Additional file 11: Figure S6. of Anisotropic Müller glial scaffolding supports a multiplex lattice mosaic of photoreceptors in zebrafish retina

Expression of cdh2 transcripts in differentiating photoreceptors (A-A”’) In situ hybridization for cdh2 transcripts (A, white or A”, A”’, magenta) in a retinal cross-section from the Müller glial reporter line, Tg(gfap:EGFP) (A’-A”’, green). (PDF 2467 kb

Additional file 5: Figure S2. of Anisotropic MĂźller glial scaffolding supports a multiplex lattice mosaic of photoreceptors in zebrafish retina

Immature UV cones have rounded apical profiles in the pre-column area. (PDF 939 kb

Additional file 9: Movie S3. of Anisotropic Müller glial scaffolding supports a multiplex lattice mosaic of photoreceptors in zebrafish retina

Müller glial apical processes provide scaffolding for differentiating photoreceptor cells. 3D–reconstruction (maximum intensity z-projection) of live multiphoton confocal imaging from a double transgenic ruby zebrafish, with reporters for Müller glia (gfap:EGFP in green) and Red cones (trß2:tdTomato in red). Müller glial processes extend laterally at the level of OLM to surround profiles of individual Red cones and other photoreceptors. (See also Fig. 4A–D.) (AVI 4930 kb

Additional file 8: Figure S5 of Anisotropic MĂźller glial scaffolding supports a multiplex lattice mosaic of photoreceptors in zebrafish retina

MĂźller glial apical processes are preferentially distributed into parallel, inter-column bands. (PDF 1085 kb

Additional file 10: Movie S4. of Anisotropic Müller glial scaffolding supports a multiplex lattice mosaic of photoreceptors in zebrafish retina

Red cones in the pre-column zone (magenta dots) and in the mature mosaic (white dots) are surrounded by Müller glial scaffolding. Higher magnification of a portion of the field shown in Fig. 4A–D and Additional file 9: Movie S3: Müller glia (green) and Red cones (red). Weak GFP signals of immature Müller glia first appear in the proliferative zone (toward the right). The intensity of the GFP signal increases in differentiating Müller glia and their processes surround photoreceptors, including differentiating Red cones (magenta dots) at the level of the OLM. Emergence of the hexagonal distribution of Red cones (white dots) is accompanied by morphological maturation of Müller glia. (AVI 4600 kb

Additional file 4: Movie S2. of Anisotropic Müller glial scaffolding supports a multiplex lattice mosaic of photoreceptors in zebrafish retina

Live imaging of the photoreceptor mosaic emerging at the retinal margin in rapidly growing juvenile zebrafish. Confocal z-stack series of live multiphoton confocal imaging at the dorsal retinal margin in a double transgenic ruby zebrafish; Tg(trß2:tdTomato) in red and Tg(crx:mCFP) in cyan. The crx promoter is expressed in all cone and rod photoreceptors; the mCFP reporter localizes to the plasma membrane. The tdTomato+ proliferative retinal progenitors are randomly distributed in the most peripheral region of the germinal zone (asterisks). The first cone column (arrow and inset) is composed of tdTomato+ Red cones alternately separated by mCFP-labeled profiles of one or three immature cones (white dots), as predicted by the organization of cone types in a column in the mature mosaic. Mature cones develop long apical projections, including conical-shaped outer segments that are strongly labeled by the crx:mCFP reporter. (See also Fig. 2D.) (AVI 1851 kb

Additional file 2: Figure S1. of Anisotropic MĂźller glial scaffolding supports a multiplex lattice mosaic of photoreceptors in zebrafish retina

Maximum intensity z-projection and lateral slice view of retinal margin illustrating shape and position of mitotic figures. (PDF 1495 kb

The rod phenotype in <i>tbx2b</i> mutant embryos is partially compensated in adults.

<p>Rods and cones were counted in wild-type (wt) and <i>tbx2b</i> mutant (mut) embryos at 3 days post-fertilization (dpf) and in adult retinas and plotted as planimetric density (#/10³ µm²). Photoreceptor profiles were identified by ZO-1 immunostaining and the rod opsin transgenic reporter was used to distinguish rods. Means ± standard deviation are plotted for n = 11 samples from 3 retinas (wt embryo), n = 8 samples from 4 retinas (mut embryo), n = 12 samples from 3 retinas (wt and mutant adult). *** p<0.001; ** p<0.01.</p

Infrequent three-fold coordination of mutant retina cone photoreceptors suggests strongly directional interaction.

<p>(See Methods section for details of image processing.) A) To identify rods, we superimposed on the ZO-1 label (white) the signal in the rod GFP reporter channel (green) from a single z-slice per cell chosen to coincide with the level of the OLM. B) Same retina as panel A. Adjacent cone photoreceptors in ventral-temporal retina of adult <i>tbx2b</i> mutant. Red lines join pairs of cones that are classified as adjacent at both low and high threshold (see Methods), while yellow lines indicate pairs that were identified as adjacent only with the less stringent threshold. (C) Histogram showing the fraction of cone photoreceptors with the specified number of identified neighbors in a sample of ventral-temporal retina. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085325#pone-0085325-t001" target="_blank">Table 1</a> for additional data.) D) Segmented image from the region outlined by the dashed box in panels A and B. Pixels between adjacent cells that were filled in by the morphological closing procedure are colored either red (high threshold) or blue (low threshold); at this magnification, 1 pixel corresponds to 0.1 µm. The two cells flanking each red region were classified as adjacent at the high threshold, but the blue region is too long (along the axis joining the centers of the two cells) and too narrow (along the orthogonal axis) to meet the adjacency criteria at the high threshold. The blue region meets the adjacency criteria at the low threshold. Stars indicate cone cells that are three-fold coordinated, <i>i.e.</i> have 3 adjacent cells. Scale bars are 20 µm for A and B, and 4 µm for D.</p

Patterning the Cone Mosaic Array in Zebrafish Retina Requires Specification of Ultraviolet-Sensitive Cones

<div><p>Cone photoreceptors in teleost fish are organized in precise, crystalline arrays in the epithelial plane of the retina. In zebrafish, four distinct morphological/spectral cone types occupy specific, invariant positions within a regular lattice. The cone lattice is aligned orthogonal and parallel to circumference of the retinal hemisphere: it emerges as cones generated in a germinal zone at the retinal periphery are incorporated as single-cell columns into the cone lattice. Genetic disruption of the transcription factor Tbx2b eliminates most of the cone subtype maximally sensitive to ultraviolet (UV) wavelengths and also perturbs the long-range organization of the cone lattice. In the <i>tbx2b</i> mutant, the other three cone types (red, green, and blue cones) are specified in the correct proportion, differentiate normally, and acquire normal, planar polarized adhesive interactions mediated by Crumbs 2a and Crumbs 2b. Quantitative image analysis of cell adjacency revealed that the cones in the <i>tbx2b</i> mutant primarily have two nearest neighbors and align in single-cell-wide column fragments that are separated by rod photoreceptors. Some UV cones differentiate at the dorsal retinal margin in the <i>tbx2b</i> mutant, although they are severely dysmorphic and are eventually eliminated. Incorporating loss of UV cones during formation of cone columns at the margin into our previously published mathematical model of zebrafish cone mosaic formation (which uses bidirectional interactions between planar cell polarity proteins and anisotropic mechanical stresses in the plane of the retinal epithelium to generate regular columns of cones parallel to the margin) reproduces many features of the pattern disruptions seen in the <i>tbx2b</i> mutant.</p></div