PMC

Representative optical sections of phosphohistone-3 (PH3) labeled retinas at E16.5 and P0. Green: PH3. Red: PI. Panels D, E, and F are enlarged from the boxed area of panels A, B, and C and rotated so that the ganglion cell layers are at the bottom. All PH3-positive cells are detected at the ventricular margin at the mitotic zone. At E16.5, both Math5- and Math5/Brn3b-deficient mice have higher numbers of PH3 positive cells than that of a wildtype control. Panels J, K, and L are enlarged from the boxed area of panels G, H, and I. At P0, a Math5-deficient retina has fewer PH3-positive cells than a wildtype control. A Math5/Brn3b-deficient retina has a further reduction in PH3-positive cells.

Projections of optical sections labeled with glutamine synthetase (GS) and Sox9 revealing distribution of Müller cells. Panels D, E, and F are enlarged from the boxed area of panels A, B, and C. A and D, Sox9 labels the nuclei of Müller cells, as indicated by positive GS signals. Sox9-positive nuclei are nicely aligned in the middle of the INL in the wildtype retina. Sox9-positive cells are not found in other locations. B and E, the distribution of Müller cells is disturbed. Sox9-positive cells are scattered throughout the outer nuclear layer and ganglion cell layer (arrows). C and F, the number of Müller cells is reduced in the Math5/Brn3b-deficient retina. Müller cells can still be found in the ONL and GCL (arrows).

Representative optical sections of TUNEL labeled retinas at E16.5 and P0. Green: TUNEL signal. Red: PI. Panels D, E, and F are enlarged from the boxed area of panels A, B, and C and rotated so that the putative ganglion cell layers are at the bottom. At E16.5, most of the cell death appears in the putative ganglion cell layer. The Math5-deficient retina has more apparent apoptotic cells than the wildtype retina. The Math5/Brn3b-deficient retina has even more apoptotic cells than the Math5-deficient retina. Panels J, K, and L are enlarged from the boxed area of panels G, H, and I. The overall trend of cell death among the three genotypes persisted at P0. In the Math5/Brn3b-deficient retina, cell death in the newly-separated ganglion cell layer appears to be intensified.

The non-linear relationship between percentage of RGC loss and percentage of remaining retinal cell number. The Brn3b-deficient retina has ~80% RGC loss. Reduction of other retinal cell types may be too trivial to be detected. A minor, but significant, reduction of INL cells can be detected in the Math5-deficient retina that 95% of RGCs being depleted. Total retinal cell loss becomes obvious in the 98% RGC depleted, brn3b-dta, retina. This data point is a rough estimation according to the result of . A slight more RGC depletion (>99%) results in much more dramatic total retinal cell loss (~55%). The highest amount of retinal cell loss observed to date.

Projections of optical sections of flat-mounted adult retinas. Green: Neurofilament-light chain (NF-L). Red: Propidium Iodide (PI). Panels E, F, G, and H are enlarged from the boxed area of panels A, B, C, and D and rotated so that the optic discs or putative optic discs are at the bottom. A and E, Wildtype NF-L positive axons are abundant and well bundled. B and F, a Brn3b-deficient retina has significant RGC loss, as indicated by the NF-L positive axons. C and G, a Math5-deficient retina has further RGC loss. Many of the RGC axons appear to be disoriented. D and H, only a few NF-L positive axons can be detected. These axons appear to be disoriented and do not enter the optic disc. Many of them traverse the retinal area more than once by turning at the retinal periphery. The putative optic disc, indicated by an arrow in D, contains only capillaries. Other capillaries are indicated by arrowheads.

Electroretinograms (ERGs) of brn3b^−/−, math5^−/−, and brn3b^−/−/math5^−/− mice, compared to ERGs from age-matched wildtype mice. A) The a-waves and b-waves of scotopic (dark-adapted: left) and photopic (light-adapted: right) full field ERGs of Brn3b-deficient mice were hardly different from those of the wildtype mice, except for very weak stimuli under dark-adapted conditions where the positive scotopic threshold response (pSTR), thought to be generated by retinal ganglion cells, was not detectable in the Brn3b-deficient mice. The insets show a-waves on an expanded time scale in response to a high energy flash. B) Both a-waves and b-waves were reduced in the dark-adapted and light-adapted ERGs of the Math5-deficient mice. The change in dark-adapted a-waves was not significant for the entire group. C) In the Math5/Brn3b-deficient mice, a-waves and b-waves of both light-adapted and dark adapted ERGs were greatly reduced. Under dark-adapted conditions, the a-wave was reduced at times in the leading edge when the response was dominated by rod photoreceptor activity, indicating effects on the rods. The photopic a-wave for both Math5-deficient and Math5/Brn3b-deficient mice was affected at times when Off-pathway neurons were contributing to the response.

Cross sections showing different cell populations in the inner nuclear layer (INL) in the adult retinas. A, B, and C, Pax6 and ChAT labeling reveals an amacrine cell population in the INL of three different genotypes. There is a significant reduction of Pax6-positive cells in the Math5-deficient retina. These cells are further reduced in number in the Math5/Brn3b-deficient retina. However, the number of ChAT-positive cells is slightly increased. These cells are unevenly distributed in the Math5/Brn3b-deficient retina. D, E, and F, PKCα labeling reveals a rod bipolar cell population. There is no significant difference of rod bipolar cells between Math5-deficient and wildtype retinas. In the double null retina, not only is the rod bipolar cell number significantly reduced, they also form small aggregates. G, H, and I, Calbindin and recoverin labeling show horizontal cells and cone bipolar cells, respectively. H & I were collected from the central retina. H′ & I′ were collected from the peripheral retina. Uneven planar distribution of both cell types can be seen in both Math5- and Math5/Brn3b-deficient retinas.

Projections of optical sections of flat-mounted and cross sectioned retinas at P0. Green: NF-L. Red: PI. Panels D, E, and F are enlarged from the boxed area of panels A, B, and C and rotated so that the optic discs or putative optics disc are at the bottom. Panels J, K, and L are enlarged from the boxed area of panels G, H, and I. A and D, well organized wildtype RGC axons can be seen at this stage. B and E, the number of NF-L-positive axons is drastically reduced. However, a significant amount of unorganized ones can be readily detected. C and F, scattered NF-L-positive cell bodies with short neurites can only be detected with high magnification. G and J, a cross sectioned wildtype retina showing newly-separated RGC layer. RGCs have distinct polygonal shapes of NF-L labeling, as indicated by the arrows. Amacrine cells above the emerging inner plexiform layer are also NF-L positive at this stage. The NF-L signal forms a thin circle, suggesting a unique cytoplasmic shape of amacrine cells. H and K, a Math5-deficient retina does not appear to be thinner than a wildtype control at this stage. However, besides a few possible RGCs (arrows), most of the cells in the INL have the shape of amacrine cells. I and L, the Math5/Brn3b-deficient retina is obviously thinner than the wildtype control. Cells exhibiting RGC shape cannot be found. Cell number in the RGC layer and NF-L positive cell number above the inner plexiform layer are severely reduced.