Malformation of Tear Ducts Underlies the Epiphora and Precocious Eyelid Opening in Prickle 1 Mutant Mice: Genetic Implications for Tear Duct Genesis

Abstract

Purpose: Obstruction of the tear drainage causes a range of ocular surface disorders. Hitherto, the genetics of tear duct development and obstruction has been scarcely explored, and related animal models are lacking. This study aims to study the potential role of the Wnt/PCP pathway mediated by Prickle 1 in tear duct development and diseases.

Methods: A severe hypomorphic Prickle 1 mutant was generated. Histology and immunohistochemistry were performed to compare wild type, Prickle 1 hypomorphic, and null mutant tear ducts. In situ hybridization was conducted to identify the signaling components in the developing tear ducts. Three-dimensional (3D) reconstruction was used to detect the human embryonic tear duct.

Results: Here, we report that a severe Prickle 1 hypomorph mouse line exhibited epiphora. This phenotype was due to the blockage of the tear drainage by incompletely formed nasolacrimal duct (NLD) and lacrimal canaliculi (LC), which also causes precocious eyelid opening. We observed a dose-dependent requirement of Prickle 1 for tear duct outgrowth. An investigation of the expression of Wnt/PCP core genes demonstrated a subset of PCP signaling components expressed in the developing tear duct. Furthermore, Prickle 1 is not required for the expression of Fgfr2/Fgf10 and p63 genes, which are associated with the NLD and LC hypoplasia in humans. Last, we showed that Prickle 1 expression in the developing tear drainage system is conserved between mice and humans.

Conclusions: The study suggests that malformed tear ducts caused by disruption of Prickle 1 underlies the epiphora and precocious eyelid opening.

Figure 1.

The tear duct histology and epiphora in the severe Prickle 1 hypomorph mutants. Abbreviations apply to all figures unless otherwise noted. Fresh frozen sections were subjected to H&E and immunohistochemistry. (A) Normal cornea surface. (B) Mutant cornea surface exhibiting epiphora. (C) Section positions in (D–F) and (J–-L). D to F Wild type nasal sections. Boxed areas are the nasolacrimal duct (NLD). (G–I) Magnified from D to F. Bracket and lines indicate the NLD. J–L Mutant nasal sections positionally correspond to that of the wild type in D to F). Boxed areas are magnified in M to O. M–O Dashed brackets and lines indicate the presumptive NLD. (P–W) The E-cadherin staining revealed the NLD and the canaliculi. (P–S) Representative sections of wild type tear duct from different positions. Sectioning direction is from temporal to nasal as illustrated in (T). SLC, superior lacrimal canaliculus; ILC, inferior lacrimal canaliculus; Asterisks, canaliculi openings at eyelids. (U–W) Mutant tear duct sections from temporal to nasal.

Figure 2.

Tear duct obstruction, tear fluid retention and ruptured eyelid in the Prickle 1 hypomorphic mutants. (A–E) H&E-stained fresh frozen sections. NC, nasal cavity; Arrows and lines indicate the NLD positions. (F–I) The E-cadherin staining revealed the NLD and LC. Note openings of the lacrimal canaliculi in the lower F and the upper G eyelid of wild type P8 mice were missing in the mutants. The furthest extended mutant SLC on the section is shown in right panel to H. Arrows point to different parts of the tear duct. (J) Section direction for A to E) (left) and F to I (right). a, anterior; p, posterior; t, temporal; n, nasal. (K) Whole-eyeball coronal sections at P8. The asterisk indicates the mutant ocular fluid accumulation under the eyelid. (L–N) Eyeballs viewed from the top. Arrows point to the mutant eyelid. (O–Q) H&E stained coronal sections of the eyelid.

Figure 3.

Accumulated ocular fluid in the Prickle 1 hypomorphic mutants is primarily of tears. (A) SDS-page profiling the mutant and wild type tears at P8 and 6-week old (6W) animals. Asterisks indicate differential intensities of similar molecular shifts in the mutant and wild type tears. (B) Immunoblotting of the mutant and wild type tears using anti-transferrin antibody. (C) Wild type and the mutant P8 Harderin glands (HGs). (D) Lacrimal glands (LGs). (E) Meibomian glands (MGs) indicated by arrows and brackets respective for wild type and the mutant mice.

Figure 4.

Prickle 1 dose-dependent loss of the nasolacrimal duct. (A–O), H&E-stained fresh-frozen sections. Yellow lines indicate the existing NLD, and red brackets indicate missing of the NLD. Interval distances are shown between each panel. A to E) Wild type NLD at P1. F to J The Prickle 1 hypomorphic mutant duct with null (Prickle 1^b) and hypomorph (Prickle 1^a) alleles. K to O The Prickle 1^b/b null mutant duct. (P) Schematic illustration of dose-dependent loss of the NLD (white lines) on Prickle 1 hypomorphic and null mutant alleles. (Q) Quantification of the missing NLD of the Prickle 1^a/b and Prickle 1^b/b mutants. The missing NLD was quantified as vertical distances (number of sections × section thickness) from the presumptive nasal ends to sections that first appear NLD. Student t-test was performed to detect significance.

Figure 5.

Expression of the Wnt/PCP and Fgf signaling components in the developing tear duct. All panels are in situ hybridization (purple) followed by p63 immunostaining (red). Dashed lines demarcate the tear duct, arrows indicate gene expression. (A, B), Fgf10. (C, D), Fgfr2. (E, F), Wnt6. (G, H), Fzd3. (I, J), Fzd6. (K, L), Celsr1. (M, N), Celsr2. (O–R), Prickle 1. (S) Schematic illustration of a horizontal sectioning plane through the developing tear duct at E11.

Figure 6.

Expression of Fgfr2, p63, Fgf10, and Wnt6. Dashed lines demarcate the tear duct, arrows indicate gene expression. All panels are in situ hybridization (purple) followed by p63 immunostaining (red). The right column of panels is from wild type embryos, whereas the left column of panels is from the Prickle 1 null mutants. (A, B), p63. (C, D), Fgf10/p63. (E, F), Fgfr2. (G, H), Wnt6.

Figure 7.

Expression of Prickle family in the developing human tear duct. (A–L) All panels are in situ hybridization (purple) followed by p63 immunostaining (red) at gestation week 8 (GW8). Brackets and arrows indicate areas of the developing tear duct (TD) and in situ hybridization signal. A, C, and E are three consecutive sections with 30 µm intervals. B, D, and F are same sections respective to A, C, and E. A to F PRICKLE1/p63. G and H) PRICKLE2/p63. I and J) PRICKLE3/p63. K and L PRICKLE4/p63. M and N) The 3D reconstruction of the human embryonic tear duct with p63 staining. The tear duct is drawn as green, whereas the head surface epithelium is drawn as red. M Top view of the developing tear duct. N Inside view of the developing tear duct. cj, conjunctiva; NLD, nasolacrimal duct; SLC, superior lacrimal canaliculus; ILC, inferior lacrimal canaliculus. (O) Schematic illustration of developing TD in the context of the GW8 embryonic head. Red dashed lines and black arrows indicate sectioning region and direction, respectively. man, mandibulary.