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Pax6 is required for establishing naso-temporal and dorsal characteristics of the optic vesicle

Nicole Bäumer, Till Marquardt^*,, Anastassia Stoykova, Ruth Ashery-Padan, Kamal Chowdhury and Peter Gruss

Max-Planck-Institute of Biophysical Chemistry, Department of Molecular Cell Biology, Am Fassberg 11, D-37077 Göttingen, Germany
^* Present address: The Salk Institute for Biological Studies, Gene Expression Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
Present address: Sackler Faculty of Medicine, Department of Human Genetics and Molecular Medicine, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel
These authors contributed equally to this work

View larger version (35K): [in a new window]   Fig. 1. The -enhancer is required for establishing a distalhigh-proximallow gradient of Pax6 activity in the neuroretina (NR). (A, top) Wild-type Pax6 genomic locus. Positions of the 1.8 kb fragment containing the -enhancer and the 0.4 kb P0 promoter used in the transgene constructs shown in B and Fig. 3I are indicated. (A, bottom) In the Pax6lacZ allele lacZ/neo was inserted in frame with the translation start, replacing exon 4 to exon 6 (St-Onge et al., 1997), thereby eliminating the -enhancer. (B) Transgene driving gfp (and Cre) expression under control of -enhancer via P0 (see Marquardt et al., 2001). (C) Immunohistochemical detection of Pax6 in an E15.5 Pax6lacZ/+ eye reveals normal distalhigh-proximallow gradient of Pax6 in the NR (arrowhead indicates distal high end of gradient). (D) In the same eye, ß-gal is uniformly distributed throughout the NR (open arrowhead). Relatively higher ß-gal levels are still found in corneal (ce) and lens epithelium (le), matching endogenous relative Pax6 levels in these tissues (see C). (E) gfp expression in transgenic mice carrying the construct shown in B. At E15.5, drives a sharp distoproximal gradient of gfp expression in the NR, matching the high end of the Pax6 gradient (arrowheads). onh, optic nerve head.

View larger version (45K): [in a new window]   Fig. 2. Pax6 activity is required for initiating and upregulating, but not for maintaining -enhancer-mediated expression. (A-C) Whole-mount views of E11.5 transgenic embryos in which the -enhancer drives gfp expression. (A) Normal gfp expression in -Cre-gfp; Pax6+/+ (wild-type) embryo. Inset: 10 µm horizontal section revealing gfp expression in distal NR. (B) Reduced gfp levels in -Cre-gfp; Pax6flox/flox where Pax6 is specifically eliminated from the distal (gfp+Cre+) NR. Note that the low fluorescence levels in B and inset, as well as in C were digitally enhanced to reveal expression. (C) No gfp expression can be detected in -Cre-gfp; Pax6–/– (Pax6-null mutant) embryos. Arrowhead indicates eye rudiment in mutant embryo.

View larger version (81K): [in a new window]   Fig. 3. The -enhancer directs expression in a subset of Pax6 positive retinal ganglion cells (RGCs) in the distal ventronasal and ventrotemporal retina. (A) Whole-mount view of E11.5 X-gal stained embryo carrying the -tau-lacZ transgene (see I) reveals specific tau-ß-gal in the nasal and temporal retina (see Fig. 2A). (B) Horizontal section through E12.5 -tau-lacZ eye (counterstained with neutral red) reveals tau-ß-gal expression in the distal nasal and temporal NR (see Fig. 2A, inset). (C) Horizontal section of P20 -tau-lacZ eye reveals tau-ß-gal mainly in the distal ganglion cell layer (gcl), as well as tau-ß-gal+ axons in the nerve fiber layer (nfl, see F). (D-F) Immunohistochemical detection of tau-ß-gal (green) and Pax6 (red) in P20 -tau-lacZ eye. (D) In the distal nasal NR, Pax6 is found in the nuclei of inl and gcl, while tau-ß-gal labels the cytoplasm (arrows) and axons (see F) of RGCs, as well as dendritic processes of RGCs and amacrine cells (AC) in the inner plexiform layer (ipl). (E) In the dorsal NR, tau-ß-gal is only detected in the ipl, representing long dendritic processes of nasal/temporal tau-ß- gal+ ACs and RGCs. (F) Nasal tau-ß-gal+ RGC fibers can be detected passing through the optic nerve head (onh). Note that proximal RGCs are tau-ß-gal–, but Pax6+. (G) Plane of sections depicted in D,E. (H) Plane of section shown in F. (I) -tau-lacZ transgene: drives expression of tau-lacZ from the P0 promoter. inl, inner nuclear layer; D, dorsal; V, ventral; T, temporal; N, nasal.

View larger version (91K): [in a new window]   Fig. 4. The -enhancer directs expression in RGCs that project to two concise domains in the lateral geniculate nucleus (LGN) and superior colliculus (SC). (A-C,E-F): Whole-mount views of P0 (A) and P20 (B-C,E-F) -tau-lacZ brains stained for tau-ß-gal activity. (A) At P0, tau-ß-gal+ axons can be followed from the eyes through the optic nerve (on), via the optic chiasm (oc) and optic tract (ot) up to the LGN (latter not shown in A). Ventral view of brain to reveal on, oc and ot. (B) At P20, tau-ß-gal activity allows tracing of entire extent of the ot, up to the superior colliculus (SC; lateral view of brain; overlying brain tissue was removed). (C) Ventral view of chiasm showing tau-ß-gal+ fibers crossing to the ot (anterior towards the top; posterior towards the bottom). (D) Schematic view of the topographic map drawn by tau-ß-gal+ axons in the -tau-lacZ line. tau-ß-gal+ axons project the distal nasal (4) and temporal (1) NR to the posterior (4) and anterior (1) LGN and superior colliculus, respectively. Proximal RGC axons (2,3) project to the remaining portions of LGN and superior colliculus. (E) Dorsal view on P20 -tau-lacZ brain showing tau-ß-gal+ axons terminating in two sickle shaped domains in the superior colliculus and LGN. Note that central portion of superior colliculus is devoid of staining, as is the inferior colliculus (IC) and surrounding tissue. (F) Close-up lateral view of the LGN of a P20 brain -tau-lacZ brain revealing tau-ß-gal+ axons terminating mainly in two domains in the ventral and dorsal LGN, respectively. (G) Sagittal section through the superior colliculus revealing tau-ß-gal+ axons terminating in the anterior and posterior regions, while omitting the central region (axons projecting to the posterior superior colliculus can be detected). (H) Transversal section through the -tau-lacZ diencephalon revealing tau-ß-gal+ axons terminating in the ventral and dorsal LGN. The additional focus of termination presumably corresponds to the binocular segment (bs). Lateral of the LGN, tau-ß-gal+ axons can be seen traversing the optic tract.

View larger version (79K): [in a new window]   Fig. 5. Retinal Pax6 expression is regulated in two complementary topographic domains. Staining for tau-ß-gal/ß-gal activity of P20 -tau-lacZ (A,C,D,G,I) and Pax6lacZ/+ (B,E,F,H,J) eyes and brains. (A) Horizontal section of a -tau-lacZ eye reveals steep distal-to-proximal gradient of expression in the gcl, while Pax6lacZ/+ eyes display a precisely complementary pattern of ß-gal activity (B). (C-F) Higher magnifications of the distal (C,E) and proximal (D,F) NR of -tau-lacZ and Pax6lacZ/+ eyes shown in A and B, respectively. The -tau-lacZ NR displays strong expression in distal (C), but not in proximal (D) RGCs, while the Pax6lacZ/+ shows strong expression in proximal (F), but no expression in distal (E) RGCs. (G-H) Dorsal view of the superior colliculus and ot of -tau-lacZ (G) and Pax6lacZ/+ (H) brains. (G) In the -tau-lacZ brain, tau-ß-gal+ axons omit the central region of the superior colliculus (dotted circle), which is innervated by ß-gal+ axons in the Pax6lacZ/+ brain (H, dotted circle), while the sickle-shaped domains stained in -tau-lacZ are devoid of ß-gal+ axons (compare G with H). (I,J) Sagittal sections through the of -tau-lacZ (I) and Pax6lacZ/+ (J) mesencephalon reveals complementary innervation of the superior colliculus by tau-ß-gal+ and ß-gal+ axons. Arrowheads in I indicate single stained axons. -tlz, -tau-lacZ; cb, cerebellum; l, lens; onl, outer nuclear layer; pc, posterior commissure.

View larger version (82K): [in a new window]   Fig. 6. Pax6 is required for initiating and maintaining the expression of the nasal and temporal retinal determinants BF-1 and BF-2. Radioactive situ hybridization of 6 µm horizontal sections. (A) In wild type at E12.5, BF-1 is expressed in the nasal (ns) NR and in the olfactory epithelium (oe), as well as the telencephalon (see C), while BF-2 is detected in the temporal (tm) NR (B). (C) In Pax6–/– embryos, no BF-1 can be detected in the optic vesicle (open arrowheads), but normal levels in the telencephalon (tel) and low levels in the neuroepithelium around the chiasm (asterisk). (D) Likewise, no BF-2 expression is detectable in the Pax6–/– optic vesicle, while normal levels are found in the surrounding mesenchyme (white arrowhead). (E,F) Absence of retinal BF-1 (E) and BF-2 expression (open arrowheads) after conditional Pax6 inactivation in the distal NR of E14.5 -Cre; Pax6flox/flox embryos. Normal levels of BF-1 are still detected in the oe (E), as well as normal BF-2 levels in the dura mater surrounding the optic nerve (F, white arrowhead). Note that in E and F, signal in the lens is due to reflection of lens fibers in dark-field microscopy. (G) In the wild-type embryo, Tbx5 is expressed specifically in the dorsal NR, while Tbx5 fails to be expressed in the Pax6–/– optic vesicle (I). Vax1 whose expression at E12.5 is found in the very ventral NR (H), is expressed throughout the whole Pax6–/– optic vesicle (J). le, lens; nr, neural retina; ov, optic vesicle; rpe, retinal pigmented epithelium; ch, chiasm.

View larger version (35K): [in a new window]   Fig. 7. (A) Schematic representation of the NR (distal is towards the left). The -enhancer () alone drives gradient expression in the distal half of the NR (red), while upon deletion of (Pax6 =Pax6lacZ) the remaining Pax6 elements mediate evenly distributed expression throughout the proximodistal extent of the NR (pink). In the wild type NR, the composite activity of both regulatory systems lead to the full retinal expression pattern of Pax6 (Pax6WT). (B) Nasal (red), temporal (green) and ventral (blue) characteristics are established around E9 in the wild-type optic vesicle (see OVH) and maintained in the optic cup (E12), while dorsal characteristics (yellow) are established slightly later (shown in OVS). In Pax6-null mutants (Pax6–/–) the distinction between nasal and temporal optic vesicle fails to be established, while after Cre/loxP mediated inactivation (-Cre; Pax6flox/flox) at E10.5 nasal and temporal patterning of the optic cup is lost. In addition, in the Pax6–/– optic vesicle ventral characteristics (Vax1, Vax2) expand dorsally (dotted arrows) and, possibly as a consequence, dorsal determinants (Tbx5) are lost. See discussion for details. D, dorsal; le, lens; N, nasal; NR, neuroretina; OVH, horizontally sectioned optic vesicle; OVS, sectioned sagittally optic vesicle; T, temporal; V, ventral.