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First published online 11 September 2008
doi: 10.1242/dev.024612

Development 135, 3379-3388 (2008)
Published by The Company of Biologists 2008
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Rewiring the retinal ganglion cell gene regulatory network: Neurod1 promotes retinal ganglion cell fate in the absence of Math5

Chai-An Mao1, Steven W. Wang2, Ping Pan1 and William H. Klein1,3,*

1 Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
2 Department of Ophthalmology and Visual Science, The University of Texas Houston Medical School, Houston, TX 77030, USA.
3 Training Program in Genes and Development, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA.


Figure 1
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  		Fig. 1. Replacing endogenous Math5 with Neurod1 or Math3. (A) Sequence relationships among bHLH domains of representative proneural bHLH genes [method described by Ledent et al. (Ledent et al., 2002Go)]. Mash1 was chosen as the outgroup. Branch lengths are proportional to the distance between the sequences. (B) Genome structure for Math5, the targeting construct and the predicted structure of the targeted Neurod1 and Math3 knock-in alleles. The single Math5 exon is depicted as a black box. The black bars underneath indicate the DNA fragments amplified from genomic DNA for the targeting construct. Red boxes depict FRT recombination sites. Blue boxes indicate SV40-pA sequence. Black arrows indicated the PCR primers used to amplify Math5 and Math5Math3-KI for qRT-PCR analysis. The primer sequences are described in the Materials and methods. (C) Representative Southern blot analysis using the 5' probe to distinguish Math5 wild-type and Math5Neurod1-KI alleles from genomic DNA of targeted ES cells. O indicates a targeted ES cell. (D) Representative Southern blot analysis using the Southern probe depicted in B to distinguish Math5 wild-type, Math5Neurod1-KI and Math5lacZ-KI alleles from tail genomic DNA of littermates resulting from a Math5Neurod1-KI/lacZ-KI x Math5lacZ-KI/+ cross. (E-H) Misexpression of Neurod1 and Math3 from the Math5 locus. Retinal sections from E12.5 wild-type (E,G) and Math5Neurod1-KI/Math3-KI (F,H) embryos were immunostained with anti-Neurod1 antibody (E,F) or labeled with a Math3 antisense probe by in situ hybridization (G,H). The images in G and H have been enhanced using Photoshop. (I) qRT-PCR analysis of Math5 and Math5Math3-KI alleles. Gene expression levels were normalized to the expression of endogenous GAPDH transcripts. Scale bar: 100 µm.

 

Figure 2
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  		Fig. 2. Neurod1 and Math3 partially restore the optic nerve in Math5-null mice. (A) Dissected eyes from P30 mice. From left to right: Math5lacZ-KI/+, Math5Neurod1-KI/lacZ-KI and Math5lacZ-KI/lacZ-KI. (A1,A2) Cross-sections of the optic nerves from Math5lacZ-KI/+ and Math5Neurod1-KI/lacZ-KI dissected eyes shown in A were stained with Cresyl Fast Violet. (B) Histological sections of P30 eyes from wild-type and Math5Neurod1-KI/Math3-KI littermates. Arrowheads indicate the optic nerves. The double arrowhead indicates a rosette structure sometimes seen in Math5Neurod1-KI/Math3-KI retinas (inset). (B1,B2) Pou4f2 expression in wild-type and Math5Neurod1-KI/Math3-KI retinal sections. Representative lateral regions were used for comparison. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars: 50 µm in A1; 200 µm in B.

 

Figure 3
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  		Fig. 3. Neurod1 partially restores RGC axons and RGC subtypes in the absence of Math5. (A-D) Immunostaining of flat-mounted retinas from P30 mice with anti-NFL antibody to reveal RGC axons. Genotypes are indicated on the lower left of each panel. Insets in C and D highlight the misoriented axons. OD, optic disk. (E,F) Representative images of immunostaining of flat-mount retinas from P30 mice using anti-melanopsin antibody to reveal the mosaic distribution pattern of melanopsin-positive RGCs. The insets in E and F show representative dendritic arborization of melanopsin-positive RGCs in layer 1 of the IPL (arrowheads). Nuclei of the three nuclear layers are stained with DAPI (blue). Scale bar: 50 µm. (G,H) Immunostaining of flat-mount retinas with anti-SMI32 antibody to show the mosaic distribution of the SMI32-positive RGC subtype.

 

Figure 4
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  		Fig. 4. Neurod1 activates early markers of RGC differentiation in the absence of Math5. (A-D3) Immunostaining of retinas from E13.5 embryos with anti-Pou4f2/Brn3b (A-D), anti-Isl1 (A1-D1), merged Pou4f2-Isl1 images (A2-D2) and anti-NF160 (A3-D3). Insets show higher magnification of indicated areas. (A-A3) Math5lacZ-KI/+, (B-B3) Math5lacZ-KI/lacZ-KI, (C-C3) Math5Neurod1-KI/Math3-KI and (D-D3) Math5Neurod1-KI/lacZ-KI. Scale bar: 100 µm.

 

Figure 5
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  		Fig. 5. Neurod1 is more effective than Math3 in activating Eomes in the absence of Math5. (A1-D3) Retinas from E14.5 embryos were immunostained with anti-Eomes (A1-D1) or anti-Pou4f2/Brn3b (A2-D2) antibodies. Merged images are shown in A3-D3. (A1-A3) Wild type, (B1-B3) Math5Neurod1-KI/Math3-KI, (C1-C3) Math5Neurodi-KI/lacZ-KI and (D1-D3) Math5Math3-KI/lacZ-KI.

 

Figure 6
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  		Fig. 6. Neurod1 and Math3 activate the RGC gene regulatory network in the absence of Math5. Math5Neurod1-KI/Math3-KI retinas from E14.5 (or E13.5 for G1,G2) embryos were analyzed by in situ hybridization (purple) or immunostaining (green). (A1-H1) Wild-type retinas. (A2-H2) Math5Neurod1-KI/Math3-KI retinas. In situ hybridization probes and antibodies for immunostaining representing genes downstream of Math5 are indicated in the lower left-hand corner in A1-H1.

 

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