Targeted disruption of Aldh1a1 (Raldh1) provides evidence for a complex mechanism of retinoic acid synthesis in the developing retina

Genetic studies have shown that retinoic acid (RA) signaling is required for mouse retina development, controlled in part by an RA-generating aldehyde dehydrogenase encoded by Aldh1a2 (Raldh2) expressed transiently in the optic vesicles. We examined the function of a related gene, Aldh1a1 (Raldh1), expressed throughout development in the dorsal retina. Raldh1(-/-) mice are viable and exhibit apparently normal retinal morphology despite a complete absence of Raldh1 protein in the dorsal neural retina. RA signaling in the optic cup, detected by using a RARE-lacZ transgene, is not significantly altered in Raldh1(-/-) embryos at embryonic day 10.5, possibly due to normal expression of Aldh1a3 (Raldh3) in dorsal retinal pigment epithelium and ventral neural retina. However, at E16.5 when Raldh3 is expressed ventrally but not dorsally, Raldh1(-/-) embryos lack RARE-lacZ expression in the dorsal retina and its retinocollicular axonal projections, whereas normal RARE-lacZ expression is detected in the ventral retina and its axonal projections. Retrograde labeling of adult Raldh1(-/-) retinal ganglion cells indicated that dorsal retinal axons project to the superior colliculus, and electroretinography revealed no defect of adult visual function, suggesting that dorsal RA signaling is unnecessary for retinal ganglion cell axonal outgrowth. We observed that RA synthesis in liver of Raldh1(-/-) mice was greatly reduced, thus showing that Raldh1 indeed participates in RA synthesis in vivo. Our findings suggest that RA signaling may be necessary only during early stages of retina development and that if RA synthesis is needed in dorsal retina, it is catalyzed by multiple enzymes, including Raldh1.     

Differential expression and regulation of myristoylated alanine-rich C kinase substrate (MARCKS) in the hippocampus of C57/BL6J and DBA/2J mice

The myristoylated alanine-rich C kinase substrate (MARCKS) is a major protein kinase C (PKC) substrate in brain that binds the inner surface of the plasma membrane, calmodulin, and cross-links filamentous actin, all in a PKC phosphorylation-reversible manner. MARCKS has been implicated in hippocampal-dependent learning and long-term potentiation (LTP). Previous studies have shown DBA/2 mice to exhibit poor spatial/contextual learning, impaired hippocampal LTP, and hippocampal mossy fiber hypoplasia, as well as reduced hippocampal PKC activity and expression relative to C57BL/6 mice. In the present study, we assessed the expression (mRNA and protein) and subcellular distribution (membrane and cytolsol) of MARCKS in the hippocampus and frontal cortex of C57BL/6 and DBA/2 mice using quantitative western blotting. In the hippocampus, total MARCKS mRNA and protein levels in C57BL/6J mice were significantly lower ( approximately 45%) compared with DBA/2J mice, and MARCKS protein was observed predominantly in the cytosolic fraction. MARCKS expression in frontal cortex did not differ significantly between strains. To examine the dynamic regulation of MARCKS subcellular distribution, mice from each strain were subjected to 60 min restraint stress and MARCKS subcellular distribution was determined 24 h later. Restraint stress resulted in a significant reduction in membrane MARCKS expression in C57BL/6J hippocampus but not in the DBA/2J hippocampus despite similar stress-induced increases in serum corticosterone. Restraint stress did not affect cytosolic or total MARCKS levels in either strain. Similarly, restraint stress (30 min) in rats also induced a significant reduction in membrane MARCKS, but not total or cytosolic MARCKS, in the hippocampus but not in frontal cortex. In rats, chronic lithium treatment prior to stress exposure reduced hippocampal MARCKS expression but did not affect the stress-induced reduction in membrane MARCKS. Collectively these data demonstrate higher resting levels of MARCKS in the hippocampus of DBA/2J mice compared to C57BL/6J mice, and that acute stress leads to a long-term reduction in membrane MARCKS expression in C57BL/6J mice and rats but not in DBA/2J mice. These strain differences in hippocampal MARCKS expression and subcellular translocation following stress may contribute to the differences in behaviors requiring hippocampal plasticity observed between these strains.     

Transgenic Complementation of MARCKS Deficiency with a Nonmyristoylatable, Pseudo-Phosphorylated Form of MARCKS: Evidence for Simultaneous Positive and Dominant-Negative Effects on Central Nervous System Development

MARCKS is a widely expressed protein kinase C substrate that is essential for normal prenatal development of the central nervous system in mice. MARCKS-deficient mice exhibit universal perinatal mortality and numerous developmental abnormalities of the brain and retina. To determine which domains of the protein were important in complementing these neurodevelopmental anomalies, we have interbred MARCKS knockout mice with transgenic mice expressing an epitope-tagged human MARCKS transgene that can completely correct the MARCKS-deficient phenotype. Previous structure–function studies showed that a nonmyristoylatable form of MARCKS could correct all of the neuroanatomical abnormalities, and resulted in approximately 25% viable pups that grew to adulthood and were fertile. The present experiment attempted a similar complementation strategy in which a nonmyristoylatable, “pseudo-phosphorylated” form of the protein was used, which has been shown to be almost completely cytosolic in cell expression studies. Surprisingly, this transgene was able to complement almost all of the cerebral anatomical abnormalities characteristic of the knockout mice. However, these mice also exhibited a universal, novel phenotype: profound retinal ectopia, in which retinal tissue was often found in the vitreous humor as well as extraocularly. Retrospective evaluation of the original MARCKS knockout phenotype revealed that this anomaly was present in about 43% of the knockout mice, and was clearly detectable as early as embryonic day 12.5, before retinal cell differentiation begins. These data suggest that a nonmyristoylatable, pseudo-phosphorylated form of MARCKS can complement most if not all cerebral aspects of the MARCKS-deficient phenotype, but that it appears to worsen a retinal phenotype, perhaps by exerting a dominant-negative effect on a coexpressed MARCKS homologue.     

Vsx1, a rapidly evolving paired-like homeobox gene expressed in cone bipolar cells.

The paired-like homeodomain (HD) protein Chx10 is distinguished by the presence of the CVC domain, a conserved 56 amino acid sequence C-terminal to the HD. In mammals, Chx10 is essential both for the proliferation of retinal progenitor cells and for the formation or survival of retinal bipolar interneurons. We describe the cloning and characterization of a mouse Chx10 homologue, Vsx1; phylogenetic analysis suggests that Vsx1 and its putative vertebrate orthologues have evolved rapidly. Vsx1 expression in the adult is predominantly retinal. Whereas Chx10 is expressed both in retinal progenitors in the developing eye and apparently in all bipolar cells of the mature retina, Vsx1 expression is first detected in the eye at postnatal day 5, where it is restricted to cone bipolar cells.     

Growth hormone action in the developing neural retina: a proteomic analysis

The possible presence and action of growth hormone (GH) in the neural retina was investigated in newborn mice. The neural retina was found to be a site of GH gene expression, as GH mRNA was abundant in cells of the retinal ganglion cell layer, in which GH was also detected. It was also a site of GH action, since GH receptor (GHR) immunoreactivity mirrored that of GH. Actions of GH within the eye were indicated by a reduction in its axial length and retinal width (its neuroblastic, inner plexiform, and optic fiber layers) in GHR gene disrupted mice (GHR-/-), in comparison with wild type (GHR+/+) littermates. In the absence of GH signaling, four proteins in the retinal proteome of the GHR-/- mice (identified by 2-D gels and MS) differed in abundance with those in the wild type mice. Brain abundant membrane attached signal protein-1 (BASP-1) was down-regulated, whereas protein kinase C inhibitor 1, cyclophilin A, KH domain-containing, RNA-binding, signal transduction-associated protein 3 were up-regulated in GHR-/- mice. These proteins are involved in retinal vascularization, neural proliferation and neurite outgrowth. GH might thus have hitherto unsuspected roles in these processes during retinal development.     

Phorbol Ester- and Retinoic Acid-Induced Regulation of the Protein Kinase C Substrate MARCKS in Immortalized Hippocampal Cells

Abstract: The expression of MARCKS, a major protein kinase C (PKC) substrate, was examined in the immortalized hippocampal cell line HN33, following differentiation using phorbol esters or retinoic acid. In cells exposed to phorbol esters, MARCKS protein levels were reduced through an apparent PKC-dependent mechanism. Exposure to 1 µ M phorbol 12-myristate 13-acetate (PMA) for 10 min resulted in a rapid loss of PKC activity in the soluble fraction with a concurrent increase in membrane-associated PKC activity. PKC activity was reduced to <20% of control values in both soluble and membrane fractions following 1 h of PMA exposure. Significant reductions in MARCKS protein levels were initially observed in membrane and soluble fractions following PMA exposure for 4 and 8 h, respectively. The reduction in MARCKS protein levels was maximal following 24 h of PMA exposure. MARCKS protein expression was also down-regulated in a dose-dependent manner on exposure of HN33 cells to retinoic acid. In cells exposed to 10 µ M retinoic acid, the MARCKS protein level was reduced in the membrane fraction within 4 h. Reduction of MARCKS protein levels was maximal (>90%) by 12 h with no evidence for any alteration in PKC activity. Reduced levels of MARCKS protein were also observed in the soluble fraction of retinoic acid-exposed cells, but to a significantly lesser extent. Addition of the PKC inhibitor GF109203X blocked the down-regulation of MARCKS protein in PMA-treated cultures but not in retinoic acid-treated cells. These findings suggest that the down-regulation of MARCKS may play an important role in both phorbol ester- and retinoic acid-induced differentiation in cells of neuronal origin.     

Dissociation of Phosphorylation and Translocation of a Myristoylated Protein Kinase C Substrate (MARCKS Protein) in C6 Glioma and N1E-115 Neuroblastoma Cells

Abstract: An 80-kDa protein labeled with [³H]myristic acid in C6 glioma and N1E-115 neuroblastoma cells has been identified as the myristoylated alanine-rich C kinase substrate (MARCKS protein) on the basis of its calmodulin-binding, acidic nature, heat stability, and immunochemical properties. When C6 cells preincubated with [³H]myristate were treated with 200 n M 4β-12- O -tetradecanoylphorbol 13-acetate (β-TPA), labeled MARCKS was rapidly increased in the soluble digitonin fraction (maximal, fivefold at 10 min) with a concomitant decrease in the Triton X-100–soluble membrane fraction. However, phosphorylation of this protein was increased in the presence of β-TPA to a similar extent in both fractions (maximal, fourfold at 30 min). In contrast, β-TPA–stimulated phosphorylation of MARCKS in N1E-115 cells was confined to the membrane fraction only and no change in the distribution of the myristoylated protein was noted relative to α-TPA controls. These results indicate that although phosphorylation of MARCKS by protein kinase C occurs in both cell lines, it is not directly associated with translocation from membrane to cytosol, which occurs in C6 cells only. The cell-specific translocation of MARCKS appears to correlate with previously demonstrated differential effects of phorbol esters on stimulation of phosphatidylcholine turnover in these two cell lines.     

Distinct patterns of expression of the RB gene family in mouse and human retina

Although RB1 function is disrupted in the majority of human cancers, an undefined cell of developing human retina is uniquely sensitive to cancer induction when the RB1 tumor suppressor gene is lost. Murine retinoblastoma is initiated only when two of the RB family of genes, RB1 and p107 or p130, are inactivated. Although whole embryonic retina shows RB family gene expression by several techniques, when E14 developing retina was depleted of the earliest differentiating cells, ganglion cells, the remaining proliferating murine embryonic retinal progenitor cells clearly did not express RB1 or p130, while the longer splice form of p107 was expressed. Each retinal cell type expressed some member of the RB family at some stage of differentiation. Rod photoreceptors stained for the RB1 protein product, pRB, and p107 in only a brief window of postnatal murine development, with no detectable staining for any of the RB family proteins in adult human and mouse rod photoreceptors. Adult mouse and human Muller glia, ganglion and rare horizontal cells, and adult human, but not adult mouse, cone photoreceptors stained for pRB. The RB gene family is dynamically and variably expressed through retinal development in specific retinal cells.     

Conditional deletion of activating protein 2alpha (AP-2alpha) in the developing retina demonstrates non-cell-autonomous roles for AP-2alpha in optic cup development

Activating protein 2alpha (AP-2alpha) is known to be expressed in the retina, and AP-2alpha-null mice exhibit defects in the developing optic cup, including patterning of the neural retina (NR) and a replacement of the dorsal retinal pigmented epithelium (RPE) with NR. In this study, we analyzed the temporal and spatial retinal expression patterns of AP-2alpha and created a conditional deletion of AP-2alpha in the developing retina. AP-2alpha exhibited a distinct expression pattern in the developing inner nuclear layer of the retina, and colocalization studies indicated that AP-2alpha was exclusively expressed in postmitotic amacrine cell populations. Targeted deletion of AP-2alpha in the developing retina did not result in observable retinal defects. Further examination of AP-2alpha-null mutants revealed that the severity of the RPE defect was variable and, although defects in retinal lamination occur at later embryonic stages, earlier stages showed normal lamination and expression of markers for amacrine and ganglion cells. Together, these data demonstrate that, whereas AP-2alpha alone does not play an intrinsic role in retinogenesis, it has non-cell-autonomous effects on optic cup development. Additional expression analyses showed that multiple AP-2 proteins are present in the developing retina, which will be important to future studies.     

Characterization of MARCKS (Myristoylated alanine-rich C kinase substrate) identified by a monoclonal antibody generated against chick embryo neural retina

To identify molecular markers of cell differentiation in developing nervous tissue, monoclonal antibodies against chick embryo neural retina were made. One of them, 3C3mAb, recognized a developmentally regulated antigen present in several organs of the CNS. Data from MALDI-TOF mass spectrometry and peptide sequencing of the immuno-affinity purified protein indicated identity of the antigen with MARCKS. The immunoreactive material was always found as a unique polypeptide (Mr 71 kDa) in SDS-PAGE, however isoelectrofocusing revealed the existence of several bands (pI ranging from 4.0 to 4.5). Interestingly some retinal cell types, as photoreceptors, exhibited an extremely significant decrease in the intensity of the immunoreactive material during the final phases of terminal differentiation while others, as some retinal neurons, maintained the immunoreactivity when fully differentiated. Taken together these results indicate that MARCKS, a protein susceptible of several posttranslational modifications as myristoylation and phosphorylation at variable extent, may act differently in neural retina cell types.     

Retinal stem/progenitor properties of iris pigment epithelial cells

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina and ciliary epithelium of the adult eye. Here, we first demonstrate the characterization of neural stem/progenitors in postnatal iris pigment epithelial (IPE) cells. Pure isolated IPE cells could form spheres that contained cells expressing retinal progenitor markers in non-adherent culture. The spheres grew by cell proliferation, as indicated by bromodeoxyuridine incorporation. When attached to laminin, the spheres forming IPE derived cells were able to exhibit neural phenotypes, including retinal-specific neurons. When co-cultured with embryonic retinal cells, or grafted into embryonic retina in vivo, the IPE cells could also display the phenotypes of photoreceptor neurons and Muller glia. Our results suggest that the IPE derived cells have retinal stem/progenitor properties and neurogenic potential without gene transfer, thereby providing a novel potential source for both basic stem cell biology and therapeutic applications for retinal diseases.     

Lamin B1 and lamin B2 are long-lived proteins with distinct functions in retinal development

Lamin B1 and lamin B2 are essential building blocks of the nuclear lamina, a filamentous meshwork lining the nucleoplasmic side of the inner nuclear membrane. Deficiencies in lamin B1 and lamin B2 impair neurodevelopment, but distinct functions for the two proteins in the development and homeostasis of the CNS have been elusive. Here we show that embryonic depletion of lamin B1 in retinal progenitors and postmitotic neurons affects nuclear integrity, leads to the collapse of the laminB2 meshwork, impairs neuronal survival, and markedly reduces the cellularity of adult retinas. In stark contrast, a deficiency of lamin B2 in the embryonic retina has no obvious effect on lamin B1 localization or nuclear integrity in embryonic retinas, suggesting that lamin B1, but not lamin B2, is strictly required for nucleokinesis during embryonic neurogenesis. However, the absence of lamin B2 prevents proper lamination of adult retinal neurons, impairs synaptogenesis, and reduces cone photoreceptor survival. We also show that lamin B1 and lamin B2 are extremely long-lived proteins in rod and cone photoreceptors. OF interest, a complete absence of both proteins during postnatal life has little or no effect on the survival and function of cone photoreceptors.     

Neurothelin: molecular characteristics and developmental regulation in the chick CNS.

Neurothelin has recently been identified as a cell surface protein specific for chick endothelial cells forming the blood-brain barrier. Neurons of the adult brain are essentially devoid of neurothelin. In contrast, neurons of the chick retina, which lack blood vessels and accessory astrocytes, express neurothelin. Here we demonstrate that during chick brain development initially neurothelin is expressed probably in all neuroblasts. With proceeding cytodifferentiation, such as vascularization and gliogenesis, brain neurons become neurothelin negative. Coincidentally the endothelial cells forming the blood-brain barrier start to synthesize neurothelin. In contrast to brain neurons, in retina neurons, neurothelin expression increases by one order of magnitude during the course of histogenesis. Coculturing of chick retinal cells with purified rat astrocytes in vitro results in reduction of neural neurothelin expression as quantified by ELISA. Conversely, disruption of the glia-neuron interactions by culturing brain neurons as individualized cells in vitro leads to a reexpression of neurothelin. This is consistent with the hypothesis that astrocytes inhibit neurothelin expression in neurons. Biochemical characterization classifies neurothelin as an integral membrane protein. Temperature-induced-detergent phase separation, phospholipase C digestion and sodium carbonate treatment were employed to distinguish between integral membrane proteins, lipid-anchored proteins and peripheral membrane proteins. Two-dimensional gel electrophoresis reveals an isoelectric point of about 6.4 for neurothelin. Polysaccharide analysis by glycosidase digestion and lectin binding indicates that neurothelin is highly glycosylated. The relative molecular mass of glycosylated neurothelin is 41 x 10(3), whereas the peptide backbone is only 25 x 10(3). The very strict spatiotemporal regulation of neurothelin expression in the central nervous system suggests that neurothelin fulfils possibly a crucial function such as transport of low relative molecular mass components that are essential for neuronal metabolism. The proposed biological activity of neurothelin might be specifically affected by some of its distinct biochemical features.