Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation.

Ocular retardation (or) is a murine eye mutation causing microphthalmia, a thin hypocellular retina and optic nerve aplasia. Here we show that mice carrying the OrJ allele have a premature stop codon in the homeobox of the Chx10 gene, a gene expressed at high levels in uncommitted retinal progenitor cells and mature bipolar cells. No CHX10 protein was detectable in the retinal neuroepithelium of orJ homozygotes. The loss of CHX10 leads both to reduced proliferation of retinal progenitors and to a specific absence of differentiated bipolar cells. Other major retinal cell types were present and correctly positioned in the mutant retina, although rod outer segments were short and retinal lamination was incomplete. These results indicate that Chx10 is an essential component in the network of genes required for the development of the mammalian eye, with profound effects on retinal progenitor proliferation and bipolar cell specification or differentiation. off

Human microphthalmia associated with mutations in the retinal homeobox gene CHX10.

Isolated human microphthalmia/anophthalmia, a cause of congenital blindness, is a clinically and genetically heterogeneous developmental disorder characterized by a small eye and other ocular abnormalities. Three microphthalmia/anophthalmia loci have been identified, and two others have been inferred by the co-segregation of translocations with the phenotype. We previously found that mice with ocular retardation (the or-J allele), a microphthalmia phenotype, have a null mutation in the retinal homeobox gene Chx10 (refs 7,8). We report here the mapping of a human microphthalmia locus on chromosome 14q24.3, the cloning of CHX10 at this locus and the identification of recessive CHX10 mutations in two families with non-syndromic microphthalmia (MIM 251600), cataracts and severe abnormalities of the iris. In affected individuals, a highly conserved arginine residue in the DNA-recognition helix of the homeodomain is replaced by glutamine or proline (R200Q and R200P, respectively). Identification of the CHX10 consensus DNA-binding sequence (TAATTAGC) allowed us to demonstrate that both mutations severely disrupt CHX10 function. Human CHX10 is expressed in progenitor cells of the developing neuroretina and in the inner nuclear layer of the mature retina. The strong conservation in vertebrates of the CHX10 sequence, pattern of expression and loss-of-function phenotypes demonstrates the evolutionary importance of the genetic network through which this gene regulates eye development.

Visualization of centrioles and basal bodies by fluorescent staining with nonimmune rabbit sera.

Several sera from nonimmunized rabbits have been found which stain centrioles and basal bodies by indirect immunofluorescence in a wide variety of cell types. So far, approximately 10% of the rabbit sera that we have examined gave strong positive staining of centrioles and basal bodies. Cytoplasmic networks, mitotic spindles, and ciliary axonemes, however, remain unstained. This specific fluorescent staining of centrioles and basal bodies could not be abolished by absorption of sera with purified brain tubulin. This technique is superior to previous methods for the visualization of basal bodies and centrioles at the light microscopic level and should be useful for rapid and convenient detection of these organelles in large populations of cells.

Microtubule-organizing centers and cell migration: effect of inhibition of migration and microtubule disruption in endothelial cells.

We have previously shown that microtubule-organizing centers (MTOC's) become preferentially oriented towards the leading edge of migrating endothelial cells (EC's) at the margin of an experimentally induced wound made in a confluent EC monolayer. To learn more about the mechanism responsible for the reorientation of MTOC's and to determine whether a similar reorientation takes place when cell migration is inhibited, we incubated the wounded cultures with colcemid (C) and cytochalasin B (CB), which disrupt microtubules (MT's) and microfilaments (MF's), respectively. The results obtained showed that the MTOC reorientation can occur independent of cell migration since MTOC's reoriented preferentially toward the wound edge in the CB-treated cultures, even though forward migration of the EC was inhibited. In addition, the MTOC reorientation is inhibited by C, indicating that it requires an intact system of MT's and/or other intracellular structures whose distribution is dependent on that of MT's.

Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence.

Microtubule accessory proteins were isolated from porcine brain microtubules by phosphocellulose chromatography, and the high molecular weight protein (HMW protein), purified from this microtubule-associated fraction by electrophoretic elution from SDS gels, was used to raise antisera in rabbits. In agarose double diffusion tests, the antiserum obtained forms precipitin lines with purified HMW protein but not with tau protein or tubulin. When rat glial cells (strain C6) are examined by indirect immunofluorescence, this serum specifically stains a colchicine-sensitive filamentous cytoplasmic network in interphase cells, a network indistinguishable from that seen when cells are treated with antitubulin serum. In dividing cells, specific staining of the mitotic spindle and the stem body is observed with the antiserum to HMW protein. These studies indicate that HMW protein, like tau protein, is associated with microtubules in intact cells.

Distribution of microtubule organizing centers in migrating sheets of endothelial cells.

This study was designed to investigate the relationship between the position of the microtubule organizing center (MTOC) and the direction of migration of a sheet of endothelial cells (EC). Using immunofluorescence and phase microscopy the MTOC's of migrating EC were visualized as the cells moved into an in vitro experimental wound produced by mechanical denudation of part of a confluent monolayer culture. Although the MTOC's in nonmigrating EC were randomly positioned in relation to the nucleus, in migrating cells the position of the MTOC's changed so that 80% of the cells had the MTOC positioned in front of the nucleus toward the direction of movement of the endothelial sheet. This repositioning of the MTOC occurred within the first 4 h after wounding and was associated with the beginning of migration of EC's into the wounded area as seen by time-lapse cinemicrophotography. These studies focus attention on the MTOC as a cytoskeletal structure that may play a role in determining the direction of cell movement.

Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells.

Murine embryonal carcinoma cells can differentiate into a varied spectrum of cell types. We observed the abundant and precocious development of neuronlike cells when embryonal carcinoma cells of various pluripotent lines were aggregated and cultured in the presence of nontoxic concentrations of retinoic acid. Neuronlike cells were also formed in retinoic acid-treated cultures of the embryonal carcinoma line, P19, which does not differentiate into neurons in the absence of the drug. The neuronal nature of these cells was confirmed by their staining with antiserum directed against neurofilament protein in indirect immunofluorescence experiments. Retinoic acid-treated cultures also contained elevated acetylcholinesterase activity. Glial cells, identified by immunofluorescence analysis of their intermediate filaments, and a population of fibroblastlike cells were also present in retinoic acid-treated cultures of P19 cells. We did not observe embryonal carcinoma, muscle, or epithelial cells in these cultures. Neurons and glial cells appeared in cultures exposed to retinoic acid for as little as 48 h. We found no evidence for retinoic acid toxicity, suggesting that the effect of the drug was to induce the development of neurons and glia rather than to select against cells differentiating along other developmental pathways.

Assembly of the sarcoplasmic reticulum. Localization by immunofluorescence of sarcoplasmic reticulum proteins in differentiating rat skeletal muscle cell cultures.

Immunofluorescent staining techniques were used to study the distribution of the Ca(2) + Mg(2+)-dependent ATPase and calsequestrin in primary cultures of differentiating rat skeletal muscle cells, grown for different periods of time under various culture conditions. In mononucleated myoblasts calsequestrin was detected after 45 h in culture whereas the ATPase was not detected until 60 h. After cell fusion began, both proteins could be identified in all multinucleated cells. Myoblasts grown for longer than 60 h in low Ca(2+) medium contained calsequestrin and the ATPase, even though they were unable to fuse. These studies at the cellular level confirm biochemical findings on the biosynthesis of calsequestrin and the ATPase. Immunofluorescent staining of myoblasts showed that calsequestrin first appears in a well-defined region of the cell near one end of the nucleus. At later times, the staining occupied progressively larger regions adjacent to the nucleus and took on a fibrous appearance. This suggests that calsequestrin first accumulates in the Golgi region and then gradually spreads throughout the cell. In contrast, the ATPase appeared to be concentrated in many small patches or foci throughout the cytoplasm and was never confined to one particular region, although some parts of the cell often stained more intensely than others. In multinucleated cells, alternating dark and fluorescent strands parallel to the longitudinal axis of the cells were evident.

Cloning of the cDNA for a novel photoreceptor membrane protein (rom-1) identifies a disk rim protein family implicated in human retinopathies.

The molecules essential to the continual morphogenesis and shedding of the opsin-containing disks of vertebrate photoreceptors are largely unknown. We describe a 37 kd protein, rom-1, which is 35% identical and structurally similar to peripherin/retinal degeneration slow (rds). Like peripherin, rom-1 is a retina-specific integral membrane protein localized to the photoreceptor disk rim. The two proteins are similarly oriented in the membrane, and each has a highly conserved (15/16 residues) cysteine- and proline-rich domain in the disk lumen. Although both rom-1 and peripherin form disulfide-linked dimers, they do not form heterodimers with each other, but appear to associate noncovalently. These results suggest both that rom-1 and peripherin are functionally related members of a new photoreceptor-specific protein family and that rom-1, like peripherin, is likely to be important to outer segment morphogenesis. The association of mutations in RDS with retinitis pigmentosa indicates that ROM1 is a strong candidate gene for human retinopathies.

Developmental expression of a novel murine homeobox gene (Chx10): evidence for roles in determination of the neuroretina and inner nuclear layer.

Few potential regulatory proteins of vertebrate retinal development have been identified. We describe a 39 kDa murine polypeptide (Chx10) with a homeodomain 82% identical to that of the nematode protein ceh-10. In the developing mouse, the Chx10 transcript is expressed throughout the anterior optic vesicle and all neuroblasts of the optic cup. In the mature retina, the Chx10 protein is restricted to the inner nuclear layer, in which its expression decreases from the outer to the inner margin. Chx10 transcripts are also detected in regions of the developing thalamus, hindbrain, and ventral spinal cord. The data suggest that Chx10 plays critical roles in the formation of the neuroretina and in the development and maintenance of the inner nuclear layer.

Microfilament distribution and adhesion patterns in cultured cells after glutaraldehyde-formaldehyde fixation.

To study the relationship between microfilament distribution and adhesion patterns in the same cultured cell, we have employed a simple glutaraldehyde-formaldehyde fixation technique followed by permeabilization of the cells in buffered Triton X-100. This method gives an excellent preservation of cellular morphology in general and of adhesion patterns in particular for examination with surface reflection interference microscopy. It also permits the concomitant use of the actin-specific fluorescent probe NBD-phallacidin to visualize the distribution of microfilaments.

Tau and HMW microtubule-associated proteins have different microtubule binding sites in vivo.

We have previously demonstrated the immunofluorescent localization of the tau and HMW MAPS on microtubules in vivo. When cells were treated with the non-ioni detergent triton before fixation, however treated with the non-ionic detergent triton before fixation, however, the immunofluorescent staining of microtubules with antiserum to HMW protein disappears, while the staining with anti-tubulin of anti-tau serum remains unaffected. Because of these differences in sensitivity to triton, we conclude that tau protein and HMW protein have different binding sites on microtubules. Parallel ultrastructural studies indicate that this loss of staining correlates with a loss of filamentous projections from the outer wall of cytoplasmic microtubules. These results indicate that in vivo the HMW protein is most probably distributed along the outer surface of the microtubule as filamentous projections similar to those observed in vitro. The in vivo distribution of the HMW protein suggests that it may form part of the microtrabecular system and be involved in the interaction of microtubules with other cell organelles.

The reorganization of cytoskeletal fibre systems in spreading porcine endothelial cells in cultures.

Porcine aortic endothelial cells spreading on a glass substrate undergo characteristic changes in shape which can be classified into four distinct stages. To study the role of the cytoskeleton in cell spreading, we have examined the distribution of microtubules (MT), microfilaments (MF), and intermediate filaments (IF) at each of these stages by using immunofluorescence and antisera specific for tubulin, tropomyosin, myosin, and vimentin. The small round Stage I cells showed diffuse staining with four antisera. In the more flattened spreading Stage II cells, MT and IF were first observed in the perinuclear region while fibres straining positively for tropomyosin and myosin were first seen along the cell margin. Later the MT began to radiate out in all directions from the perinuclear region while the IF became localized in a region on one side of the nucleus. In the very flat Stage III cells with a circular outline, additional MT could be seen along and parallel to cell margin while the IF emanating from the perinuclear region and the tropomyosin and myosin positive fibres became concentrically distributed around the nucleus. In the very flat asymmetric Stage IV cells, both the MT and IF radiated out from the perinuclear region towards the cell periphery while most of the tropomyosin and myosin-positive fibres became reorganized so that they ran parallel to the edges of the cell. In addition several loci from which a number of the tropomyosin and myosin-containing fibres radiated also appeared at this stage. These results indicate that during spreading each of the three major fibre systems undergo extensive and specific reorganization which is well coordinated with changes in cell shape.

Centrioles are lost as embryonic myoblasts fuse into myotubes in vitro.

Embryonic chick myoblasts possess an extensive network of cytoplasmic microtubules which emanate from a single, perinuclear centrosome containing a microtubule-organizing center (MTOC) and the centrioles. However, after myoblasts fuse into myotubes the centrosome is no longer apparent, and instead long parallel arrays of microtubules are seen. From ultrastructural studies on developing muscle tissue, it has been proposed that centrioles are present in myoblasts but are absent from fused muscle fibers. We have examined this hypothesis in vitro in cultures of chick embryonic muscle cells using sera which specifically label centrioles. Almost all (90-97%) mononucleated cells in these cultures, including myoblasts aligned just prior to fusion, contain a pair of centrioles in close proximity to the nucleus. However, in newly fused multinucleated myotubes as well as in older myotubes that had developed myofibrils, centrioles were rarely found (1-10% positive cells). This study thus provides direct evidence for a loss of centrioles from muscle cells soon after they fuse to form myotubes.

Immunocytochemical studies of intermediate filament aggregates and their relationship to microtubules in cultured skin fibroblasts from patients with giant axonal neuropathy.

Giant axonal neuropathy (GAN) is a severe autosomal recessive disease affecting both the peripheral and central nervous systems. It is characterized by segmental axonal ballooning due to large neurofilamentous masses and abnormal aggregation of filaments in other cell types including glial cells. Coomassie blue staining of the detergent-resistant cytoskeleton of cultured skin fibroblasts from three patients with GAN revealed the presence of large cytoplasmic filamentous aggregates in the great majority of cells. The aggregates were birefringent when viewed under polarization microscopy and electron microscopy showed that they were composed of aggregates of 8 to 10 nm intermediate filaments. The aggregates stained with antisera specific for vimentin but did not stain with antibodies to actin, tubulin, or the high molecular weight (HMW) microtubule associated protein. Examination of the fibroblasts containing the vimentin aggregates with antibodies to tubulin and the HMW protein showed that they had a normal distribution of microtubules and that the microtubules present were normally associated with the HMW protein. The results suggest that giant axonal neuropathy is a generalized inborn error of organization of intermediate filaments and that a defect in microtubules or their association with HMW protein is not responsible for the observed aggregation of intermediate filaments in this disease. Further study of GAN may be useful in understanding the function of intermediate filaments.

A 48 kilodalton intermediate filament associated protein (IFAP) in reactive-like astrocytes induced by dibutyryl cyclic AMP in culture and in reactive astrocytes in situ.

In this paper we demonstrate that the 48 kilodalton (kDa) intermediate filament associated protein (IFAP), previously reported to be present in normal astrocytes, is also present in reactive astrocytes in situ and in reactive-like astrocytes induced by dibutyryl cyclic AMP in vitro. This IFAP is detectable by antibodies in normal rabbit serum (F2N) and is closely associated with glial fibrillary acidic protein-containing intermediate filaments (IF). The expression of 48 kDa IFAP is related to the acquisition of stellate shape by normal and reactive-like astrocytes in vitro. It is proposed that 48 kDa IFAP may be responsible for cross-linking IF into bundles and is thereby associated with cell process formation.

Organization of microfilaments in astrocytes that form in the presence of dibutyryl cyclic AMP in cultures, and which are similar to reactive astrocytes in vivo.

This paper deals with changes in the arrangement of microfilaments at various stages during the transformation of astroblasts into reactive astrocytes in the presence of dibutyryl 3',5'-cyclic adenosine monophosphate in vitro. When cultures of two-week-old mouse astroblasts are treated with dibutyryl cyclic AMP, drastic changes occur in cell shape and in the organization of microfilaments, resulting in cells that closely resemble reactive astrocytes in vivo. A thick, prominent ring of microfilaments in such cells which stains strongly with 7-nitrobenz-2 oxa-1,3-diazole-phallacidin, delineates the perikaryon. Electron microscope examination showed that the ring is composed of many smaller bundles of microfilaments running parallel to each other. Prominent bundles of microfilaments radiate from the cell body into the cell processes. Based on the observation of intermediate forms, we propose that the microfilament ring may be important in the development of cell processes in reactive astrocytes.

A method for examining the endothelial cytoskeleton in situ using immunofluorescence.

A simple technique is described for producing en face preparations of endothelial cells (EC) from large blood vessels fixed in situ that are suitable for studying the distribution of specific antigens in EC by immunofluorescence. This method has permitted us to examine the distribution of various components of the cytoskeleton, including microtubules (MT), centrioles, and microfilaments (MF) in EC in vivo.

Localization of laminin, type IV collagen, fibronectin, and heparan sulfate proteoglycan in chick retinal pigment epithelium basement membrane during embryonic development.

Type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin were localized in the basement membrane (BM) of chick retinal pigment epithelium (RPE) during various stages of eye development. At different times over a 4-17 day period after fertilization, chick embryo eyes were dissected, fixed in periodate-lysine-paraformaldehyde, and 6 micron frozen sections through the central regions of the eye were prepared. Sections were postfixed in -20 degrees C methanol and stained immediately by indirect immunofluorescence using sheep anti-mouse laminin, sheep antimouse type IV collagen, rabbit anti-mouse heparan sulfate proteoglycan, and mouse monoclonal anti-porcine plasma fibronectin. Fluorescein-labeled F(ab')2 fragments of the appropriate immunoglobulins (IgGs) were used as secondary antibodies. Laminin could be readily demonstrated in the BM of the RPE during all stages of development. The staining for type IV collagen, fibronectin, and heparan sulfate proteoglycan HSPG) was less intense than that for laminin, but was also localized in the BM along the basal side of the RPE. In addition to staining the BM, antiserum to HSPG, gave a diffuse labeling from day 9 onward, above the RPE extending into the region of the photoreceptors. Whereas the intensity of staining generally increased between day 4 and day 17 of development, the distribution of the different BM components did not change. Hence the presence of type IV collagen, laminin, fibronectin, and HSPG in the BM of RPE in vivo during all the stages of development investigated supports the concept that these macromolecules are important basic components of this, and other, BMs. Furthermore, these results indicate that the composition of the BM of RPE cells in vivo is similar to the BM material deposited by RPE cells in vitro (Turksen K, Aubin JE, Sodek JE, Kalnins VI: Collagen Rel Res, 4:413-426, 1984) and that the in vitro cultures can therefore serve as a useful model for studying BM formation.

Distribution of microtubules in cultured RPE cells from normal and dystrophic RCS rats.

Indirect immunofluorescence and antibodies to tubulin were used to visualize the distribution of microtubules (MT) in short-term primary cultures of retinal pigment epithelial (RPE) cells isolated from the eyes of control RCS rats and those with inherited retinal degeneration. At all stages of cell spreading in vitro, the pigment granules (PG) in these cells remained tightly clustered in the perinuclear region, surrounded by numerous MT. Initially, in relatively round cells, the perinuclear region was encompassed by a closely woven ring of circularly arranged MT. At later stages of spreading, the ring had disappeared and MT running in various directions could be observed. Cells that were plated singly assumed at first a discoid, then stellate, shape during spreading, whereas, those plated in small groups formed colonies of wedge-shaped cells. In well-spread single cells or colonies, a brightly fluorescent zone that appeared to contain the highest density of MT within the cells was located between the PG-containing perinuclear region and the spreading edges of the cells. A single prominent star-shaped structure from which a number of MT radiated was situated among the PG in the perinuclear region of each cell; we believe this structure corresponds to the centrioles. It was generally well-separated from the regions of highest MT density. No differences in cell shape or in the distribution of MT, centriole-containing regions or PG in spreading or fully spread cells were detected when RPE cells from normal and dystrophic RCS rats were compared. Thus, the distribution of MT and their assembly during cell spreading appear normal in RPE cells of the dystrophic rat.