A marker of early amacrine cell development in rat retina

doi:10.1016/0165-3806(85)90116-6

Developmental Brain Research

Volume 20, Issue 2, June 1985, Pages 286-290

https://doi.org/10.1016/0165-3806(85)90116-6 Get rights and content

Abstract

A monoclonal antibody, HPC-1, labels only amacrine and displaced amacrine cells in adult rat retina. Reactivity with displaced amacrine cells was demonstrated by double-label immunofluorescence with HPC-1 and ganglion cell-specific reagents. HPC-1 antibody reacts with a polypeptide of apparent molecular weight 35,000. HPC-1 antibody labels migrating amacrine cells in late embryonic retinas. The results define cell-specific gene expression in relation to migration of one subclass of CNS neurons.

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Cited by (98)

Rapidly purified ganglion cells from neonatal mouse retinas allow studies of mitochondrial morphology and autophagy
2018, Pharmacological Research
Citation Excerpt :
Thy1 negative-cells of the retina are mainly astrocytes and neuronal cells, i.e., horizontal cells, photoreceptors, amacrine and bipolar cells. Astrocytes express the selective marker glial fibrillary acidic protein (GFAP) [18,19], horizontal cells express LIM homeobox 1 (Lhx1) [20]; photoreceptors express recoverin (Rcvr) [21]; amacrine cells express syntaxin 1 A (Stx1a) [22,23], and bipolar cells express visual system homeobox 2 (Vsx2) [24,25]. Notably, all these markers were amplified in the excluded, but not in the Thy1 positive-cells selected fraction (Fig. 1D).
Retinal explants and mixed primary cultures are currently used to investigate retinal ganglion cells (RGCs) pathophysiology and pharmacology, but information on yield, quality and quantity of contaminant cells for the available RGCs enrichment techniques is lacking. Here we compare two methods of mouse primary RGCs purification and show that mitochondrial and autophagy parameters can be measured in rapidly purified RGCs. RGCs were purified from P0 mouse eyes using two methods based on the surface antigen Thy1. In a two-step immunopanning purification, a subtraction plate bound macrophage antiserum removed contaminant macrophages and endothelial cells; unbound RGCs were then affinity selected using a plate-bound antiThy1 antibody. In an immunopanning-magnetic separation, macrophage-antiserum bound cells were first subtracted and then RGCs were positively selected using an antiThy1 antibody bound to a magnetic column. The two-steps immunopanning yielded low amounts of 90% pure RGCs, whereas RGCs represented 30% of the 6-fold more cells collected by immunopanning-magnetic separation. RGCs purified with both methods could be microelectroporated to image expressed mitochondria and autophagosomes fluorescent probes and to show that expression of pathogenic Optic atrophy 1 mutants causes mitochondrial fragmentation. Thus, these two methods purify primary mouse RGCs amenable to studies of cell morphology, mitochondrial biology and autophagy.
Fatty acid amide hydrolase expression during retinal postnatal development in rats
2011, Neuroscience
Citation Excerpt :
The immunoreactivity we observed is in agreement with the reported ChAT distribution using the same antibody as well as others (Voigt, 1986; Jeon et al., 1998; Haverkamp and Wässle, 2000; Majumdar et al., 2008). The protein syntaxin-1 was recognized as a specific marker of retinal amacrine and horizontal cells by several research teams (Barnstable et al., 1985; Hirano et al., 2005). Mouse anti-syntaxin (HPC-1; Sigma-Aldrich, Oakville, ON, Canada) recognizes syntaxin-1, a 35-kDa protein, from hippocampal, retinal, and cortical neurons (Inoue et al., 1992).
The endocannabinoid (eCB) system is thought to participate in developmental processes in the CNS. The rodent retina represents a valuable model to study CNS development because it contains well-identified cell types with established developmental timelines. The distribution of cannabinoid receptor type 1 (CB1R) was recently revealed in the developing retina; however, the expression patterns of other elements of this system remain unknown. In this study, we investigated the expression pattern of the degradative enzyme fatty acid amide hydrolase (FAAH), a key regulator of the eCB system, in the rat retina during postnatal development. To identify the cells expressing the enzyme, co-stainings were carried out for FAAH and retinal cell type markers. FAAH was expressed at postnatal day (P) 1 in ganglion and cholinergic amacrine cells. In the course of development, it appeared in cones, horizontal, and bipolar cells. For most cell types (horizontal, cholinergic amacrine cells, and cone bipolar cells), FAAH was transiently expressed, suggesting an important redistribution of the enzyme during postnatal development and thus a potential role of the eCB system in developmental processes. Our results also indicated that, in the adult retina, FAAH is expressed in cones, rod bipolar cells, and some retinal ganglion cells. The presence of FAAH in adult animals supports the hypothesis that the eCB system is involved in retinal functions. Overall these results indicate that, as shown in other structures of the brain, the eCB system could play an instrumental role in the development and function of the retina.
Foxn4 is required for retinal ganglion cell distal axon patterning
2011, Molecular and Cellular Neuroscience
Citation Excerpt :
We confirmed that Foxn4−/− mice have fewer nuclei in the INL and GCL, where amacrine cells reside, compared to their wildtype littermates (Fig. 1A, B), but there was no difference in number of nuclei in the outer nuclear layer (ONL) where photoreceptors are found (Fig. 1C). When we immunostained for two pan-amacrine cell markers, syntaxin and Vc1.1 (Arimatsu et al., 1987; Barnstable et al., 1985), we found a greatly reduced staining in the inner aspect of the INL and the GCL (Fig. 1E), consistent with prior characterization (Li et al., 2004). Heterozygotes were indistinguishable from wildtypes (data not shown).
The regulation of retinal ganglion cell (RGC) axon growth and patterning in vivo is thought to be largely dependent on interactions with visual pathway and target cells. Here we address the hypothesis that amacrine cells, RGCs' presynaptic partners, regulate RGC axon growth or targeting. We asked whether amacrine cells play a role in RGC axon growth in vivo using Foxn4^−/− mice, which have fewer amacrine cells, but a normal complement of RGCs. We found that Foxn4^−/− mice have a similar reduction in most subtypes of amacrine cells examined. Remarkably, spontaneous retinal waves were not affected by the reduction of amacrine cells in the Foxn4^−/− mice. There was, however, a developmental delay in the distribution of RGC projections to the superior colliculus. Furthermore, RGC axons failed to penetrate into the retinorecipient layers in the Foxn4^−/− mice. Foxn4 is not expressed by RGCs and was not detectable in the superior colliculus itself. These findings suggest that amacrine cells are critical for proper RGC axon growth in vivo, and support the hypothesis that the amacrine cell–RGC interaction may contribute to the regulation of distal projections and axon patterning.
Mechanisms controlling Pax6 isoform expression in the retina have been conserved between teleosts and mammals
2007, Developmental Biology
The Pax6 gene plays several roles in retinal development, including control of cell proliferation, maintenance of the retinogenic potential of progenitor cells, and cell fate specification. Emerging evidence suggests that these different aspects of Pax6 gene function are mediated by different isoforms of the Pax6 protein; however, relatively little is known about the spatiotemporal expression of Pax6 isoforms in the vertebrate retina. Using bacterial artificial chromosome (BAC) technology, we modified a zebrafish Pax6a BAC such that we could distinguish paired-containing Pax6a transcripts from paired-less Pax6a transcripts. In the zebrafish, the spatial and temporal onset of expression of these transcripts suggests that the paired-less isoform is involved in the cell fate decision leading to the generation of amacrine cells; however, because of limitations associated with transient transgenic analysis, it was not feasible to establish whether this promoter was active in all amacrine cells or in a specific population of amacrine cells. By making mice transgenic for the zebrafish Pax6a BAC reporter transgene, we were able to show that paired-containing and paired-less Pax6a transcripts were differentially expressed in amacrine subpopulations. Our study also directly demonstrates the functional conservation of the regulatory mechanisms governing Pax6 transcription in teleosts and mammals.
Single molecule mechanical probing of the SNARE protein interactions
2006, Biophysical Journal
Citation Excerpt :
Success in coupling of recombinant proteins to their respective surfaces was assessed using indirect immunochemistry. Monoclonal antibody against Sx (49) revealed the presence of Sx1A-H6 recombinant protein only on functionalized cantilevers, but not on the control cantilevers, where recombinant proteins were omitted during the functionalization procedure (Fig. 1, B and C). Similarly, incubation of nickel-coated glass coverslips with recombinant Sb2-H6 resulted in functionalization of glass surface (Fig. 1 D) as detected by a monoclonal antibody directed against Sb2 (42).
Exocytotic release of neurotransmitters is mediated by the ternary soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) complex, comprised of syntaxin (Sx), synaptosome-associated protein of 25 kDa (SNAP25), and synaptobrevin 2 (Sb2). Since exocytosis involves the nonequilibrium process of association and dissociation of bonds between molecules of the SNARE complex, dynamic measurements at the single molecule level are necessary for a detailed understanding of these interactions. To address this issue, we used the atomic force microscope in force spectroscopy mode to show from single molecule investigations of the SNARE complex, that Sx1A and Sb2 are zippered throughout their entire SNARE domains without the involvement of SNAP25. When SNAP25B is present in the complex, it creates a local interaction at the 0 (ionic) layer by cuffing Sx1A and Sb2. Force loading rate studies indicate that the ternary complex interaction is more stable than the Sx1A-Sb2 interaction.
The Iroquois homeobox gene, Irx5, is required for retinal cone bipolar cell development
2005, Developmental Biology
In the mouse retina, at least ten distinct types of bipolar interneurons are involved in the transmission of visual signals from photoreceptors to ganglion cells. How bipolar interneuron diversity is generated during retinal development is poorly understood. Here, we show that Irx5, a member of the Iroquois homeobox gene family, is expressed in developing bipolar cells starting at postnatal day 5 and is localized to a subset of cone bipolar cells in the mature mouse retina. In Irx5-deficient mice, defects were observed in the expression of some, but not all, immunohistological markers that define mature Type 2 and Type 3 OFF cone bipolar cells, indicating a role for Irx5 in bipolar cell differentiation. The differentiation of these two bipolar cell types has previously been shown to require the homeodomain-CVC transcription factor, Vsx1. However, the defects observed in Irx5-deficient retinas do not coincide with a reduction of Vsx1 expression, and conversely, the expression of Irx5 in cone bipolar cells does not require the presence of a functional Vsx1 allele. These results indicate that there are at least two distinct genetic pathways (Irx5-dependent and Vsx1-dependent) regulating the development of Type 2 and Type 3 cone bipolar cells.

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Short communicationA marker of early amacrine cell development in rat retina

Abstract

Neuroscience

Neurochem. Int.

Peptides

Cell

Curr. Topics develop. Biol.

Develop. Biol.

Neuroscience

Brain Res.

Monoclonal antibodies which recognise different cell types in rat retina

Nature (Lond.)

Developmental studies of rat retinal cells using cell-type-specific monoclonal antibodies

Immunological studies of the retina

Monoclonal antibodies that label discrete cell types in the mammalian nervous system

Short communication
A marker of early amacrine cell development in rat retina