αB-Crystallin is Expressed in Myelinating Oligodendrocytes of the Developing and Adult Avian Retina

It is well known that the expression of αB-crystallin (aBC) is increased in neurons and glia under pathologic conditions. However, the expression of aBC during the normal development of the central nervous system has not been reported. This study aimed to clarify the cell type in the chick retina in which aBC is expressed and timing of aBC expression in this cell type during development. Double immunofluorescence with cell-specific markers demonstrated that aBC was selectively expressed in oligodendrocytes (OLs) in the embryonic day 20 (E20) chick retina. A small number of aBC-expressing OLs first appeared in the nerve fiber layer of the central and peripheral retina at E16. Faint aBC expression was also observed in myelin sheaths near cell bodies in the central retina. The number of aBC-expressing OLs and intensity of aBC expression in myelin sheaths were increased in the periphery as well as in the center of the E19 retina. aBC signals in the post-hatching day 120 retina were observed in the entire nerve fiber layer. The spatiotemporal expression pattern of aBC was identical to that of myelin basic protein. These data indicate that aBC-expressing OLs are myelinating OLs among OL-lineage cells. Besides, intrayolk injection of tocopherol, an antioxidant, provoked a decrease in the levels of aBC expression in myelinating OLs. These data suggest that aBC expression in myelinating OLs responds to the change of physiological oxidative stress.     

Neurochemical Characteristics of Myelin-like Structure in the Chick Retina

Abstract: Certain characteristics of myelin-like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post-hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post-hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS-polyacrylamide gel elec-trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch-Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral-structured myelin-like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin-like structure in the chick retina is other than from oligodendroglia or Schwann cells.     

Epigenetic factors up-regulate expression of myelin proteins in the dysmyelinating jimpy mutant mouse

Proteolipid protein (PLP) is a major structural component of central nervous system (CNS) myelin. Evidence exists that PLP or the related splice variant DM-20 protein may also play a role in early development of oligodendrocytes (OLs), the cells that form CNS myelin. There are several naturally occurring mutations of the PLP gene that have been used to study the roles of PLP both in myelination and in OL differentiation. The PLP mutation in the jimpy (jp) mouse has been extensively characterized. These mutants produce no detectable PLP and exhibit an almost total lack of CNS myelin. Additionally, most OLs in affected animals die prematurely, before producing myelin sheaths. We have studied cultures of jp CNS in order to understand whether OL survival and myelin formation require production of normal PLP. When grown in primary cultures, jp OLs mimic the relatively undifferentiated phenotype of jp OLs in vivo. They produce little myelin basic protein (MBP), never immunostain for PLP, and rarely elaborate myelin-like membranes. We report here that jp OLs grown in medium conditioned by normal astrocytes synthesize MBP and incorporate it into membrane expansions. Some jp OLs grown in this way stain with PLP antibodies, including an antibody to a peptide sequence specific for the mutant jp PLP. This study shows that: (1) an absence of PLP does not necessarily lead to dysmyelination or OL death; (2) OLs are capable of translating at least a portion of the predicted jp PLP; (3) the abnormal PLP made in the cultured jp cells is not toxic to OLs. These results also highlight the importance of environmental factors in controlling OL phenotype. © 1996 John Wiley & Sons, Inc.     

Peculiar and typical oligodendrocytes are involved in an uneven myelination pattern during the ontogeny of the lizard visual pathway

We studied the myelination of the visual pathway during the ontogeny of the lizard Gallotia galloti using immunohistochemical methods to stain the myelin basic protein (MBP) and proteolipid protein (PLP/DM20), and electron microscopy. The staining pattern for the PLP/DM20 and MBP overlapped during the lizard ontogeny and was first observed at E39 in cell bodies and fibers located in the temporal optic nerve, optic chiasm, middle optic tract, and in the stratum album centrale of the optic tectum (OT). The expression of these proteins extended to the nerve fiber layer (NFL) of the temporal retina and to the outer strata of the OT at E40. From hatching onwards, the labeling became stronger and extended to the entire visual pathway. Our ultrastructural data in postnatal and adult animals revealed the presence of both myelinated and unmyelinated retinal ganglion cell axons in all visual areas, with a tendency for the larger axons to show the thicker myelin sheaths. Moreover, two kinds of oligodendrocytes were described: peculiar oligodendrocytes displaying loose myelin sheaths were only observed in the NFL, whereas typical medium electron-dense oligodendrocytes displaying compact myelin sheaths were observed in the rest of the visual areas. The weakest expression of the PLP/DM20 in the NFL of the retina appears to be linked to the loose appearance of its myelin sheaths. We conclude that typical and peculiar oligodendrocytes are involved in an uneven myelination process, which follows a temporo-nasal and rostro-caudal gradient in the retina and ON, and a ventro-dorsal gradient in the OT.     

The developing avian retina expresses agrin isoforms during synaptogenesis

The localization, isoform pattern, and mRNA distribution of the synapse-organizing molecule agrin was investigated in the developing avian retina. Injection of anti-agrin Fab fragments into the vitreous humor of chick eyes of embryonic days 3 to 20, a procedure that labels only extracellular agrin, reveals staining in the inner and outer plexiform layers before, during, and after the period of synapse formation. The labeling in these layers changes from a diffuse to a punctate pattern at the time when synapses form. At all stages investigated, the inner limiting membrane (a basal lamina that separates vitreous from neural retina) is intensely labeled, as are the axonal processes of retinal ganglion cells in the optic fiber layer and in the optic nerve, although the staining intensity declines after embryonic day 10 in both retina and optic nerve. In culture, axons of retinal ganglion cells also express agrin-like immunoreactivity on their surfaces. Polymerase chain reaction analysis reveals that several different agrin isoforms are expressed in the developing neural retina. In situ hybridization studies show that agrin isoforms are expressed in the ganglion cell and inner nuclear layers, correlating well with the staining for agrin protein in the optic fiber and plexiform layers. The expression of mRNA coding for several agrin isoforms and the presence of extracellular agrin in the synapse-containing layers during the period of synapse formation is consistent with the idea that agrin isoforms might play a role during synapse formation in the central nervous system. © 1996 John Wiley & Sons, Inc.     

Emergence of three myelin proteins in oligodendrocytes cultured without neurons

Oligodendrocytes, the myelin-forming cells of the central nervous system, were cultured from newborn rat brain and optic nerve to allow us to analyze whether two transmembranous myelin proteins, myelin-associated glycoprotein (MAG) and proteolipid protein (PLP), were expressed together with myelin basic protein (MBP) in defined medium with low serum and in the absence of neurons. Using double label immunofluorescence, we investigated when and where these three myelin proteins appeared in cells expressing galactocerebroside (GC), a specific marker for the oligodendrocyte membrane. We found that a proportion of oligodendrocytes derived from brain and optic nerve invariably express MBP, MAG, and PLP about a week after the emergence of GC, which occurs around birth. In brain-derived oligodendrocytes, MBP and MAG first emerge between the fifth and the seventh day after birth, followed by PLP 1 to 2 d later. All three proteins were confined to the cell body at that time, although an extensive network of GC positive processes had already developed. Each protein shows a specific cytoplasmic localization: diffuse for MBP, mostly perinuclear for MAG, and particulate for PLP. Interestingly, MAG, which may be involved in glial-axon interactions, is the first myelin protein detected in the processes at approximately 10 d after birth. MBP and PLP are only seen in these locations after 15 d. All GC-positive cells express the three myelin proteins by day 19. Simultaneously, numerous membrane and myelin whorls accumulate along the oligodendrocyte surface. The sequential emergence, cytoplasmic location, and peak of expression of these three myelin proteins in vitro follow a pattern similar to that described in vivo and, therefore, are independent of continuous neuronal influences. Such cultures provide a convenient system to study factors regulating expression of myelin proteins.     

Myelin under construction -- teamwork required

Myelinating glial cells synthesize specialized myelin proteins and deposit them in the growing myelin sheath that enwraps axons multiple times. How do axons and myelinating glial cells coordinate this spectacular cell–cell interaction? In this issue, Trajkovic et al. (p. 937) show that neuronal signaling regulates cell surface expression of the myelin proteolipid protein in cultured oligodendrocytes in unexpected ways that may also contribute to myelination in situ.Myelination is a stunning example of how multiple cells cooperate to build a complex structure. Understanding how myelinating glia and neurons work together to achieve this feat is thus a challenging and important problem. Trajkovic et al. (p. 937) investigate the regulation of the trafficking of a major myelin protein, proteolipid protein (PLP), to the plasma membrane (PM) of cultured oligodendrocytes (OLs). When initially expressed in cultured OLs, PLP resides in a compartment with characteristics of a late endosome/lysosome (LE/L). Co-culture with neurons leads to an increase of PLP on the PM and a disappearance from the LE/L. This increased surface expression of PLP is due to at least two distinct mechanisms: a decrease in PLP endocytosis from the PM and an increase in exocytosis from the LE/L. The relative contributions of these two mechanisms (and possibly additional ones?) remain open questions for the future.The cells that produce myelin are highly specialized glial cells, Schwann cells in the peripheral nervous system (PNS) and OLs in the central nervous system (CNS). Myelin consists of many wrappings of glial cell membrane around the axon with little or no cytoplasm left between adjacent wraps. This compact myelin region insulates the axon from the extracellular medium and allows saltatory conduction along axons. Each successive myelin wrap creates at its lateral margins a membrane loop containing some cytoplasm. These so-called paranodal loops make up part of the noncompact myelin. Each paranodal loop forms a specialized cell junction with the axon, the axoglial apparatus. The paranodal loops, in turn, flank Nodes of Ranvier, gaps in the myelin where voltage-gated sodium channels cluster and regenerate the action potential (for review see Sherman and Brophy, 2005).Myelination is a supreme example of differential protein distribution. During myelination, glia elaborate distinct domains (such as soma and compact and noncompact myelin) with distinct lipids and protein components. At the same time, axonal membrane proteins also accumulate in distinct regions, such that the Node of Ranvier contains different proteins than the paranodal region (underlying the paranodal loops) or the juxtaparanode (flanking the paranode). Much work on who signaled whom, when, and why, revealed that neurons and myelinating glia communicate with each other bidirectionally in multiple ways to orchestrate myelination (Sherman and Brophy 2005). For instance, glial cells signal to neurons to influence axonal diameter, neurofilament spacing, and phosphorylation (Hsieh et al., 1994). Additionally, nodal, paranodal, and juxtaparanodal domains on axons form as a result of interactions with glial cells. Mutations in genes encoding paranodal proteins lead to aberrant paranodal loops and mislocalization of paranodal and juxtaparanodal components in the axon (for review see Poliak and Peles, 2003; Salzer, 2003). Somewhat surprisingly, nodal proteins still cluster in these mice, leading to the suggestion that nodal assembly might be intrinsic to axons or (in the CNS) driven by diffusible glial-derived factors (Kaplan et al., 1997). New work argues that glial cell processes which contact the node itself could direct nodal assembly. In the PNS, the node is contacted directly by microvilli of the myelinating Schwann cell. Mice lacking Schwann cell dystroglycan or laminin have aberrant microvilli and poorly clustered voltage-gated sodium channels (Saito et al., 2003; Occhi et al., 2005). Gliomedin, identified by the Peles lab, is expressed in Schwann cell microvilli and required for clustering of nodal axonal components (Eshed et al., 2005). In the CNS, Colman''s group localized the outgrowth-inhibitory molecule Omgp to distinct glial cells that can encircle nodes (Huang et al., 2005). Omgp knock-out mice show wider and disorganized nodes as well as aberrant sprouting of branches from nodes. These findings highlight the importance of node-encircling glial cells for organizing the axon.Do neurons in turn give instructions to glial cells? Oligodendrocyte precursor cells (OPCs) in the CNS migrate into developing white matter where they differentiate into postmitotic OLs and produce the myelin sheath. The differentiation of OPCs in terms of changes in gene expression and in morphology has been studied extensively in vitro and in vivo (for reviews see Pfeiffer et al., 1993; Barres and Raff, 1999). Because OPCs differentiate normally in axon-free culture and express myelin components, a role for neurons was not immediately apparent. In vivo, on the other hand, few OLs develop after transection of the optic nerve and subsequently, axons were shown to be required for survival and differentiation of OLs (Barres and Raff, 1999). OPCs and newly born OLs require astrocyte-derived factors such as PDGF, but OLs become dependent on axonal signals later. Axonal signaling to OLs occurs on at least two levels (Barres and Raff, 1999; Coman et al., 2005). Electrical activity (mediated by extrasynaptic release of adenosine [Stevens et al., 2002]) is required for proliferation of OPCs. Additionally, contact-mediated neuronal signals play important roles in OPC and Schwann cell differentiation and myelination (Corfas et al., 2004). Salzer and colleagues recently showed that the levels of neuregulin 1 type III expressed on axons determine the ensheathment fate of axons in the PNS (Taveggia et al., 2005).Compact myelin has a very specific composition of 70% lipids by dry weight (mostly composed of galactoceramide and cerebroside) with 80% of the protein mass comprised of only two proteins, myelin basic protein MBP and proteolipid protein PLP/DM20 (for review see Kramer et al., 2001). Studies have therefore focused on how OLs synthesize MBP and PLP and incorporate them into the growing myelin sheath. MBP is synthesized on free ribosomes, but its mRNA is localized to the myelin sheath (Colman et al., 1982). PLP on the other hand is a membrane-spanning protein that traverses the ER and Golgi. The role for axonal signaling for production of the myelin sheath is not well understood. For instance, OPCs in culture undergo differentiation and start to synthesize myelin components in the absence of neurons (Pfeiffer et al., 1993). Early reports from cultured rat OLs concluded that PLP was synthesized and incorporated into the PM without neurons (Hudson et al., 1989). Interestingly though, PM expression of PLP could not be detected for many days after intracellular pools of PLP were clearly detectable. The delayed PM expression of PLP raised the possibility that axonal signaling could speed up PM expression.The paper by Simons and colleagues in this issue demonstrates neuronal control of PLP trafficking (Trajkovic et al., 2006). Primary OLs, as well as two OL cell lines, contain PLP in a LE/L (as well as on the PM). This LE/L pool of PLP persists if neurons are absent from the culture. When OLs are cocultured with neurons, PLP is found with LE/L initially, but later disappears from there and increased amounts can be detected on the PM. When brain sections were costained against lysosomal markers and PLP, high colocalization of PLP with LE/L was detected in P7 mice while in P60 brains PLP did not colocalize with LE/L. Therefore, PLP localizes (at least partially) with LE/L in vivo and disappears from there upon myelination. This finding assuages much of the fear that PLP-containing LE/L are just a culture phenomenon or due to inappropriate expression levels (Kramer et al., 2001; Simons et al., 2002). The authors tested three explanations to account for their observations: increased proteolysis of PLP, decreased endocytosis, and/or increased exocytosis from LE/L. Proteolysis of PLP was found to be unaffected by neuronal coculture. Endocytosis (via a clathrin-independent, cholesterol-dependent, actin-dependent, and RhoA-dependent pathway), on the other hand, was decreased. Using PLP-GFP and lysotracker to mark LE/L in live OLs, the authors also found that the LE/L became much more mobile in the presence of neurons. To determine whether the moving LE/L in cocultured OLs can fuse with the PM and potentially deliver PLP sequestered in LE/L, the authors used total internal reflection fluorescence microscopy (TIRFM) on lysotracker-labeled OLs. Without neurons present, the LE/L was not found within 100 nm of the PM and was therefore invisible to TIRFM. When neurons were present, many LE/L were found near the PM and events suggestive of fusion could be observed at a rate of 1–2 events/min. Lastly, the authors determined that diffusible neuronal factors were sufficient to induce increased PLP surface expression. Addition of a membrane-permeable cAMP analogue to OLs in the absence of neurons led to increased PLP on the surface as well as high mobility of lysotracker pools containing PLP-GFP.These results suggest that diffusible neuronal factors (currently unknown) could activate cAMP signaling in OLs and regulate endocytosis and exocytosis of PLP. Exocytosis from LE/L is a regulated pathway in other cells as well (Blott and Griffiths, 2002). In OLs, at least some of the PLP could be stored in LE/L until neuronal promyelinating signals are received. Because many proteins arrive in the LE/L from the TGN, it would be interesting to investigate the potential neuronal regulation of PLP sorting events in the Golgi. Although we still await a complete quantitative account of what proportion of PLP is transported where and when, this paper presents an exciting advance in our understanding of the neuronal control of OL membrane traffic.     

Studies of the developing chick retina using monoclonal antibody 8A2 that recognizes a novel set of gangliosides

A monoclonal antibody, Mab 8A2, that recognizes a novel set of gangliosides was produced by immunizing a mouse with Embryonic Day 14 chick optic nerve. Immunohistochemical studies of the developing chick retina revealed a complex pattern of Mab 8A2 immunoreactivity. Initially, staining is concentrated in the optic fiber layer in the central retina. Later in development, the most intense staining is seen at the periphery of the retina and 8A2 immunoreactivity appears in other retina layers. In the adult retina, 8A2 immunoreactivity is lost from the optic fiber layer but persists in the inner plexiform layer, inner nuclear layer, and outer plexiform layer. Cell culture experiments showed intense staining of neurites from retinal ganglion cells but no staining of Muller cells. Biochemical characterization of the epitope recognized by Mab 8A2 suggests that it includes a 9-O-acetyl group that is present on five different gangliosides. The 8A2 immunoreactive gangliosides are distinct from and have slower mobilities on thin-layer chromatographs than those recognized by Mab D1.1 which recognizes 9-O-acetyl GD3.     

Expression of αB-Crystallin in the Peripapillary Glial Cells of the Developing Chick Retina

Peripapillary glial cells (PPGCs) are a peculiar macroglia in avian species, located in the central retina adjacent to the optic nerve head. PPGCs have a similar shape and orientation to Müller cells, which traverse the entire layer of the retina; however, there are differences in protein expression between the two cell types. In the present study, we first demonstrated that PPGCs expressed αB-crystallin, which is not expressed in Müller cells, during retinal development. αB-crystallin was first faintly expressed in PPGCs of the E5 retina, adjacent to the optic nerve head. Further, αB-crystallin was exclusively expressed in PPGCs up to E14. The shape of these cells was bipolar with vitread and ventricular processes. The vitread processes of αB-crystallin+ PPGCs became finer at E18. Double labeling analysis clearly demonstrated that only vimentin+ or GFAP+ astrocytes were located in the optic nerve head and were demarcated from the retina by αB-crystallin+ PPGCs. Furthermore, we determined that αB-crystallin+ PPGCs, with a number of processes, completely wrapped the optic nerve head and were densely located in the junction of the optic nerve head and the retina in a whole mount preparation and in vertical-sectioned retinae. The results of present study, together with reports that retinal astrocytes migrate from the optic nerve head, suggest that PPGCs prevent astrocytes from migrating into the retina in avian species.     

Origin of Oligodendrocytes in the Vertebrate Optic Nerve: A Review

One of the unsolved problems in the research field of oligodendrocyte (OL) development has been the site(s) of origin of optic nerve OLs and its precursor cells (OPCs). It is generally accepted that OLs in the optic nerve are derived from the brain, and thus optic nerve OLs are immigrant cells. We previously demonstrated the brain origin of optic nerve OPCs in chick embryos. However, the site of optic nerve OPC origin has not been examined experimentally in developing rodents for the past two decades. We have recently reported that optic nerve OPCs in mice arise in the preoptic area by E12.5 and gradually migrate caudally and enter the optic nerve. These OPCs give rise to myelinating OLs in the optic nerve in the postnatal or adult stages. Surprisingly, there are species differences with respect to the origin of optic nerve OPCs between chicks and mice. Here, we summarize the site of OPC origin in the optic nerve based on our own previous and recent results, and discuss possible mechanisms underlying these species differences.     

Role of cell adhesion molecule DM-GRASP in growth and orientation of retinal ganglion cell axons

The cell adhesion molecule (CAM) DM-GRASP was investigated with respect to a role for axonal growth and navigation in the developing visual system. Expression analysis reveals that DM-GRASP's presence is highly spatiotemporally regulated in the chick embryo retina. It is restricted to the optic fiber layer (OFL) and shows an expression maximum in a phase when the highest number of retinal ganglion cell (RGC) axons extend. In the developing retina, axons grow between the DM-GRASP-displaying OFL and the Laminin-rich basal lamina. We show that DM-GRASP enhances RGC axon extension and growth cone size on Laminin substrate in vitro. Preference assays reveal that DM-GRASP-containing lanes guide RGC axons, partially depending on NgCAM in the axonal membrane. Inhibition of DM-GRASP in organ-cultured eyes perturbs orientation of RGC axons at the optic fissure. Instead of leaving the retina, RGC axons cross the optic fissure and grow onto the opposite side of the retina. RGC axon extension per se and navigation from the peripheral retina towards the optic fissure, however, is not affected. Our results demonstrate a role of DM-GRASP for axonal pathfinding in an early phase of the formation of the higher vertebrate central nervous system.     

Tracing retinal fiber trajectory with a method of transposon-mediated genomic integration in chick embryo

We report convenient retinal fiber tracing by transfecting the tracer cDNA by in ovo electroporation. Long-term and stable expression of tracer proteins such as green fluorescent protein is achieved by transposon-mediated genome integration of the tracer protein expression cassette. We carried out coelectroporation of a plasmid containing CAGGS-tracer cDNA flanked by the Tol2 transposable element along with a transposase expression vector to the optic vesicle of chick embryos at stage 11. By selecting electrodes, we can label a large group of retinal ganglion cells, or a small group of retinal ganglion cells; parallel electrodes assure transfection of large areas of the retina, and needle type electrodes label small areas of the retina. The retinal fiber trajectory and terminal zone (TZ) could be detected in the precise retinotopic manner on the contra-lateral side of the optic tectum. The method has advantage in that we can show the retinal fiber trajectory in relation to the molecules that are responsible for pathfinding for the retinal fibers in the same specimen.     

FGF signals induce Caprin2 expression in the vertebrate lens

The lens of the eye is derived from the non-neural ectoderm situated next to the optic vesicle. Fibroblast growth factor (FGF) signals play a major role at various stages of vertebrate lens development ranging from induction and proliferation to differentiation. Less is however known about the identity of genes that are induced by FGF activity within the lens. We have isolated and characterized mouse cytoplasmic activation/proliferation-associated protein-2 (Caprin2), with domains belonging to both the Caprin family and the C1q and tumour necrosis factor (TNF) super-family. Here we show that Caprin2 is expressed in the developing vertebrate lens in mouse and chick, and that Caprin2 expression is up-regulated in primary lens fiber cells, after the induction of crystallins the earliest known markers for differentiated lens fiber cells. Caprin2 is subsequently down-regulated in the centre of the lens at the time and at the position of the first fiber cell denucleation and terminal differentiation. In vitro analyses of lens fiber cell differentiation provide evidence that FGF activity emanating from neighboring prospective retinal cells is required and that FGF8 activity is sufficient to induce Caprin2 in lens fiber cells. These results not only provide evidence that FGF signals induce the newly characterized protein Caprin2 in the lens, but also support the general idea that FGF signals are required for lens fiber cell differentiation.     

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.