitric oxide synthase in the developing rat retina

The localization of nitric oxide synthase (NOS) was investigated in the developing rat retina by immunocytochemistry and western blot analysis, using an antiserum directed against neuronal NOS. NOS-labeled cells were first detected at postnatal day 5 (P5) in the inner row of the neuroblastic layer. These cells were considered to correspond to the type 1 cell of the adult rat retina. Type 2 cells, characterized by a small soma and weak immunoreactivity, and a class of displaced amacrine cells were detected at P9 and P7, respectively. By P14 or P15, the time of eye opening, NOS immunoreactivity appeared in some bipolar cells. NOS was first expressed at the protein level at P9. Thereafter, quantitative evaluation by immunoblotting confirmed that the intensity of the immunoreactive bands increased abruptly, reaching the same value as is found in the adult retina at P21. Our results demonstrate that differentiation of NOS-labeled cells follows a discrete developmental pattern and is most active during the 2nd postnatal period in the rat retina.     

Morphometry and allometry of the postnatal lung development in the quokka wallaby (Setonix brachyurus): a light microscopic study

The postnatally developing lungs of the quokka wallaby, Setonix brachyurus, were investigated macroscopically and by light microscopic morphometry. Lung, parenchymal and non-parenchymal volumes as well as the components of the latter two were analysed by regression analysis. The lungs comprised a single undivided left lung and a right lung with an adherent accessory lobe. Septal tissue growth was most remarkable in the canalicular and saccular stages. Between mid-canalicular stage and the saccular stage, the lung volume increased 2-fold, mainly due to airspace expansion, coupled with septal tissue thinning. The non-parenchymal vascular volume increase accelerated in the successive developmental stages while the airway and connective tissue volumes progressed in a decreasing order, being highest in the canalicular and saccular stages and lowest in the alveolar stage. Growth and remodelling of the alveolar septa occurred simultaneously with airspace subdivision. Airspace expansion accelerated during the stage of microvascular maturation, when most other parameters showed the least rate of increase.     

Temperature regulation and oxygen consumption in the developing macropod marsupial Setonix brachyurus.

1. When kept at ambient temperatures of 17-5 and 24 degrees C the colonic temperatures of joeys younger than 166 days declined to near ambient temperature. Pouch joeys of 166 days and older were however able to maintain their colonic temperatures at about 35 degrees C. 2. Joeys first developed the ability to sustain high O2 consumption rates in response to cooling, when aged between 144 and 169 days. Only when this latter facility was fully developed in ontogeny could body temperature be maintained in a cool environment. 3. When joeys older than 130 days were kept in a metabolism chamber at pouch temperature (37-5 degrees C) and at high humidity their body temperatures quickly rose to lethal levels, demonstrating the need for cooling mechanisms whilst still contained within the pouch.     

钠尿肽受体A在小鼠视网膜发育过程中的表达

目的检测钠尿肽受体(NPR)在不同年龄小鼠视网膜内的表达,探讨其在视网膜发育过程中的作用。方法收集从受孕16日(E16)到出生90日(P90)小鼠眼球标本共127只,对NPR-A进行免疫荧光检测。结果NPR-A广泛存在于视网膜神经元中,例如,在外核层,NPR-A于P7开始高表达在视锥、视杆细胞内、外突起上,于P14减弱,P30之后持续稳定弱表达;在内核层,从P7开始NPR-A持续弱表达在双极细胞的突起中,而在水平细胞中未见NPR-A表达;在神经节细胞层,NPR-A于E16开始高表达在神经节细胞胞体中,P14明显减弱,而在神经纤维层,即神经节细胞的轴突中,NPR-A从胚胎期至成年持续高表达;在外网状层和内网状层,NPR-A于P14均高表达,但于P30之后逐渐减弱。此外,NPR-A还广泛的存在于Müller细胞的突起中。结论 NPR-A参与了视网膜的发育,可能是小鼠视网膜神经元发育过程中的关键分子,并对Müller细胞的功能活动起着重要的调节作用。     

Morphological patterns in the developing vertebrate retina

Summary Changes in the morphology of the early optic cup were observed in embryos of two distantly-related vertebrate species, a teleost fish, northern pike (Esox lucius), and chicken (Gallus gallus). A similar morphological pattern was noted to appear in both species shortly after the involution of the optic vesicle and the formation of the inner retinal layer. At a gross level, three notches were observed in the retinal margin at approximately nasal, dorsal, and temporal positions, while in histological sections a sharp constriction was found in the thickness of the dorsal retinal layer. In both species, this dorsal constriction appeared to be continuous with the central or dorsal notch. The time of appearance and configuration of this morphological pattern is intriguingly similar to the specification and polarity of retinal positional markers, and suggest a segmentation hypothesis for the origin of retinal polarity.     

Development of the rabbit retina

Summary Measures of rabbit eyes and retinal whole-mounts were used to evaluate the development of retinal area and shape. The retina is shown to have a horizontal axis about a third longer than the vertical axis just before birth, and to adopt an almost symmetrical shape during postnatal development to adulthood. In general, retinal thickness is shown to decrease after birth, but differently in particular retinal regions: the reduction is marked in the periphery, and less pronounced in the visual streak. As an exception, the myelinated region — after it becomes really myelinated, from 9 days p.p. — even increases in thickness. In all regions of the retina, the absolute and relative thickness of the nuclear layers decreases, whereas the relative thickness of plexiform and fibrous layers increases. Proliferation of cells within the rabbit retina was studied during the first three postnatal weeks. ³H-thymidine incorporation was used to demonstrate DNA synthesis autoradiographically in histological sections as well as in enzymatically isolated retinal cells. A first proliferation phase occurs in the neuroblastic cell layer and ceases shortly after birth in the retinal center, but lasts for about one week in the retinal periphery. We found, however, a few ³H-thymidine-labeled cells as late as in the third postnatal week.These late-labeled cells were found within the nerve fiber layer and in the inner plexiform layer. The latter cells were shown to express antigens detected by antibodies directed to the intermediate-sized filament protein vimentin, which are known to label Müller cells and neuroepithelial stem cells. This was confirmed in our preparation of enzymatically isolated cells; all cells with autoradiographically labeled nuclei revealed a characteristic elongated morphology typical for Müller radial glia (and also for early neuroepithelial stem cells). ³H-thymidine-labeled cells in the nerve fiber layer were most probably astrocytic. In analogy to the brain, we conclude that the mammalian retina undergoes a series of proliferation phases: first an early phase producing both neurons and glial cells, and then a late phase producing glial cells, e.g., in the nerve fiber layer. Most probably, the late phase within the inner nuclear layer is glial as well, i.e., consists of dividing Müller cells; it cannot be excluded, however, that there may remain some mitotically active stem cells.     

Subretinal macrophages in the developing eye of eutherian mammals and marsupials

Blood-borne mononuclear cells invade the developing retina via the hyaloid vasculature at the optic nerve head. Following removal of apoptotic cell debris they give rise to the network of resident microglia. The population of cells recently described in the peripheral subretinal space of developing human eyes may represent a further population of macrophages destined to become microglia. The aim of the present study was to confirm the presence of subretinal macrophages in the developing eye in other mammalian species and perform preliminary immunophenotypic analysis in rat tissues. The range of species chosen included eutherian mammals (rat and rabbit) and marsupials (wallaby and opossum). Ocular tissues from a range of developmental stages were studied by scanning electron microscopy and transmission electron microscopy. Distinctive networks of dendriform and pleomorphic macrophages were observed by scanning electron microscopy in the peripheral subretinal space of D2 rabbits, newborn and D2 rats and D75 wallaby. Transmission electron microscopic studies of D2 rabbit, newborn and D2 rat and all ages of North American opossum revealed cells with the ultrastructural features of macrophages in the peripheral subretinal space, cilio-retinal junction and between ciliary epithelial cells. Preliminary immunoperoxidase studies using a panel of anti-leukocyte monoclonal antibodies on frozen sections of rat ocular tissues (newborn, D2 and D4) revealed ED1⁺ Ox42⁺ ED2⁺ but Ox6^– cells in the peripheral subretinal space, peripheral retina and ciliary body epithelia. The data confirms that subretinal macrophages are a feature of the developing eye in a broad range of mammalian species and immunophenotypic evidence leads the author to postulate that these cells arise from the ciliary body vasculature and may migrate into peripheral neural retina and mature into resident microglia. Accepted: 23 March 1999     

The initial stages of development of the retinocollicular projection in the wallaby (Macropus eugenii): distribution of ganglion cells in the retina and their axons in the superior colliculus

The time course of ingrowth of retinal projections to the superior colliculus in the marsupial mammal, the wallaby (Macropus eugenii), was determined by anterograde labelling of axons from the eye with horseradish peroxidase, from birth to 46 days, when axons cover the colliculus contralaterally and ipsilaterally. The position of retinal ganglion cells giving rise to these projections over this period was determined in fixed tissue by retrograde labelling from the colliculus with a carbocyanine dye. Axons first reach the rostrolateral contralateral colliculus 4 days after birth and extend caudally and medially, reaching the caudal pole at 18 days and the far caudomedial pole at 46 days. The first contralaterally projecting cells are in the central dorsal and temporal retina, followed by cells in the nasal and finally the ventral retina. They are distributed closer to the periphery with increasing age. The first sign of a visual streak appears by 18 days. Axons reach the ipsilateral colliculus a day later than contralateral axons and come from a similar region of the retina. The sparser ipsilateral projection reaches the caudal and medial collicular margins by 46 days but by 16–18 days, ganglion cells giving rise to this transient projection are already concentrated in the temporoventral retina. The orderly recruitment of ganglion cells from retinotopically appropriate regions of the retina as axons advance across the contralateral colliculus suggests that the projection is topographically ordered from the beginning. The ipsilateral projection is less ordered as cells are located in the temporoventral crescent at a time when their axons are still transiently covering the colliculus prior to becoming restricted to the rostral colliculus. Features of mature retinal topography such as the visual streak and the location of ipsilaterally projecting cells begin to be established very early in development, before the period of ganglion cell loss and long before eye opening at 140 days.     

预定色素上皮视网膜化的作用机制

本文采用移植和切除的方法证明,预定水晶体外胚层、头部普通外胚层、腹部外胚层、耳囊、肾小管、体腔膜、软骨、咽壁、肌节、肝、围心肌膜和脑膜等12种器官组织接触预定色素上皮,均可使后者发生视网膜化。视网膜化频率的分析结果又揭示,上述各器官组织在作用能力方面存在明显差异。其中以预定水晶体外胚层最强,头部普通外胚层次之,腹部外胚层及一般器官组织最弱。作者推测,这种可导致预定色素上皮视网膜化的接触作用,可能就是一种诱导作用。此外,实验结果还证明,移植眼接触寄主眼,可合并调整成一个大眼;预定色素上皮接触脑组织,则一律脑化并与之融合。     

Histamine in the tissues of the marsupial Setonix brachyurus (the quokka).

Passive cutaneous anaphylactic (PCA) reactions in the marsupial Setonix brachyurus (the quokka) were completely inhibited by the histamine antagonist mepyramine maleate, but were unaffected by disodium cromoglycate or the serotonin antagonist, methysergide. Histological examination of quokka skin indicated that mast cell degranulation occurred during the PCA reaction in this marsupial and animals whose skin was relatively deficient in mast cells were poor PCA recipients. In contrast to many eutherian (placental) species, this marsupial was found to lack histamine in blood leukocytes and platelets. Also, while the peritoneal mast cells of rats and mice contain large quantities of histamine, this amine was not detected to quokka peritoneal washings, even after the induction of a peritoneal exudate or the regular intraperitoneal injection of antigen. Immunologic challenge of quokka blood or peritoneal cells did not induce the synthesis of histamine, but histamine release was elicited from sensitized quokka lung by antigenic challenge.     

Proliferation of actively migrating ameboid microglia in the developing quail retina

 Sheets containing the inner limiting membrane covered by a carpet of Müller cell endfeet were used to show that ameboid microglial cells migrating tangentially in the vitreal part of the developing retina of quail embryos underwent mitosis. Double labeling with anti-β-tubulin/QH1 or Hoechst 33342/QH1 revealed that some migroglial cells with morphological features typical of active migration were in early prophase. By anaphase and early telophase, microglial cells had retracted their lamellipodia and were ovoid in shape. Later in telophase, but well before completion of cytokinesis, both daughter cells again emitted lamellipodia, thus regaining the typical morphology of migrating cells. We concluded that ameboid microglial cells go through cycles in which migration and mitosis alternate, and that both mechanisms contribute to the spread of microglia throughout the developing retina. The mitotic spindle of dividing microglial cells showed different orientations, which probably influenced the course of subsequent migration. The expression of the proliferating cell nuclear antigen in the nucleus of most tangentially migrating ameboid microglial cells at E9–E10 confirmed their proliferative capability. However, the rate of proliferation of these cells decreased during embryonic development, and was nearly zero at E14. Accepted: 16 February 1999     

Intercellular gap junctions in the developing retina and pigment epithelium of the chick

Summary Gap junctions are found in the pigment epithelium, between retina and pigment epithelium and in the retina of 5–14 day chick embryos, they are identified using block staining and extracellular tracer techniques. In the pigment epithelium gap junctions are found between cell bodies and interdigitating processes and many change their position during development. Gap junctions between retina and pigment epithelium are only made by undiferentiated retinal ventricular cells and may provide intercytoplasmic pathways important for photoreceptor differentiation. Retinal gap junctions are found in an outer zone next to the pigment epithelium and inner zone near the vitreous, they are only seen between ventricular cells but may provide pathways for ganglion cell specification. The role of gap junctions in the generation of retinal neurons is discussed.