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W Halfter

Publications and source records attributed to W Halfter.

46 records · Page 3Linked to original sources
PubMed1988

The distribution of fibronectin and tenascin along migratory pathways of the neural crest in the trunk of amphibian embryos.

It is generally assumed that in amphibian embryos neural crest cells migrate dorsally, where they form the mesenchyme of the dorsal fin, laterally (between somites and epidermis), where they give rise to pigment cells, and ventromedially (between somites and neural tube), where they form the elements of the peripheral nervous system. While there is agreement about the crest migratory routes in the axolotl (Ambystoma mexicanum), different opinions exist about the lateral pathway in Xenopus. We investigated neural crest cell migration in Xenopus (stages 23, 32, 35/36 and 41) using the X. laevis-X. borealis nuclear marker system and could not find evidence for cells migrating laterally. We have also used immunohistochemistry to study the distribution of the extracellular matrix (ECM) glycoproteins fibronectin (FN) and tenascin (TN), which have been implicated in directing neural crest cells during their migrations in avian and mammalian embryos, in the neural crest migratory pathways of Xenopus and the axolotl. In premigratory stages of the crest, both in Xenopus (stage 22) and the axolotl (stage 25), FN was found subepidermally and in extracellular spaces around the neural tube, notochord and somites. The staining was particularly intense in the dorsal part of the embryo, but it was also present along the visceral and parietal layers of the lateral plate mesoderm. TN, in contrast, was found only in the anterior trunk mesoderm in Xenopus; in the axolotl, it was absent. During neural crest cell migration in Xenopus (stages 25-33) and the axolotl (stages 28-35), anti-FN stained the ECM throughout the embryo, whereas anti-TN staining was limited to dorsal regions. There it was particularly intense medially, i.e. in the dorsal fin, around the neural tube, notochord, dorsal aorta and at the medial surface of the somites (stage 35 in both species). During postmigratory stages in Xenopus (stage 40), anti-FN staining was less intense than anti-TN staining. In culture, axolotl neural crest cells spread differently on FN- and TN-coated substrata. On TN, the onset of cellular outgrowth was delayed for about 1 day, but after 3 days the extent of outgrowth was indistinguishable from cultures grown on FN. However, neural crest cells in 3-day-old cultures were much more flattened on FN than on TN. We conclude that both FN and TN are present in the ECM that lines the neural crest migratory pathways of amphibian embryos at the time when the neural crest cells are actively migrating. FN is present in the embryonic ECM before the onset of neural crest migration.(ABSTRACT TRUNCATED AT 400 WORDS)

Ambystoma↗
PubMed1987

Immunohistochemical localization of laminin, neural cell adhesion molecule, collagen type IV and T-61 antigen in the embryonic retina of the Japanese quail by in vivo injection of antibodies.

Antibodies against laminin (LN), fibronectin (FN), collagen type IV (Col IV), neural cell adhesion molecule (N-CAM), T-61 antigen, actin, tubulin and neurofilament protein were injected into the eyes of quail embryos (Coturnix coturnix japonica) of different ages. Twenty h after injection, the heads of the embryos were fixed and the antibodies visualized in sections with the use of fluorescein-isothiocyanate (FITC) or peroxidase-labeled second antibodies by light- and electron microscopy. Antibodies against cell surface molecules, such as N-CAM, LN, Col IV and T 61, labeled matrix and membrane components of the retinal cells in different antigen-specific patterns. Antibodies against intracellular antigens, such as actin, tubulin and neurofilament protein labeled nonspecifically the vitreous body and the inner basal lamina of the retina, but resulted in only a very weak and diffuse labeling of retinal cells. N-CAM was detected in high concentration in the optic fiber layer on the surface of axons and on the membranes of all retinal cells. Col IV, LN and T 61 antigen were found predominantly in the optic fiber layer. LN and Col IV were located on the surface of axons and the endfeet of ventricular (neuroepithelial) cells in a patchy distribution. The T-61 antigen was found in early stages in the cell-free space of the optic fiber layer, on the surface of ventricular cells and axons, and at later stages also in high-density patches between nerve fibers. The distribution of LN and T-61 antigen together with data from in vitro experiments suggests a crucial role of these proteins in axon extension in the avian retina during early development of the optic fiber layer.

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PubMed1987

Anterograde tracing of retinal axons in the avian embryo with low molecular weight derivatives of biotin.

Several reactive biotin esters were injected into the eyes of chick and quail embryos at various stages of development. Four of the biotin esters reacted with molecules of the eye tissue and were detected with light and electron microscopy in fluorescein isothiocyanate and peroxidase-avidin incubated sections and whole mounts. Intra and extracellular components of the lens, the vitreous body, and the retina were labeled to different degrees. Three of the biotin esters (biotin-N-hydroxysuccinimidester, biotin-epsilon-aminocaproic acid-N-hydroxysuccinimidester, and desthiobiotin-N-hydroxysuccinimidester) prominently marked the optic fiber layer in the retina and the biotin labels were transported along the optic pathway. The tracers were detected up to the growth cone of axons 24 to 36 hr after injection. Explants from biotin marked retinas were cultured on collagen or basal laminae. During culturing axons grew out from these explants into the substratum showing that labeled tissue and nerve fibers were viable. The development of the optic pathway at the chiasma of quail embryos was studied using the biotin/avidin tracing. The bulk of fibers emerging from the retina crossed as shown by double labeling of both optic nerves in a complex pattern of segregated and interdigitizing axon bundles at the chiasma toward the contralateral side of the brain. From stage 25 onward a minor ipsilateral projection was found. At the same developmental stage a few fibers traveled into the contralateral optic nerve and grew retrogradely toward the contralateral eye. The percentage of specimens having this retino-retinal projection increased during development from 53% (stage 24 to 27; E3.5-E5.5) to 89% (stage 29 to 35; E6-E8) and declined to 40% at late embryogenesis (stage 37 to 41; E9-E12). The fact that all retinal axons were found within predictable pathways with some of them running in the wrong direction suggests that nerve fiber pathways provide accurate positional information, but at best weak directional information for growing nerve fibers.

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PubMed1987

Nondirected axonal growth on basal lamina from avian embryonic neural retina.

The vitreous surface of the embryonic avian retinal neuroepithelium was isolated by mechanical disruption of the retina mounted between 2 adhesive substrata. The 200-micron-thick sheath covered an area of up to 1 cm2 and consisted of the vitreal basal lamina with a lamina densa, 2 laminae rarae, and a carpet of ventricular cell endfeet on top of the lamina. The vitreal endfeet were removed by detergent treatment and an extracellular basal lamina was obtained. The laminae were further characterized by immunohistochemistry and immunoblotting. A 190 kDa laminin protein was detected in laminae with and without vitreal endfeet, whereas the membrane-bound neural cell adhesion molecule (N-CAM) was detectable only on the endfeet of the ventricular cells and was absent in the detergent-treated basal laminae. Neither immunoblotting nor immunostaining revealed fibronectin in these preparations. Explants of retina, sensory ganglia, and cerebellum from chick, quail, and mouse were cultured on the basal lamina as a substratum. In all cases axonal outgrowth was excellent, with a growth rate similar to that in situ. Outgrowing axons from sensory ganglia and cerebellar explants were accompanied by migratory cells, which, in the case of sensory ganglia, were flat cells and, in the case of cerebellar explants, resembled granular neurons. Optic axons grew on the laminae in an asymmetric, explant-inherent pattern specific for the position of origin of the explant. On detergent-treated basal laminae, as well as on laminin, the retinal axons grew in a clockwise orientation. This axonal growth pattern was specific for retinal tissue and was not observed with axons from other neural explants. In spite of the excellent substrate properties provided by the substratum, cues for growing axons (toward or away from the optic disk) were not detectable in the basal lamina preparations.

Animals↗
PubMed1986

Inhibition of cell proliferation by cytosine-arabinoside and its interference with spatial and temporal differentiation patterns in the chick retina.

Incubation of chick embryos with 200 nmoles/egg of cytosine arabinoside (AraC) completely inhibits cell proliferation in the embryo. At an age older than embryonic day 4 (E4) more than 90% of the embryos survive this treatment. The drug induces various malformations; in particular the retina is heavily affected. This simple method offers the possibility to study the effect of a more or less decreased cell production on processes of further differentiation and histogenesis of retinal tissue. The following results are obtained: In spite of the inhibition of cell proliferation in the retina by AraC an abnormally thick basal lamina is found and the expansion of the eye still proceeds, indicating that eye growth is not only dependent on retinal cell numbers. Stereotyped malformations of retinal histogenesis are induced and categorized into three groups: in addition to areas of normal structure cells are found arranged in rosettes and in half-rosettes sometimes linked by areas of undefined transient cell arrangements. The results point to a strong tendency of a severely diminished cell population to form regularly laminated retinal-like structures as long as a minimal ratio of cell types is given. The spatio-temporal appearance of the type of retinal malformation in a given retinal area is dependent on the time of AraC exposure and thus on the degree of differentiation reached at a "spatio-temporal spot": Full rosettes develop at earlier, and half-rosettes at later stages of AraC interference. Furthermore, deformities first appear on temporal and ventral sides. Thus, the establishment of these malformations follows and reflects the normal sequence of differentiation within the retina. Cells within rosettes organize in specific layers and start to differentiate normally. This shows that earlier formed cells are not dependent on the influence of factors derived from cells that are formed later for their proper differentiation.

Animals↗
PubMed1986

Axonal pathfinding in organ-cultured embryonic avian retinae.

Eye cups from stage 14-28 (E2 to E5) chick and quail embryos consisting of neural retina, lens, and vitreous body were cultured for 1 or 2 days. These eyes expanded by proliferation of the retinal cells and the surface areas of the retinae increased several-fold. The area covered by ganglion cells and axons also expanded in vitro. [3H]Thymidine labeling showed extensive proliferation of the neuroepithelial cells including the formation of new ganglion cells. Culturing eyes from embryos before stage 17 results, as in vivo, in the generation of the first ganglion cells of the retina, but unlike in the in vivo situation, the outgrowing axons always formed a random fiber net in the central portion of the retina. A defined axonal pattern identical to the in vivo developed only in specimens from embryos of stage 17 and older. Some aberrant axons, however, were also observed at the retinal periphery in specimens from embryos of more advanced stages (20-24), but only during the second day of culturing. Axons in retinae from embryos of stages 23 to 26 heading toward the optic fissure often crossed the fissure and, in contrast to the situation in vivo, invaded the opposite retinal side. These axons of wrong polarity followed the pathways of axons growing centripetally but in reverse direction. This suggests that the polarity of growing nerve fibers and their course are determined by different factors. Culturing the eyes of embryos from stages 20 to 25 in the presence of antibodies showed that the antibodies penetrated the entire retina with 6 hr. Neither anti-N-CAM nor the T-61 antibody--both recognizing membrane proteins of retinal cells--affected the growth of the eyes in vitro. The development of the axonal pattern in vitro was not affected by incubation with N-CAM-antibodies at concentrations up to 500 micron/ml, whereas the T-61 antibody which is known to block neurite extention in vitro (S. Henke-Fahle, W. Reckhaus, and R. Babiel (l984). "Developmental Neuroscience: Physiological, Pharmacological, and Clinical Aspects," pp. 393-398. Elsevier, Amsterdam/New York) showed inhibition of axonal growth in retina cultures at 50 micron/ml. These results indicate that the eye cultures can be used as a test system for antibodies against antigens which could be involved in axon extension and neurite pathfinding in situ.

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PubMed1985

The formation of the axonal pattern in the embryonic avian retina.

Both the polarity of the axonal growth and the formation of the optic fiber pattern early in retinal morphogenesis were studied in silver stained whole mounts of embryonic chick, quail, and pigeon retinae. The surface area of the retina and of the optic fiber layer increases in size exponentially, the optic fiber layer expanding faster than the retina. The optic fiber layer covers the retinal surface at E5 in quail and at E6 in chick and pigeon. In all species studied, the retinal fiber layer does not expand homogeneously with the optic nerve head as the center. Instead, the retinal fiber layer enlarges with polarities in the dorsal to ventral and nasal to temporal direction. The very first axon bearing ganglion cells appear at stage 16 in the dorsal and central portion of the retina and grow ventrally to merge at the optic disk. From stage 23 on, the optic fiber layer expands faster in the temporal than in the nasal side. Measurements on the initial polarization of young axonal processes show that the axonal growth is directed toward the optic fissure and the optic nerve head. This growth polarization is found at the onset of growth cone formation and in axons far from the nearest ganglion cells or ganglion cell axons. Therefore axon-axon interaction cannot be involved in the initial axon orientation early in retinal morphogenesis.

Animals↗
PubMed1984

Axon growth in embryonic chick and quail retinal whole mounts in vitro.

Whole retinae of 4- to 10-day-old chick and quail embryos were spread on membrane filters and kept in culture for up to 4 days. Axon growth during culture was demonstrated by silver staining, anterograde labeling of fibers with RITC, time-lapse recording, and SEM. Fiber growth was observed in specimens from chick embryos up to 7 days old, with a growth maximum at E6 and from quail embryos up to E6 with the maximum at E5. Newly growing axons followed the optic fiber pattern already existing and, like axons in vivo, grew predominantly toward the optic fissure. Directional and orientational adaptation of newly growing axons to the preexisting fibers increased with the donor age. Retinae from donors up to E5 in chick and up to E4 in quail showed a high proportion of axons which crossed the optic fissure during the culture period and invaded the opposite retinal fiber layer. These fibers showed a correct radial orientation while growing in the opposite direction to normal. Likewise, in cultures from these young donors some fibers grew out initially in the diametrically opposite direction to normal toward the tissue periphery. Since all of the wrongly directed axons grew at the same rate as normal and adapted correctly to the already formed axon pattern, this suggests independent signals for the direction and orientation of growing fibers. Treatment of mounted retinae with collagenase or trypsin removed the vitreal retinal surface, leaving the existing axon pattern intact. Subsequently, new axons grew profusely in culture, but lost both their orientational and directional characteristics.

Animals↗
PubMed1983

Oriented axon outgrowth from avian embryonic retinae in culture.

The neural retina of avian embryos was spread on a membrane filter and cut in any desired orientation. Strips cut across the retina of 4- to 7-day chick or 3- to 6-day quail embryos were explanted onto collagen gels. Vigorous neurite outgrowth was seen for about 3 days, by which time many neurites were 3 mm long. Horseradish peroxidase (HRP) labeling showed that the cells producing the neurites were large and formed a layer near the inner limiting membrane, indicating that the neurites in vitro were axons of retinal ganglion cells. The size of the neurite population and the regions from which neurites emerged varied with the donor age, while most neurites sprouted from the side of the explant formerly closest to the optic fissure. This pattern closely resembled that of axon growth in the normal retina, as revealed by SEM, silver staining, and HRP labeling. Mitotic inhibitors (Ara-C and FUdR) did not alter the neurite outgrowth. Pretreatment of retinae with trypsin or collagenase did not disorganize axons at the time of explanation, but tended to equalize neurite emergence on each side of the retinal strips. We suggest that microenvironmental factors, especially the enzyme-labile inner limiting membrane, are important for axon guidance in the retina.

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