Irene E. Whitney

Spatial patterning of cholinergic amacrine cells in the mouse retina

The two populations of cholinergic amacrine cells in the inner nuclear layer (INL) and the ganglion cell layer (GCL) differ in their spatial organization in the mouse retina, but the basis for this difference is not understood. The present investigation examined this issue in six strains of mice that differ in their number of cholinergic cells, addressing how the regularity, packing, and spacing of these cells varies as a function of strain, layer, and density. The number of cholinergic cells was lower in the GCL than in the INL in all six strains. The nearest neighbor and Voronoi domain regularity indexes as well as the packing factor were each consistently lower for the GCL. While these regularity indexes and the packing factor were largely stable across variation in density, the effective radius was inversely related to density for both the GCL and INL, being smaller and more variable in the GCL. Consequently, despite the lower densities in the GCL, neighboring cells were more likely to be positioned closer to one another than in the higher‐density INL, thereby reducing regularity and packing. This difference in the spatial organization of cholinergic cells may be due to the cells in the GCL having been passively displaced by fascicles of optic axons and an expanding retinal vasculature during development. In support of this interpretation, we show such displacement of cholinergic somata relative to their dendritic stalks and a decline in packing efficiency and regularity during postnatal development that is more severe for the GCL. J. Comp. Neurol. 508:1–12, 2008.

Dendritic spread and functional coverage of starburst amacrine cells.

The network of starburst amacrine cells plays a fundamental role in the neural circuitry underlying directional selectivity within the retina. Individual sectors of the starburst dendritic field are directionally selective by virtue of a mutually inhibitory relationship between starburst amacrine cells with overlapping dendrites. These features of the starburst amacrine cell network suggest that starburst cells regulate their dendritic overlap to ensure a uniform coverage of the retinal surface. The present study has compared the dendritic morphology of starburst amacrine cells in two different strains of mice that differ in starburst amacrine cell number. The A/J (A) strain contains about one‐quarter fewer starburst amacrine cells than does the C57BL/6J (B6) strain, although the mosaics of starburst amacrine cells in both strains are comparably patterned. Dendritic field size, however, does not compensate for the difference in density, the A strain having a slightly smaller dendritic field relative to the B6 strain, yielding a significantly larger dendritic coverage factor for individual cells in the B6 strain. The area of the distal (output) annulus of the dendritic field occupies a comparable proportion of the overall field area in the two strains, but overlapping annuli establish a finer meshwork of co‐fasciculating processes in the B6 strain. These results would suggest that the architecture of the dendritic network, rather than the overall size of the dendritic field, is dependent on the density of starburst amacrine cells. J. Comp. Neurol. 505:539–546, 2007.

Multiple Genes on Chromosome 7 Regulate Dopaminergic Amacrine Cell Number in the Mouse Retina

PURPOSE The size of neuronal populations is modulated by gene variants that influence cell production and survival, in turn influencing neuronal connectivity, function, and disease risk. The size of the dopaminergic amacrine (DA) cell population is a highly heritable trait exhibiting sixfold variation among inbred strains of mice and is used here to identify genes that modulate the number of DA cells. METHODS The entire population was counted in retinal wholemounts from 37 genetically defined lines of mice, including six standard inbred strains, 25 recombinant inbred strains (AXB/BXA), reciprocal F1 hybrids, a chromosome (Chr) 7 consomic line, and three additional genetically modified lines. RESULTS Much of this variation was mapped to a broad locus on Chr 7 (Dopaminergic amacrine cell number control, Chr 7 [Dacnc7]). The Dacnc7 locus is flanked by two candidate genes known to modulate the number of other types of retinal neuron-the proapoptotic gene, Bax, and tyrosinase. The Tyr mutation was shown to modulate DA cell number modestly, though in the direction opposite that predicted. In contrast, Bax deficiency increased the population fourfold. Bax expression was significantly greater in the A/J than in the C57BL/6J strain, an effect that may be attributed to an SNP in a p53 consensus binding site known to modulate transcription. Finally, we note a strong candidate situated at the peak of the Dacnc7 locus, Lrrk1, a Parkinsons disease gene exhibiting missense mutations segregating within the AXB/BXA cross. CONCLUSIONS Multiple polymorphic genes on Chr 7 modulate the size of the population of DA cells.

Genetic modulation of horizontal cell number in the mouse retina

Neuronal populations display conspicuous variability in their size among individuals, but the genetic sources of this variation are largely undefined. We demonstrate a large and highly heritable variation in neuron number within the mouse retina, affecting a critical population of interneurons, the horizontal cells. Variation in the size of this population maps to the distal end of chromosome (Chr) 13, a region homologous to human Chr 5q11.1–11.2. This region contains two genes known to modulate retinal cell number. Using conditional knock-out mice, we demonstrate that one of these genes, the LIM homeodomain gene Islet-1 (Isl1), plays a role in regulating horizontal cell number. Genetic differences in Isl1 expression are high during the period of horizontal cell production, and cis-regulation of Isl1 expression within the retina is demonstrated directly. We identify a single nucleotide polymorphism in the 5′ UTR of Isl1 that creates an E-box sequence as a candidate causal variant contributing to this variation in horizontal cell number.

Independent Genomic Control of Neuronal Number across Retinal Cell Types

The sizes of different neuronal populations within the CNS are precisely controlled, but whether neuronal number is coordinated between cell types is unknown. We examined the covariance structure of 12 different retinal cell types across 30 genetically distinct lines of mice, finding minimal covariation when comparing synaptically connected or developmentally related cell types. Variation mapped to one or more genomic loci for each cell type, but rarely were these shared, indicating minimal genetic coregulation of final number. Multiple genes, therefore, participate in the specification of the size of every population of retinal neuron, yet genetic variants work largely independent of one another during development to modulate those numbers, yielding substantial variability in the convergence ratios between pre- and postsynaptic populations. Density-dependent cellular interactions in the outer plexiform layer overcome this variability to ensure the formation of neuronal circuits that maintain constant retinal coverage and complete afferent sampling.

Involvement of OA1, an Intracellular GPCR, and Gαi3, Its Binding Protein, in Melanosomal Biogenesis and Optic Pathway Formation

PURPOSE Ocular albinism type 1 (OA1) is characterized by abnormalities in retinal pigment epithelium (RPE) melanosomes and misrouting of optic axons. The OA1 gene encodes a G-protein-coupled receptor (GPCR) that coimmunoprecipitates with the G alpha i-subunit of heterotrimeric G-proteins from human melanocyte extracts. This study was undertaken to test whether one of the G alpha i proteins, G alpha i3, signals in the same pathway as OA1 to regulate melanosome biogenesis and axonal growth through the optic chiasm. METHODS Adult G alpha i3(-/-) and Oa1(-/-) mice were compared with their respective control mice (129Sv and B6/NCrl) to study the effects of the loss of G alpha i3 or Oa1 function. Light and electron microscopy were used to analyze the morphology of the retina and the size and density of RPE melanosomes, electroretinograms to study retinal function, and retrograde labeling to investigate the size of the uncrossed optic pathway. RESULTS Although G alpha i3(-/-) and Oa1(-/-) photoreceptors were comparable to those of the corresponding control retinas, the density of their RPE melanosomes was significantly lower than in control RPEs. In addition, the RPE cells of G alpha i3(-/-) and Oa1(-/-) mice showed abnormal melanosomes that were far larger than the largest 129Sv and B6/NCrl melanosomes, respectively. Although G alpha i3(-/-) and Oa1(-/-) mice had normal results on electroretinography, retrograde labeling showed a significant reduction from control in the size of their ipsilateral retinofugal projections. CONCLUSIONS These results indicate that G alpha i3, like Oa1, plays an important role in melanosome biogenesis. Furthermore, they suggest a common Oa1-G alpha i3 signaling pathway that ultimately affects axonal growth through the optic chiasm.

A QTL on Chromosome 10 Modulates Cone Photoreceptor Number in the Mouse Retina

PURPOSE This investigation examines the genetic sources of marked variation in cone photoreceptor number among inbred lines of mice, identifying candidate genes that may control the proliferation, differentiation, or survival of this neuronal population. METHODS Cone photoreceptor populations were counted in C57BL/6J (B6/J) and A/J strains, and 26 recombinant inbred (RI) strains derived from them. Eyes from RI strains were also collected for microarray analysis. Quantitative trait locus (QTL) analysis was carried out by simple and composite interval mapping and validated using a consomic line. Candidate genes were evaluated based on genetic variance between the parental strains and analysis of gene expression. Expression data, deposited in GeneNetwork (www.GeneNetwork.org), were used to generate a coexpression network of established cone photoreceptor genes as a reference standard. RESULTS B6/J has 70% more cone photoreceptors than A/J. A significant QTL was mapped to chromosome 10 (Chr 10) and confirmed using B6.A<10> mice. Of 19 positional candidate genes, one-the myeloblastosis oncogene (Myb)-stood out. Myb has a potentially damaging missense mutation, high retinal expression, and a known role in cell proliferation. The ectonucleotide pyrophosphatase/phosphodiesterase 1 gene (Enpp1) was a second strong candidate, with an expression pattern that covaried with cone photoreceptors and that was differentially expressed between the parental strains. Enpp1 and several other candidate genes covaried with multiple genes within the cone photoreceptor gene network. CONCLUSIONS The mouse retina shows marked variation in cone photoreceptor number, some of which must be controlled by polymorphisms in a gene or genes on Chr 10.

Developmental plasticity of dendritic morphology and the establishment of coverage and connectivity in the outer retina.

Developing retinal neurons differentiate their distinctive dendritic morphologies through cell‐intrinsic instructions and cellular interactions within the local environment. This review examines the contributions of interactions with afferents and with homotypic neighbors upon the dendritic morphogenesis of retinal bipolar cells in four different mouse models that modulate the frequency of these interactions. Comparisons with horizontal cell differentiation are discussed, and differences between the dendritic plasticity within the outer versus inner plexiform layers are highlighted. Finally, the developmental plasticity of the bipolar and horizontal cells is considered in light of thenatural variation in afferent and target cell number, ensuring a uniformity of coverage and connectivity across the retinal surface.

Sox2 Regulates Cholinergic Amacrine Cell Positioning and Dendritic Stratification in the Retina

The retina contains two populations of cholinergic amacrine cells, one positioned in the ganglion cell layer (GCL) and the other in the inner nuclear layer (INL), that together comprise ∼1/2 of a percent of all retinal neurons. The present study examined the genetic control of cholinergic amacrine cell number and distribution between these two layers. The total number of cholinergic amacrine cells was quantified in the C57BL/6J and A/J inbred mouse strains, and in 25 recombinant inbred strains derived from them, and variations in their number and ratio (GCL/INL) across these strains were mapped to genomic loci. The total cholinergic amacrine cell number was found to vary across the strains, from 27,000 to 40,000 cells, despite little variation within individual strains. The number of cells was always lower within the GCL relative to the INL, and the sizes of the two populations were strongly correlated, yet there was variation in their ratio between the strains. Approximately 1/3 of that variation in cell ratio was mapped to a locus on chromosome 3, where Sex determining region Y box 2 (Sox2) was identified as a candidate gene due to the presence of a 6-nucleotide insertion in the protein-coding sequence in C57BL/6J and because of robust and selective expression in cholinergic amacrine cells. Conditionally deleting Sox2 from the population of nascent cholinergic amacrine cells perturbed the normal ratio of cells situated in the GCL versus the INL and induced a bistratifying morphology, with dendrites distributed to both ON and OFF strata within the inner plexiform layer.

Genomic Control of Retinal Cell Number: Challenges, Protocol, and Results

This chapter considers some of the challenges in obtaining accurate and consistent estimates of neuronal population size in the mouse retina, in order to identify the genetic control of cell number through QTL mapping and candidate gene analysis. We first discuss a variety of best practices for analyzing large numbers of recombinant inbred strains of mice over the course of a year in order to amass a satisfactory dataset for QTL mapping. We then consider the relative merits of using average cell density versus estimated total cell number as the target trait to be assessed, and why estimates of heritability may differ for these two traits when studying the retina in whole-mount preparations. Using our dataset on cell number for 12 different retinal cell types across the AXB/BXA recombinant inbred strain set as an example, we briefly review the QTL identified and their relationship to one another. Finally, we discuss our strategies for parsing QTL in order to identify prospective candidate genes, and how those candidates may in turn be dissected to identify causal regulatory or coding variants. By identifying the genetic determinants of nerve cell number in this fashion, we can then explore their roles in modulating developmental processes that underlie the formation of the retinal architecture.