Multiple roles for Pax2 in the embryonic mouse eye

Abstract

The vertebrate eye anlage grows out of the brain and folds into bilayered optic cups. The eye is patterned along multiple axes, precisely controlled by genetic programs, to delineate neural retina, pigment epithelium, and optic stalk tissues. Pax genes encode developmental regulators of key morphogenetic events, with Pax2 being essential for interpreting inductive signals, including in the eye. PAX2 mutations cause ocular coloboma, when the ventral optic fissure fails to close. Previous studies established that Pax2 is necessary for fissure closure and to maintain the neural retina -- glial optic stalk boundary. Using a Pax2^GFP/+ knock-in allele we discovered that the mutant optic nerve head (ONH) lacks molecular boundaries with the retina and RPE, rendering the ONH larger than normal. This was preceded by ventronasal cup mispatterning, a burst of overproliferation and followed by optic cup apoptosis. Our findings support the hypothesis that ONH cells are tripotential, requiring Pax2 to remain committed to glial fates. This work extends current models of ocular development, contributes to broader understanding of tissue boundary formation and informs the underlying mechanisms of human coloboma.

Keywords: Coloboma; Foxg1; Optic stalk; Pax2; Pax6; RPE; Retina.

Figure 1.. Comparison of Pax2^GFP and endogenous Pax2 ocular expression.

(A) At E8 EGFP fluorescence corresponds to known Pax2 mRNA and protein domains (Nornes et al., 1990; Schwarz et al., 2000). (B) Live Pax2^GFP expression in the E9.5 optic cup, otic vesicle, midbrain-hindbrain boundary and the developing kidneys. (C,C’) Pax2^GFP expression in E11.5 live embryos in the ventral optic cup (arrow in C’). (D-D”) Antibody colabeling of E13.5 ocular sections shows overlap of GFP endogenous Pax2 proteins in the ONH. (E) SLO live image of 6 month old Pax2^GFP/+ eye fundus. Arrow points to a GFP+ retinal astrocyte. pP – presumptive prosencephalon, pM – presumptive mesencephalon, R – rhombencephalon; MHB – midbrain-hindbrain boundary; OV – optic vesicle; OtV – otic vesicle, N - nephros; NR = neural retina; ONH = optic nerve head; OS = optic stalk; Anterior is up in A; to left in B,C,C';nasal is up in D-D". Scalebar = 250μm in A;500 μm in B,C;50 μm in D.

Figure 2.. Brain and eye deformities of Pax2^GFP/GFP embryos.

(A,A’,C,C’) Lateral views of E16.5 live embryos. Pax2^GFP/GFP animals with exencephaly and ocular colobomas (arrow in C,C’). (B,B’,D,D’) The optic fissure remains open as a ventral cleft in Pax2^GFP/GFP mutant eyes (arrow in D), with GFP fluorescence visible (arrow in D’) (n=4/genotype). L – lens, Rostral is left in A,C; distal up in B,D. Scalebar = 500 μm

Figure 3.. Downregulated Pax2 mRNA but total loss of Pax2 protein in Pax2^GFP/GFP eyes.

All panels have horizontal sections, with nasal up. (A,A’,E,E’) Pax2 mRNA expression shown by in situ hybridization, with boxed areas shown at higher mag in A',E'. At E13.5, Pax2 transcripts are normally visible in sections containing both eye and hindbrain (arrow) (A). E13.5 Pax2^GFP/GFP sections contain residual Pax2 mRNA in hindbrain (arrow), but loss of specific signal in the mutant ONH and OS (E, E'). This embryo had non-specific background (e.g., lens), but other embryos analyzed in parallel show near total loss of Pax2 mRNA (Suppl Fig 1). (B, F) Anti-Pax2 labeling of E13.5 Pax2^GFF/+ and Pax2^GFP/GFP sections demonstrate complete loss of Pax2 protein in the mutant ONH and optic stalk. (C,G) EGFP mRNA expression is expanded into retina in Pax2^GFP/GFP eyes. (D,H) Anti-GFP labeling of sections nearby to C,G highlight expanded GFP protein domain in mutants (n=3 biologic replicates/genotype). Panels A,E are stitched composites of 9 image tiles. Scalebar: A =250 μm, A’,B = 50 μm.

Figure 4.. Nasotemporal and optic fissure defects of Pax2^GFP/GFP mutants

All panels show sagittal view of eye. (A,A',F,F') Vsx2-Mitf colabeling of E11.5 sections. In the ventronasal optic, Mitf domain is expanded at the expense of Vsx2/Chx10. (B,F) At E11.5, Foxg1 immunostaining also highlights proportionally smaller nasal side of Pax2^GFP/GFP optic cup. (C,G) In E13.5 Pax2 mutants, optic cup size and the Foxg1 domain better resemble controls, but nasal tip is abnormally positioned, emphasizing failed fissure closure (D,H) E13.5 Anti-laminin labeling of the basement membrane also highlights failed fissure closure of Pax2^GFP/GFP eyes (arrow in H). n = 3 biologic replicates/genotype Scalebar = 50μm

Figure 5.. Abnormal marker expression in the ONH of Pax2^GFP/GFP eyes

All panels oriented with rostral left and nasal up. (A,E) Vsx2 + Mitf colabeling normally highlights the retina-RPE boundary at E13.5, along with typical weak Mitf expression in ONH cells. Pax2 mutants have a truncated Vsx2 domain, with the RPE extending continuously down the optic stalk. (B,F) By contrast, Rax expression is derepressed in the GFP+ domain Pax2 mutant eyes (C,G) E13.5 Hes1+ GFP colabeling normally highlights higher, sustained Hes1 expression in the Pax2-GFP domain. The separation of these domains is no longer discernible in Pax2^GFP/GFP eyes. (D,H) Vax1 mRNA is normally confined to the ONH and OS at E13.5, but abnormally expanded into the optic cup of Pax2^GFP/GFP eyes. n = 3 biologic replicates/genotype Scalebar = 50μm

Figure 6.. Coordinate derepression of Pax6 isoforms in Pax2 mutants

(A) In situ hybridization shows Pax6 mRNA derepression into E13.5 ONH and optic stalk of Pax2^GFP/GFP eyes. (B,F) Immunostaining using C-terminal polyclonal Pax6 antibody that recognizes multiple protein isoforms (FL + ΔPD, see Suppl Fig 3) also shows expanded expression into the optic stalk of Pax2^GFP/GFP eyes (n=3/3 mutants) (C,G) Pax6 paired-domain specific antibody confirms FL Pax6 isoform derepression in Pax2 mutants (n=3/3 mutants) (D,H) All B-G sections were colabeled with anti-GFP to confirm Pax6 isoforms spread into the Pax2^GFP domain. (I) Triplex RT-PCR strategy to compare the relative abundance of Pax6 mRNA isoforms in Pax2^GFP/+ and Pax2^GFP/GFP eyes. The FAM labeled (*) 3 ’ primer resides in sequences common to both isoforms; while unlabeled 5’ primers are specific for the FL or ΔPD isoform. Competitive amplification of each product reflects isoform abundance. (J) Representative capillary electrophoresis profiles of PCR products from Pax2^GFP/+ and Pax2^GFP/GFP cDNAs. The average ratio of FL (206nt) and ΔPD (202nt) cDNA peak areas reflect the relative abundance of each isoform. The 200nt peak (pink) is the ROX-500 size standard. (K) The average ratio of Pax6 FL/ΔPD cDNA peak areas plotted show no significant difference between Pax2^GFP/+ and Pax2^GFP/GFP ONH domains (n = 3 biologic replicates/genotype; p=0.054). Graph displays individual replicate data points, mean and S.E.M. Scalebar = 50μm

Figure 7.. Early proliferation increase followed by apoptosis in the absence of Pax2

(A,B,F,G) E11.5 and E13.5 sections with the ONH/OS from EdU pulse-labeled embryos. (C,D,H,I) Anti PhosphoHistone-H3 (PHH3) M-phase cell labeling at E11.5 and E13.5 sections (E,J) Anti-cPARP labeling at E16.5 to assess apoptosis. (K-M) Quantification of S-Phase, M-Phase or apoptotic cells. K,L) Both EdU pulse labeling and PHH3 expression indicate overproliferation of GFP-labeled population in Pax2^GFP/GFP eyes at E11.5, followed by a significant decline by E13.5 (K,L). At E16.5 the number of cPARP+ cells in Pax2^GFP/GFP mutant sections was dramatically increased, many located in the nascent RGC inner layer (J). Graphs display individual replicate data points, the mean and S.E.M;* = p<0.05; ** = p<0.01; Scalebar in A,E = 50μm; n = ≥2 sections from 3 biologic replicates/genotype.

Figure 8.. Loss of the neural-glial boundary impacts RGC axon guidance.

(A,D) Atoh7 and GFP colabeling of sections at the ONH/OS. In Pax2 mutants, the Atoh7 domain relative to that of expanded GFP suggests a loss of retinal neurogenic territory with the nasal more affected (D, G). However, a proportion of Pax2 mutant GFP+ cells remain Atoh7+. (B,E) TUBB3 + GFP colabeling is consistent with mutant GFP+ cells retaining neuronal characteristics, plus highlights RGC axon misrouting to the subretinal space (arrows). (C,F) netrin1 mRNA ONH domain is more diffuse in Pax2^GFP/GFP retina. (G) Atoh7 and Pax2^GFP domain diagram. (G') The measured relative area of DAPI, Pax2^GFP or Atoh7 image channels mark with white dotted lines for nasal and temporal cup. (H,I) Graphical depiction of increased GFP territory and smaller Atoh7+ territory on both sides of Pax2^GFP/GFP eyes. Individual data points, the mean and S.E.M are shown;* = p<0.05; ** = p<0.01; Scalebar = 50μm; n = 3 biologic replicates per genotype.