Embryonic Mouse Retina Research Articles

Light activates Ube3a, an Angelman syndrome-associated gene, by mediating the chromatin structures during postnatal development of mouse retina.

Angelman syndrome, a severe neurodevelopmental disorder, is primarily caused by mutations or deletions of maternally inherited ubiquitin protein ligase E3A (UBE3A). Activation of the silenced paternal copy of UBE3A can occur with pharmacological perturbation; however, an environmental approach has not been examined. Here, we found Ube3a is highly expressed in embryonic and early neonatal mouse retina and is maternally-, but not paternally-, expressed in ganglion cells, amacrine cells, and horizontal cells. Moreover, we analyzed UBE3A expression in the retina and visual cortex of postnatal day 28 mice (P28) following exposure to light emissions from white compact-fluorescent bulbs or blue light-emitting diodes from postnatal day 0 (P0) to 28 (P28), encompassing a crucial phase of visual system development. We found higher levels of Ube3a RNA and protein in the retina, but not visual cortex compared with tissues from P28 mice exposure to typical lighting (controls). Levels of both paternal- and maternal-UBE3A protein in mouse retina were higher than controls in P28 mice exposed to white or blue light. Moreover, levels of open and repressive chromatin structures, indicated by histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3), respectively, were increased in the Ube3a promoter from mouse retina exposed to white or blue light. Our findings strongly suggest that extended exposure to white or blue light constitutes a substantial environmental factor that can effectively promote UBE3A expression within the central nervous system.

Sstr2 Defines the Cone Differentiation-Competent Late-Stage Retinal Progenitor Cells in the Developing Mouse Retina.

Cone cell death is a characteristic shared by various retinal degenerative disorders, such as cone-rod dystrophy, Stargardt disease, achromatopsia, and retinitis pigmentosa. This leads to conditions like color blindness and permanently impaired visual acuity. Stem cell therapy focused on photoreceptor replacement holds promise for addressing these conditions. However, identifying surface markers that aid in enriching retinal progenitor cells (RPCs) capable of differentiating into cones remains a complex task. In this study, we employed single-cell RNA sequencing to scrutinize the transcriptome of developing retinas in C57BL/6J mice. This revealed the distinctive expression of somatostatin receptor 2 (Sstr2), a surface protein, in late-stage RPCs exhibiting the potential for photoreceptor differentiation. In vivo lineage tracing experiments verified that Sstr2+ cells within the late embryonic retina gave rise to cones, amacrine and horizontal cells during the developmental process. Furthermore, Sstr2+ cells that were isolated from the late embryonic mouse retina displayed RPC markers and exhibited the capability to differentiate into cones in vitro. Upon subretinal transplantation into both wild-type and retinal degeneration 10 (rd10) mice, Sstr2+ cells survived and expressed cone-specific markers. This study underscores the ability of Sstr2 to enrich late-stage RPCs primed for cone differentiation to a large extent. It proposes the utility of Sstr2 as a biomarker for RPCs capable of generating cones for transplantation purposes.

A potential role for somatostatin signaling in regulating retinal neurogenesis

Neuropeptides have been reported to regulate progenitor proliferation and neurogenesis in the central nervous system. However, these studies have typically been conducted using pharmacological agents in ex vivo preparations, and in vivo evidence for their developmental function is generally lacking. Recent scRNA-Seq studies have identified multiple neuropeptides and their receptors as being selectively expressed in neurogenic progenitors of the embryonic mouse and human retina. This includes Sstr2, whose ligand somatostatin is transiently expressed by immature retinal ganglion cells. By analyzing retinal explants treated with selective ligands that target these receptors, we found that Sstr2-dependent somatostatin signaling induces a modest, dose-dependent inhibition of photoreceptor generation, while correspondingly increasing the relative fraction of primary progenitor cells. These effects were confirmed by scRNA-Seq analysis of retinal explants but abolished in Sstr2-deficient retinas. Although no changes in the relative fraction of primary progenitors or photoreceptor precursors were observed in Sstr2-deficient retinas in vivo, scRNA-Seq analysis demonstrated accelerated differentiation of neurogenic progenitors. We conclude that, while Sstr2 signaling may act to negatively regulate retinal neurogenesis in combination with other retinal ganglion cell-derived secreted factors such as Shh, it is dispensable for normal retinal development.

H3K27me3 demethylase UTX regulates the differentiation of a subset of bipolar cells in the mouse retina.

Di- and trimethylation of lysine 27 on histone 3 (H3K27me2/3) is a critical gene repression mechanism. We previously showed that down-regulation of the H3K27 demethylase, Jumonji domain-containing protein 3 (JMJD3), resulted in a reduced number of protein kinase C (PKC)α-positive rod ON-bipolar cells. In this work, we focused on the role of another H3K27 demethylase, ubiquitously transcribed tetratricopeptide repeat X chromosome (UTX), in retinal development. UTX was expressed in the retinal progenitor cells of the embryonic mouse retina and was observed in the inner nuclear layer during late retinal development and in the mature retina. The short hairpin RNA-mediated knockdown of Utx in a mouse retinal explant led to a reduced number of PKCα-positive rod ON-bipolar cells. However, other retinal subtypes were unaffected by this knockdown. Using a retina-specific knockout of Utx in mice, the in vivo effects of UTX down-regulation were examined. Again, the number of PKCα-positive rod ON-bipolar cells was reduced, and no other apparent phenotypes, including retinal progenitor proliferation, apoptosis or differentiation, were observed. Finally, we examined retina-specific Utx and Jmjd3 double-knockout mice and found that although the number of rod ON-bipolar cells was reduced, no additional effects from the loss of Utx and Jmjd3 were observed. Taken together, our data show that UTX contributes to retinal differentiation in a lineage-specific manner.

Simultaneous Requirements for Hes1 in Retinal Neurogenesis and Optic Cup-Stalk Boundary Maintenance.

The bHLH transcription factor Hes1 is a key downstream effector for the Notch signaling pathway. During embryogenesis neural progenitors express low levels of Hes1 in an oscillating pattern, whereas glial brain boundary regions (e.g., isthmus) have high, sustained Hes1 levels that suppress neuronal fates. Here, we show that in the embryonic mouse retina, the optic nerve head and stalk express high Hes1, with the ONH constituting a boundary between the neural retina and glial cells that ultimately line the optic stalk. Using two Cre drivers with distinct spatiotemporal expression we conditionally inactivated Hes1, to delineate the requirements for this transcriptional repressor during retinal neurogenesis versus patterning of the optic cup and stalk. Throughout retinal neurogenesis, Hes1 maintains proliferation and blocks retinal ganglion cell formation, but surprisingly we found it also promotes cone photoreceptor genesis. In the postnatal eye, Hes1 inactivation with Rax-Cre resulted in increased bipolar neurons and a mispositioning of Müller glia. Our results indicate that Notch pathway regulation of cone genesis is more complex than previously assumed, and reveal a novel role for Hes1 in maintaining the optic cup-stalk boundary.SIGNIFICANCE STATEMENT The bHLH repressor Hes1 regulates the timing of neurogenesis, rate of progenitor cell division, gliogenesis, and maintains tissue compartment boundaries. This study expands current eye development models by showing Notch-independent roles for Hes1 in the developing optic nerve head (ONH). Defects in ONH formation result in optic nerve coloboma; our work now inserts Hes1 into the genetic hierarchy regulating optic fissure closure. Given that Hes1 acts analogously in the ONH as the brain isthmus, it prompts future investigation of the ONH as a signaling factor center, or local organizer. Embryonic development of the ONH region has been poorly studied, which is surprising given it is where the pan-ocular disease glaucoma is widely believed to inflict damage on RGC axons.

Complement Targets Newborn Retinal Ganglion Cells for Phagocytic Elimination by Microglia.

Microglia play important roles in shaping the developing CNS, and at early stages they have been proposed to regulate progenitor proliferation, differentiation, and neuronal survival. However, these studies reveal contradictory outcomes, highlighting the complexity of these cell-cell interactions. Here, we investigate microglia function during embryonic mouse retina development, where only microglia, progenitors, and neurons are present. In both sexes, we determine that microglia primarily interact with retinal neurons and find that depletion of microglia via conditional KO of the Csf1 receptor results in increased density of retinal ganglion cells (RGCs). Pharmacological inhibition of microglia also results in an increase in RGCs, with no effect on retinal progenitor proliferation, RGC genesis, or apoptosis. We show that microglia in the embryonic retina are enriched for phagocytic markers and observe engulfment of nonapoptotic Brn3-labeled RGCs. We investigate the molecular pathways that can mediate cell engulfment by microglia and find selective downregulation of complement pathway components with microglia inhibition, and further show that C1q protein marks a subset of RGCs in the embryonic retina. KO of complement receptor 3 (CR3; Itgam), which is only expressed by microglia, results in increased RGC density, similar to what we observed after depletion or inhibition of microglia. Thus, our data suggest that microglia regulate neuron elimination in the embryonic mouse retina by complement-mediated phagocytosis of non-apoptotic newborn RGCs.SIGNIFICANCE STATEMENT Microglia are emerging as active and important participants in regulating neuron number in development, during adult neurogenesis, and following stem cell therapies. However, their role in these contexts and the mechanisms involved are not fully defined. Using a well-characterized in vivo system, we provide evidence that microglia regulate neuronal elimination by complement-mediated engulfment of nonapoptotic neurons. This work provides a significant advancement of the field by defining in vivo molecular mechanisms for microglia-mediated cell elimination. Our data add to a growing body of evidence that microglia are essential for proper nervous system development. In addition, we elucidate microglia function in the developing retina, which may shed light on microglia involvement in the context of retinal injury and disease.

Rbpj direct regulation of Atoh7 transcription in the embryonic mouse retina

In vertebrate retinal progenitor cells, the proneural factor Atoh7 exhibits a dynamic tissue and cellular expression pattern. Although the resulting Atoh7 retinal lineage contains all seven major cell types, only retinal ganglion cells require Atoh7 for proper differentiation. Such specificity necessitates complex regulation of Atoh7 transcription during retina development. The Notch signaling pathway is an evolutionarily conserved suppressor of proneural bHLH factor expression. Previous in vivo mouse genetic studies established the cell autonomous suppression of Atoh7 transcription by Notch1, Rbpj and Hes1. Here we identify four CSL binding sites within the Atoh7 proximal regulatory region and demonstrate Rbpj protein interaction at these sequences by in vitro electromobility shift, calorimetry and luciferase assays and, in vivo via colocalization and chromatin immunoprecipitation. We found that Rbpj simultaneously represses Atoh7 transcription using both Notch-dependent and –independent pathways.

Delayed neurogenesis leads to altered specification of ventrotemporal retinal ganglion cells in albino mice.

BackgroundProper binocular vision depends on the routing at the optic chiasm of the correct proportion of retinal ganglion cell (RGC) axons that project to the same (ipsilateral) and opposite (contralateral) side of the brain. The ipsilateral RGC projection is reduced in mammals with albinism, a congenital disorder characterized by deficient pigmentation in the skin, hair, and eyes. Compared to the pigmented embryonic mouse retina, the albino embryonic mouse retina has fewer RGCs that express the zinc-finger transcription factor, Zic2, which is transiently expressed by RGCs fated to project ipsilaterally. Here, using Zic2 as a marker of ipsilateral RGCs, Islet2 as a marker of contralateral RGCs, and birthdating, we investigate spatiotemporal dynamics of RGC production as they relate to the phenotype of diminished ipsilateral RGC number in the albino retina.ResultsAt embryonic day (E)15.5, fewer Zic2-positive (Zic2+) RGCs are found in the albino ventrotemporal (VT) retina compared with the pigmented VT retina, as we previously reported. However, the reduction in Zic2+ RGCs in the albino is not accompanied by a compensatory increase in Zic2-negative (Zic2−) RGCs, resulting in fewer RGCs in the VT retina at this time point. At E17.5, however, the number of RGCs in the VT region is similar in pigmented and albino retinae, implicating a shift in the timing of RGC production in the albino. Short-term birthdating assays reveal a delay in RGC production in the albino VT retina between E13 and E15. Specifically, fewer Zic2+ RGCs are born at E13 and more Zic2− RGCs are born at E15. Consistent with an increase in the production of Zic2− RGCs born at later ages, more RGCs at E17.5 express the contralateral marker, Islet2, in the albino VT retina compared with the pigmented retina.ConclusionsA delay in neurogenesis in the albino retina is linked to the alteration of RGC subtype specification and consequently leads to the reduced ipsilateral projection that characterizes albinism.

In utero Electroporation of the Embryonic Mouse Retina

In utero Electroporation of the Embryonic Mouse Retina

Early Neural Cell Death Is an Extensive, Dynamic Process in the Embryonic Chick and Mouse Retina

Orchestrated proliferation, differentiation, and cell death contribute to the generation of the complex cytoarchitecture of the central nervous system, including that of the neuroretina. However, few studies have comprehensively compared the spatiotemporal patterns of these 3 processes, or their relative magnitudes. We performed a parallel study in embryonic chick and mouse retinas, focusing on the period during which the first neurons, the retinal ganglion cells (RGCs), are generated. The combination of in vivo BrdU incorporation, immunolabeling of retinal whole mounts for BrdU and for the neuronal markers Islet1/2 and βIII-tubulin, and TUNEL allowed for precise cell scoring and determination the spatiotemporal patterns of cell proliferation, differentiation, and death. As predicted, proliferation preceded differentiation. Cell death and differentiation overlapped to a considerable extent, although the magnitude of cell death exceeded that of neuronal differentiation. Precise quantification of the population of recently born RGCs, identified by BrdU and βIII-tubulin double labeling, combined with cell death inhibition using a pan-caspase inhibitor, revealed that apoptosis decreased this population by half shortly after birth. Taken together, our findings provide important insight into the relevance of cell death in neurogenesis.

An Essential Role for RAX Homeoprotein and NOTCH–HES Signaling inOtx2Expression in Embryonic Retinal Photoreceptor Cell Fate Determination

The molecular mechanisms underlying cell fate determination from common progenitors in the vertebrate CNS remain elusive. We previously reported that the OTX2 homeoprotein regulates retinal photoreceptor cell fate determination. While Otx2 transactivation is a pivotal process for photoreceptor cell fate determination, its transactivation mechanism in the retina is unknown. Here, we identified an evolutionarily conserved Otx2 enhancer of ∼500 bp, named embryonic enhancer locus for photoreceptor Otx2 transcription (EELPOT), which can recapitulate initial Otx2 expression in the embryonic mouse retina. We found that the RAX homeoprotein interacts with EELPOT to transactivate Otx2, mainly in the final cell cycle of retinal progenitors. Conditional inactivation of Rax results in downregulation of Otx2 expression in vivo. We also showed that NOTCH-HES signaling negatively regulates EELPOT to suppress Otx2 expression. These results suggest that the integrated activity of cell-intrinsic and -extrinsic factors on EELPOT underlies the molecular basis of photoreceptor cell fate determination in the embryonic retina.

Smad4 Is Required Predominantly in the Developmental Processes Dependent on the BMP Branch of the TGF-β Signaling System in the Embryonic Mouse Retina

The present study was aimed at defining developmental roles of Smad4, a key mediator of the TGF-β superfamily signaling system, in the embryonic mouse retina. Using a Cre/loxP-mediated conditional gene targeting approach, Smad4 gene function was deleted from the embryonic mouse retina. Mutant phenotypes were morphologically and molecularly examined. Loss of Smad4 in the developing retina led to varying degrees of microphthalmia at birth, presumably because of elevated apoptosis observed transiently at embryonic day 12.5 in the developing retina. This was also associated with an apparent delay in accumulation of retinal ganglion cells. Smad4 conditional mutants also exhibited alterations of retinal spatial patterning along the dorsal-ventral axis, consistent with a known function of BMP signaling in the embryonic retina. However, despite a known role for BMP signaling in retinal cell survival, proliferation, and differentiation, Smad4 mutant retinal progenitor cells were capable of maintaining growth and neurogenesis throughout embryonic development. We also found that the loss of Smad4 led to abnormal targeting of retinal ganglion cell axons to the optic nerve head, a phenotype consistent with reduced BMP signaling in the developing retina. These results suggest that Smad4 is essential for a subset of, but not all, TGF-β/BMP-dependent developmental processes in the embryonic retina. In addition, genetic requirements for Smad4 in the embryonic retina are evident predominantly in the developmental events regulated by the BMP branch of the TGF-β signaling pathway.

A Potential Role for Shed Soluble Major Histocompatibility Class I Molecules as Modulators of Neurite Outgrowth

The neurobiological activities of classical major histocompatibility class I (MHCI) molecules are just beginning to be explored. To further examine MHCI's actions during the formation of neuronal connections, we cultured embryonic mouse retina explants a short distance from wildtype thalamic explants, or thalami from transgenic mice (termed “NSE-Db”) whose neurons express higher levels of MHCI. While retina neurites extended to form connections with wildtype thalami, we were surprised to find that retina neurite outgrowth was very stunted in regions proximal to NSE-Db thalamic explants, suggesting that a diffusible factor from these thalami inhibited retina neurite outgrowth. It has been long known that MHCI-expressing cells release soluble forms of MHCI (sMHCI) due to the shedding of intact MHCI molecules, as well as the alternative exon splicing of its heavy chain or the action proteases which cleave off it's transmembrane anchor. We show that the diffusible inhibitory factor from the NSE-Db thalami is sMHCI. We also show that COS cells programmed to express murine MHCI release sMHCI that inhibits neurite outgrowth from nearby neurons in vitro. The neuroinhibitory effect of sMHCI could be blocked by lowering cAMP levels, suggesting that the neuronal MHCI receptor's signaling mechanism involves a cyclic nucleotide-dependent pathway. Our results suggest that MHCI may not only have neurobiological activity in its membrane-bound form, it may also influence local neurons as a soluble molecule. We discuss the involvement of complement proteins in generating sMHCI and new theoretical models of MHCI's biological activities in the nervous system.

DNA-PK promotes the survival of young neurons in the embryonic mouse retina

Programmed cell death is a crucial process in neural development that affects mature neurons and glial cells, as well as proliferating precursors and recently born neurons at earlier stages. However, the regulation of the early phase of neural cell death and its function remain relatively poorly understood. In mouse models defective in homologous recombination or nonhomologous end-joining (NHEJ), which are both DNA double-strand break (DSB) repair pathways, there is massive cell death during neural development, even leading to embryonic lethality. These observations suggest that natural DSBs occur frequently in the developing nervous system. In this study, we have found that several components of DSB repair pathways are activated in the developing mouse retina at stages that coincide with the onset of neurogenesis. In short-term organotypic retinal cultures, we confirmed that the repair pathways can be modulated pharmacologically. Indeed, inhibiting the DNA-dependent protein kinase (DNA-PK) catalytic subunit, which is involved in NHEJ, with NU7026 increased caspase-dependent cell death and selectively reduced the neuron population. This observation concurs with an increase in the number of apoptotic neurons found after NU7026 treatment, as also observed in the embryonic scid mouse retina, a mutant that lacks DNA-PK catalytic subunit activity. Therefore, our results implicate the generation of DSB and DNA-PK-mediated repair in neurogenesis in the developing retina.

Distinct Effects of Hedgehog Signaling on Neuronal Fate Specification and Cell Cycle Progression in the Embryonic Mouse Retina

Cell-extrinsic signals can profoundly influence the production of various neurons from common progenitors. Yet mechanisms by which extrinsic signals coordinate progenitor cell proliferation, cell cycle exit, and cell fate choices are not well understood. Here, we address whether Hedgehog (Hh) signals independently regulate progenitor proliferation and neuronal fate decisions in the embryonic mouse retina. Conditional ablation of the essential Hh signaling component Smoothened (Smo) in proliferating progenitors, rather than in nascent postmitotic neurons, leads to a dramatic increase of retinal ganglion cells (RGCs) and a mild increase of cone photoreceptor precursors without significantly affecting other early-born neuronal cell types. In addition, Smo-deficient progenitors exhibit aberrant expression of cell cycle regulators and delayed G(1)/S transition, especially during the late embryonic stages, resulting in a reduced progenitor pool by birth. Deficiency in Smo function also causes reduced expression of the basic helix-loop-helix transcription repressor Hes1 and preferential elevation of the proneural gene Math5. In Smo and Math5 double knock-out mutants, the enhanced RGC production observed in Smo-deficient retinas is abolished, whereas defects in the G(1)/S transition persist, suggesting that Math5 mediates the Hh effect on neuronal fate specification but not on cell proliferation. These findings demonstrate that Hh signals regulate progenitor pool expansion primarily by promoting cell cycle progression and influence cell cycle exit and neuronal fates by controlling specific proneural genes. Together, these distinct cellular effects of Hh signaling in neural progenitor cells coordinate a balanced production of diverse neuronal cell types.

Dll1 and Dll4 function sequentially in the retina and pV2 domain of the spinal cord to regulate neurogenesis and create cell diversity

Signalling mediated by Notch receptors is known to have multiple functions during vertebrate neural development, regulating processes like progenitor differentiation and cell type diversification. Various Notch ligands are expressed in the developing nervous system and their activities might contribute to this multiplicity of functions. Here, we show that two Delta-like genes, Dll1 and Dll4, are sequentially expressed in differentiating neurons of the embryonic mouse retina and spinal cord's pV2 domain, with Dll1 starting to be expressed before Dll4. Analysis of Dll1 mutants reveals this gene is necessary and sufficient to maintain a pool of progenitors in the embryonic neuroepithelium. Accordingly, in the spinal cord domains where Dll1 is the only expressed Notch ligand, its inactivation leads to an increased rate of neurogenesis and premature differentiation of neural progenitors. In contrast, in the pV2 domain and retina where Dll1 is co-expressed with Dll4, progenitors are not exhausted and cell diversity is maintained. Together, our results support a model where Dll1 and Dll4 are part of a unique genetic circuitry that regulates subsequent steps of neurogenesis in the retina and pV2 domain: while Dll1 serves to prevent the untimely differentiation of neural progenitors, Dll4 might function to generate diversity within the population of differentiating neurons.

Hedgehog signals received by retinal progenitor cells regulate cell cycle progression and neuronal fate specification in the embryonic mouse retina

Hedgehog signals received by retinal progenitor cells regulate cell cycle progression and neuronal fate specification in the embryonic mouse retina

Characterization of a Transient TCF/LEF-Responsive Progenitor Population in the Embryonic Mouse Retina

High mobility group (HMG) transcription factors of the T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) family are a class of intrinsic regulators that are dynamically expressed in the embryonic mouse retina. Activation of TCF/LEFs is a hallmark of the Wnt/beta-catenin pathway; however, the requirement for Wnt/beta-catenin and noncanonical Wnt signaling during mammalian retinal development remains unclear. The goal of the study was to characterize more fully a TCF/LEF-responsive retinal progenitor population in the mouse embryo and to correlate this with Wnt/beta-catenin signaling. TCF/LEF activation was analyzed in the TOPgal (TCF optimal promoter) reporter mouse at embryonic ages and compared to Axin2 mRNA expression, an endogenous readout of Wnt/beta-catenin signaling. Reporter expression was also examined in embryos with a retina-specific deletion of the beta-catenin gene (Ctnnb1), using Six3-Cre transgenic mice. Finally, the extent to which TOPgal cells coexpress cell cycle proteins, basic helix-loop-helix (bHLH) transcription factors, and other retinal cell markers was tested by double immunohistochemistry. TOPgal reporter activation occurred transiently in a subpopulation of embryonic retinal progenitor cells. Axin2 was not expressed in the central retina, and TOPgal reporter expression persisted in the absence of beta-catenin. Although a proportion of TOPgal-labeled cells were proliferative, most coexpressed the cyclin-dependent kinase inhibitor p27/Kip1. TOPgal cells give rise to the four earliest cell types: ganglion, amacrine, horizontal, and photoreceptor. TCF/LEF activation in the central retina does not correlate with Wnt/beta-catenin signaling, pointing to an alternate role for this transcription factor family during retinal development.

MARCKS-like protein, a membrane protein identified for its expression in developing neural retina, plays a role in regulating retinal cell proliferation

Membrane proteins are expressed in a specific manner in developing tissues, and characterization of these proteins is valuable because it allows them to be used as cell surface markers. Furthermore, they are potentially important for the regulation of organogenesis because some may participate in signal transduction. In the present study, we used proteomics to examine the comprehensive protein expression profile of the membrane fraction in the embryonic and adult mouse retina. We purified the retinal membrane fraction by sucrose-density-gradient centrifugation and analysed total proteins using shotgun analysis on a nanoflow LC-MS/MS (liquid chromatography tandem MS) system. Approximately half of the 326 proteins from the adult retina and a quarter of the 310 proteins from the embryonic retina (day 17) appeared to be membrane-associated proteins. Among these, MLP [MARCKS (myristoylated alanine-rich C-kinase substrate)-like protein], which shares approx. 50% amino acid identity with MARCKS, was selected for further characterization. The mRNA and surface protein expression of MLP decreased as retinal development progressed. Overexpression of MLP by retrovirus-mediated gene transfer enhanced the proliferation of retinal progenitor cells without affecting differentiation or cell migration in a retinal explant culture system. In contrast, MLP overexpression did not promote proliferation in fibroblasts (NIH 3T3 cells). Mutation analysis of MLP demonstrated that myristoylation was necessary to promote proliferation and that phosphorylation inhibited proliferation, indicating the functional importance of membrane localization.

Retinal and olfactory bulb precursor cells show distinct responses to FGF‐2 and laminin

We analyzed whether the embryonic (E12.5-E14.5) mouse retina possesses genuine neural stem cells and how they respond to defined growth factors and extracellular matrix molecules. Whereas most combinations produced no or limited cell survival and proliferation in culture, FGF-2 plus heparin and laminin stimulated proliferation and the formation of aggregates composed, after two days, of 95.2% nestin-positive cells. However, cells in these aggregates could only be passaged poorly, lost nestin expression and proliferative capacity, and differentiated into neurons. Under the same conditions, olfactory bulb precursor cells divided efficiently and could be expanded. These data suggest that, in addition to FGF-2 and laminin, embryonic retinal neuroepithelial cells need additional extrinsic and/or intrinsic regulators to maintain cell proliferation and self-renewal.