Arf-like Protein 2 (ARL2) Controls Microtubule Neogenesis during Early Postnatal Photoreceptor Development

Abstract

Arf-like protein 2 (ARL2) is a ubiquitously expressed small GTPase with multiple functions. In a cell culture, ARL2 participates with tubulin cofactor D (TBCD) in the neogenesis of tubulin αβ-heterodimers, the building blocks of microtubules. To evaluate this function in the retina, we conditionally deleted ARL2 in mouse retina at two distinct stages, either during the embryonic development (^retArl2^-/-) or after ciliogenesis specifically in rods (^rodArl2^-/-). ^retArl2^-/- retina sections displayed distorted nuclear layers and a disrupted microtubule cytoskeleton (MTC) as early as postnatal day 6 (P6). Rod and cone outer segments (OS) did not form. By contrast, the rod ARL2 knockouts were stable at postnatal day 35 and revealed normal ERG responses. Cytoplasmic dynein is reduced in ^retArl2^-/- inner segments (IS), suggesting that dynein may be unstable in the absence of a normal MTC. We investigated the microtubular stability in the absence of either ARL2 (^retARL2^-/-) or DYNC1H1 (^retDync1h1^-/-), the dynein heavy chain, and found that both the ^retArl2^-/- and ^retDync1h1^-/- retinas exhibited reduced microtubules and nuclear layer distortion. The results suggest that ARL2 and dynein depend on each other to generate a functional MTC during the early photoreceptor development.

Keywords: Arf-like protein 2 (ARL2); dynein heavy chain (DYNC1H1); microtubule cytoskeleton (MTC); retina; rod photoreceptors; tubulin αβ-heterodimers.

Figure 1

Generation of Arl2 conditional knockouts. (A) schematic of mouse ARL2 protein and its functional domains. CC, coiled coil domain; G, guanine nucleotide binding domain; S1 and S2, switch 1 and switch 2; R15L, mutation linked to retina disease. (B) the mouse Arl2 gene with 5 exons. (C) the gene trap is located in intron 1. The gene trap is flanked by FRT sites. LoxP sites are flanking the NEO cassette and a third loxP site is placed in intron 3. Horizontal blue arrows delineate approximate positions of genotyping primers. (D) floxed allele. (E) null allele. (F) genotyping floxed allele in tail DNA with arl2-F and arl2-R yielding a WT amplicon of 520 bp and a floxed amplicon of 400 bp. (G) presence of Six3Cre in tail DNA using Six3Cre–F and Six3Cre–R yielding an amplicon of 450 bp. The bands of 200 bp are an internal positive control (see Methods). (H) genotyping the Six3Cre (ret) and iCre65 (rod) knockout allele with CSDF and CSDR using retina DNA as a template. The diagnostic fragment of 740 bp is absent in Arl2^F/F. (I,J) Western blots (left panels) with anti-ARL2 antibody using P10 ^retArl2^−/− retina lysate (I) and P15 ^rodArl2^−/− retina lysate (J). Polypeptides marked with asterisks are nonspecific polypeptides serving as loading controls. (I,J) right panels are Image J density scans of ARL2.

Figure 2

Histology of ^retArl2^−/− sections at P10–15. (A–C), plastic sections show histology of control (left panels) and ^retArl2^−/− retina (right panels) at days P10, P12 and P15. Note significant ONL/INL distortions, absence of OSs, abnormal ISs and displaced INL neurons in the P15 KO retina, reminiscent of retinas from Dync1h1 knockouts.

Figure 3

Immunohistochemistry with Arl2 cryosections. (A–D), ^retArl2^−/− (right panels) and control cryosections (left panels) were probed with anti-DYNC1H1 (A–D), anti-GC1 (B), anti-PSD95 (C), and anti-RIBEYE (D) antibodies as indicated. (A–C) are postnatal day P6 cryosections, D is P10. Note distorted ONL, poorly developed OPL and suppression of DYNC1H1 fluorescence.

Figure 4

ERG of retina and rod ARL2 knockouts. (A,B) averaged (n = 3–5) pan-retina ERGs at P15 and P18. Average scotopic a- and photopic b-wave amplitudes revealed diminished amplitudes. Control ERGs are shown in blue, ^retArl2^−/− ERGs in red. (C,D) scotopic a- and b-wave amplitudes as a function of light intensity. Note near complete extinction of responses. (E,F) P35 rod knockout ERGs. Average scotopic a-wave (E) and photopic (F) b-wave amplitudes (n = 5) of control (blue) and rod KO (red) as a function of flash intensity. Control and knockout scotopic a- and b-wave amplitudes are nearly identical. (G,H) scotopic a-wave (G) and photopic b-wave (H) as a function of light intensity. Rod knockouts shown in red, controls in blue.

Figure 5

Basal body and CC are mislocalizing into the ONL. (A–D) Immunohistochemistry of P10 control (A,A′,C,C′) and ^retArl2^−/− cryosections (B,B′,D,D′) probed with anti-CEP164 antibody (A–B′), and anti-CEP250 (C-NAP1) (C–D′). (A′–D′) are enlargements of (A–D) as indicted by yellow hatched boxes. To visualize individual rods, enlargements are shown next to (A′–D′). In Arl2 knockout panels, the BB–CC structures are mislocalized into the ONL. CEP164 still enables docking of the basal body and extension of CC, and CEP250 still connects mother and daughter centrioles. Mice were kept on an EGFP-CETN2 transgenic background to mark centrioles and CC. Image was post-processed with Airy Scan of the LSM800 confocal microscope.

Figure 6

Immunohistochemistry of ^retArl2^−/− sections at P6 and P10. (A–D) ^retArl2^F/F (rows (A,C)) and ^retArl2^−/− (rows (B,D)) retina cryosections probed with anti-acetylated α-tubulin (Ac-Tub, white, left column), anti-polyglutamylated tubulin (PolyE-Tub, red, middle column). The right column shows merged images of Ac-Tub and PolyE-Tub contrasted with DAPI to show the ONL. Note severe distortions of the ONL at P6 (row (B)) when ciliogenesis begins, but lesser distortions at P10 when OSs form (row (D)). A dashed rectangle in (C) (right panel) indicates the area of enlargements shown in row (C) (upper right corner).

Figure 7

Retina-specific deletion of Arl2 and DYNC1H1 damages the MTC. (A–D), P10 Arl2^F/F (row (A)), ^retArl2^−/− (row (B)), P6 Dync1H1^F/F (row (C)) and ^retDync1h1^−/− cryosections (row (D)) probed with anti-TUBB3 (green) ((A–D), left and right columns) and anti-TUBA1A antibodies (red) (middle and right columns). Note reduction in microtubules in knockout sections (B,D).