Cdc42- and IRSp53-dependent contractile filopodia tether presumptive lens and retina to coordinate epithelial invagination

Abstract

The vertebrate lens provides an excellent model with which to study the mechanisms required for epithelial invagination. In the mouse, the lens forms from the head surface ectoderm. A domain of ectoderm first thickens to form the lens placode and then invaginates to form the lens pit. The epithelium of the lens placode remains in close apposition to the epithelium of the presumptive retina as these structures undergo a coordinated invagination. Here, we show that F-actin-rich basal filopodia that link adjacent presumptive lens and retinal epithelia function as physical tethers that coordinate invagination. The filopodia, most of which originate in the presumptive lens, form at E9.5 when presumptive lens and retinal epithelia first come into close contact, and have retracted by E11.5 when invagination is complete. At E10.5--the lens pit stage--there is approximately one filopodium per epithelial cell. Formation of filopodia is dependent on the Rho family GTPase Cdc42 and the Cdc42 effector IRSp53 (Baiap2). Loss of filopodia results in reduced lens pit invagination. Pharmacological manipulation of the actin-myosin contraction pathway showed that the filopodia can respond rapidly in length to change inter-epithelial distance. These data suggest that the lens-retina inter-epithelial filopodia are a fine-tuning mechanism to assist in lens pit invagination by transmitting the forces between presumptive lens and retina. Although invagination of the archenteron in sea urchins and dorsal closure in Drosophila are known to be partly dependent on filopodia, this mechanism of morphogenesis has not previously been identified in vertebrates.

Fig. 1.

Transient, F-actin-filled processes connect lens and retina during lens pit-optic cup invagination. (A) The inter-epithelial process in the early human eye [redrawn from Mann (Mann, 1928)]. (B-G) Phalloidin (F-actin, green) and nuclear (Hoechst 33258, blue) labeling of eye region cryosections from mouse embryos of the indicated ages. Cytoplasmic protrusions containing filamentous actin are indicated by arrowheads. (H) Quantification of lens-retina inter-epithelial processes from E9.5 to E11.0. At each time-point, n=6. lp, lens pit; lpl, lens placode; ov, optic vesicle; pl, presumptive lens; pr, presumptive retina. Scale bars: 20 μm.

Fig. 2.

Many inter-epithelia filopodia originate in the lens pit. (A-K) Eye region cryosections from E10.5 (A,B,D-K) or E9.5 (C) mouse embryos labeled for F-actin (phalloidin, green), nuclei with Hoechst 33258 (blue) and DiI in the lens pit (A,B, red), GFP from the Le-Cre transgene (C,D, red), β-catenin (E-H, red, white), tubulin (I, red), acetylated tubulin (J, red) or keratin 18 (K, red). The position of filopodia is indicated by white arrowheads; red arrowheads indicate filopodial position according to F-actin labeling. A gray line between panels indicates that they are separated color channels of the same image. Scale bars: 20 μm.

Fig. 3.

Generation and phenotype of Cdc42^flox/flox; Le-Cre mice. (A) Schematic of the Cdc42^flox allele. Exons are represented by numbered light green boxes, loxP sites by green arrowheads and Frt sites by blue arrowheads. The positively selectable expression unit TK-Neo-pA is shown in gray. The Le-Cre transgene was used to delete the Cdc42^flox allele in presumptive lens cells. (B) Confirmation of Cdc42^flox/flox deletion by Le-Cre in dissected lens pit (E10.5), lens vesicle (E12.5) and adult lens (Ad). The undeleted Cdc42^flox allele shows a 760 bp band that changes to 190 bp after recombination. IL is an internal control amplifying a region from the interleukin 1 gene. (C-J) Eye region cryosections from E9.5 (C,D), E10.5 (E,F,I,J) or E11.5 (G,H) embryos labeled for nuclei with Hoechst 33258 (blue), GFP (D,F,H,J, green), Cdc42 (C-H, red), or ZO1 (I,J, red). Scale bars: 20 μm.

Fig. 4.

Filopodia are dependent on Cdc42, IRSp53 and FAK. (A-I) Eye region cryosections from E10.5 mouse embryos labeled for nuclei with Hoechst 33258 (blue), F-actin (A-E, green) or laminin (F-I, green). In A-E, the position of filopodia is indicated by red arrowheads. (J-M) Quantification of filopodial index (J), inter-epithelial distance (K), lens pit depth (L), and lens pit cell number (M) in E10.5 embryos of the indicated genotypes. (N) Quantification of filopodial index in E9.5 embryos of the indicated genotypes. All data points represent n=10. Statistical significance as indicated. Scale bars: 20 μm.

Fig. 5.

Inter-epithelial filopodia contain active actin-myosin complexes. (A-H) Eye region cryosections from E10.5 mouse embryos labeled for nuclei with Hoechst 33258 (blue), F-actin (green), myosin II (A-D, red) or phospho-myosin II (E-H, red). In C,D, the position of red channel myosin labeling is indicated on the green channel (F-actin) by the white-outlined regions. In G,H, the position of red channel phospho-myosin labeling is indicated on the green channel (F-actin) by the white-outlined regions. In G, phospho-myosin labeling in filopodia that appear to originate in presumptive retina is indicated by arrowheads. A gray line between panels indicates that they are separated color channels of the same image. Scale bars: 20 μm.

Fig. 6.

Lens-retina filopodia control inter-epithelial distance and lens pit curvature via actin-myosin contractile activity. (A-F) Eye region cryosections from E10.5 mouse embryo heads cultured for 3 hours in the presence of vehicle (A,B), blebbistatin (C,D) or calyculin A (E,F) and labeled for nuclei with Hoechst 33258 (blue), F-actin (green), and phospho-myosin II (red). The position of phospho-myosin labeling in B, D and F is indicated by arrowheads. A gray line between panels indicates that they are separated color channels of the same image. (G-I) Quantification of lens pit F-actin (green bars) and phospho-myosin (red bars) fluorescence intensity (G), filopodial index (H) and inter-epithelial distance (I) in the eyes of explanted embryo heads after vehicle, blebbistatin or calyculin A treatment. For all data points, n=5. In H and I, data from Le-Cre; FAK^flox/flox mutants is represented from Fig. 4 for comparison purposes. (J) Quantification of lens pit F-actin labeling intensity at apical, middle and basal cell positions for E10.5 control and Le-Cre; Cdc42^flox/flox mutants as labeled. For all data points, n=5. (K) Quantification of basal position rate of change of curvature from the nasal to temporal side in E10.5 control and Le-Cre; Cdc42^flox/flox lens pits. n=6. Scale bars: 20 μm.

Fig. 7.

A model: Cdc42/IRSp53-dependent filopodia fine-tune coordinated invagination of presumptive lens and retina. This schematic summarizes the function, mechanism of formation and contractile activity of lens-retina inter-epithelial filopodia. (A) The filopodia form at ∼E9.0 to connect the lens placode within the embryonic surface ectoderm to the presumptive retina within the optic cup. (B) Formation of the filopodia requires Cdc42 and IRSp53 (red). We propose that phosphorylated myosin II (green) positioned in the base of the filopodia is one component of a larger actin-myosin contractile complex that regulates the inter-epithelial distance via filopodial length. We further suggest that FAK-dependent integrin adhesion might be required to anchor filopodia to the ECM (blue). (C) Based on the failure of full lens pit invagination in mutants that are deficient in filopodia and on the increased inter-epithelial distance when myosin II activity is inhibited, it is likely that the filopodia and the actin-myosin complexes within are crucial to transmitting the physical forces that partly mediate a coordinated morphogenetic process.