Analysis of Pax6 contiguous gene deletions in the mouse, Mus musculus, identifies regions distinct from Pax6 responsible for extreme small-eye and belly-spotting phenotypes

Abstract

In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca(+2)-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.

Figure 1.—

Eye morphology and histology in E15 embryos. (A–C) Gross morphology. (A) Pax6 +/+ with well-developed eye. (B) Pax6^12Neu −/+ with the typical eye phenotype associated with Pax6 null mutations; microphthalmia and triangular shaped pupil. Iris pigmentation is normal. (C) Pax6^11Neu −/+ expressing microphthalmia, reduced iris pigmentation and iris coloboma (arrowhead). (D–F) Eye histology. (D) Pax6 +/+ with well-developed cornea (co), lens (le), retina (ret), and an intact retinal pigmented epithelium (arrowhead). There is a distinct anterior chamber separating the cornea and the anterior surface of the lens. (E) Pax6^12Neu −/+ with a thickened cornea, adhesion of the lens to the cornea resulting in the absence of an anterior chamber, remnants of epithelial cells in the cornea (arrow) and vacuoles in the anterior region of the lens (arrowhead). The retinal pigmented epithelium is normal. (F) Pax6^11Neu −/+ with a thickened cornea, adhesion of the lens to the cornea, and absence of the anterior chamber. Posterior coloboma is present, indicated by an interruption of the retinal pigmented epithelium in the region between the arrows. The orientation of the eye is rotated ∼45° ventrally. (G–I) Head overview documenting the eye orientation. (G) Pax6 +/+ and (H) Pax6^12Neu −/+ in which the medial-lateral eye axes are perpendicular to the dorsal-ventral axis of the head. (I) Pax6^11Neu −/+ in which the medial-lateral axes of both eyes are orientated at a 45° angle to the dorsal-ventral axis of the head. (J–L) Higher magnification of F. (J) The extent of the retinal pigmented epithelium in the ventral region is indicated by the arrows. (K and L) The retinal pigmented epithelium in the dorsal region is indicated by the arrows. Bars in D–K, 200 μm; bar in L, 100 μm.

Figure 2.—

Slit lamp microscopy documenting eye phenotypes in P35 mice. (A) Pax6 +/+. (B) Pax6^3Neu −/+, an intragenic null mutation expressing microphthalmia, a central opacity, lens-corneal adhesion and corneal opacity. (C) Pax6^12Neu −/+ expressing microphthalmia and total lens opacity. The degree of eye abnormality is similar to that observed in Pax6^3Neu heterozygotes. (D) Pax6^11Neu −/+ expressing extreme microphthalmia. All eyes were photographed at 32× magnification.

Figure 3.—

Chr 2 sequences flanking the Pax6^11Neu, Pax6^12Neu, and Pax6^13Neu deletions. The deleted regions are depicted by the black triangles. Pax6^11Neu: The chimeric sequence across the Pax6^11Neu deletion contained in the 5′ end a portion of genomic sequence defined by the BAC RP23-8C14 joined in the 3′ end to a portion of genomic sequence defined by the BAC RP23-431C3. The proximal deletion breakpoint is after the BAC RP23-8C14 position 20,431, and the distal breakpoint is after the BAC RP23-431C3 position 132,356. Pax6^12Neu: The chimeric sequence across the Pax6^12Neu deletion contained in the 5′ end a portion of genomic sequence defined by the BAC RP23-290H11 joined in the 3′ end to a portion of genomic sequence defined by the BAC RP23-35G10. The proximal deletion breakpoint is after the BAC RP23-290H11 position 2388, and the distal breakpoint is after the BAC RP23-35G10 position 5858. Pax6^13Neu: The chimeric sequence across the Pax6^13Neu deletion contained in the 5′ end a portion of genomic sequence defined by the BAC RP23-431C3 joined in the 3′ end to a portion of genomic sequence defined by the BAC RP23-146D23. The proximal deletion breakpoint is after the BAC RP23-431C3 position 78,814, and the distal breakpoint is after the BAC RP23-146D23 position 148,187.

Figure 4.—

Schematic overview of the deleted regions in the Pax6^Sey-Dey, Pax6^Sey-H, Pax6^11Neu, Pax6^12Neu, and the Pax6^13Neu deletions. Pax6 and the proximal genes Wt1 and Rcn1 as well as the distal genes Elp4 and Immp1L are shown with their proximal end position in Mb (Ensembl build 51). The Pax6^Sey-Dey deletion begins most proximal, includes Wt1, Rcn1, Pax6, and Elp4, and is estimated to be 1.2 Mb. The Pax6^Sey-H deletion includes Wt1, Rcn1, Pax6, Elp4, and Immp1L, extends much further distally, and is estimated to be 2.9 Mb. The Pax6^11Neu deletion begins distal to Wt1 and proximal to Rcn1, extends into Elp4, and is 540 kb. Since the Elp4 gene is orientated tail to tail to the Pax6 gene the 3′ end of the Elp4 gene is deleted. The Pax6^12Neu deletion begins distal to Rcn1 and proximal to Pax6, extends furthest distally, and is 6.08 Mb long. The Pax6^13Neu deletion is 238 kb, begins within intron 6-7 of Pax6, extends through Elp4, and ends within the 5′ region of Immp1L. The critical region responsible for the extreme eye phenotype is marked by the broken lines.

Figure 5.—

Pax6 isoforms and Pax6-Immp1L fusion transcript in mutant and wild-type eyes of P1 mice. (A) PCR amplification products of a region of the Pax6 transcript spanning exon 5a. In all samples both a shorter 182-bp band, which represents the amplification of the canonical Pax6 transcript, and a longer 224-bp band, which represents the amplification of the alternatively spliced Pax6(5a) isoform, are present, indicating that alternative splicing was not affected by the deletions. (Lanes 1–3) Pax6^11Neu −/+. (Lanes 4 and 5) Pax6^12Neu −/+. (Lanes 6–8) Pax6 +/+. (Lanes 9 and 10) Pax6^3Neu −/+. (B) Pax6 canonical, Pax6(5a) and Pax6-Immp1L fusion transcripts in Pax6^13Neu heterozygotes. (Lanes 1–3) Pax6^13Neu −/+ amplified for the region of Pax6 spanning exon 5a. (Lanes 4–6) Pax6^13Neu −/+ amplified for the predicted Pax6-Immp1L fusion transcript.