Developmental expression of the zebrafish Arf-like small GTPase paralogs arl13a and arl13b

Abstract

Members of the Arf-like (Arl) family of small GTP-binding proteins regulate a number of cellular functions and play important roles in cilia structure and signaling. The small GTPase Arl13a is a close paralog to Arl13b, a small GTPase required for normal cilia formation that causes Joubert Syndrome when mutated. As mutation of arl13b causes a slow retinal degeneration in zebrafish (Song et al., 2016), we hypothesized that expression of arl13a may provide functional redundancy. We determined the expression domains of arl13a and arl13b during zebrafish development and examined subcellular localization by expression of fluorescence fusion proteins. Both genes are widely expressed during early cell division and gastrulation and Arl13a and Arl13b both localize to microtubules in ciliated and dividing cells of the early zebrafish embryo. Between 2–5 days post fertilization (dpf), arl13b is expressed in neural tissues while expression of arl13a is downregulated by 2 dpf and restricted to craniofacial structures. These results indicate that arl13a and arl13b have evolved different roles and that arl13a does not function in the zebrafish retina.

Introduction

Cilia are microtubule-based organelles that protrude from the surface of most eukaryotic cells (Satir and Christensen, 2007) and can be classified as motile cilia or non-motile (primary) cilia. Motile cilia function to drive locomotion in single-celled eukaryotes and gametes, or to propel fluid across the cell surface (Roy, 2009). Primary cilia function as sensory organelles and monitor the extracellular environment by concentrating receptors within the ciliary membrane (Malicki and Johnson, 2016). It is now well established that cilia are necessary for detecting a diverse range of signals, including light, hormones, neurotransmitters, morphogens, and growth factors (Ishikawa and Marshall, 2011; Hilgendorf et al., 2016). Given the importance of ciliary function for normal development and physiology, it is not surprising that defects in cilia can result in a number of pleiotropic genetic diseases, termed ciliopathies (Sharma et al., 2008). Joubert Syndrome (JBTS), Leber Congenital Amaurosis (LCA), Meckel Syndrome (MKS) Bardet-Biedl Syndrome (BBS), and Nephronophthisis (NPHP) are ciliopathies with overlapping clinical features, including retinal degeneration, obesity, polydactyly, skeletal abnormalities, and defects in hepatic, respiratory, and renal function (Sharma et al., 2008).

Three members of the ADP-ribosylation-factor-like (Arl) family of small G-proteins (Arl6, Arl3, and Arl13b) play important roles in cilia biogenesis (Li et al., 2010; Li et al., 2012). Arl6 was the first member of the Arl family shown to cause disease when it was identified in a set of genes found to cause BBS (Chiang et al., 2004; Fan et al., 2004). Also known as Bbs3, Arl6 localized to the basal body in a ring-shaped pattern in hTERT cells (Wiens et al., 2010). Although not detected within the cilium of wild-type cells, it is thought that Arl6 facilitates ciliary exit of the BBSome, the large complex of BBS proteins required for ciliary membrane biogenesis (Nachury et al., 2007; Liew et al., 2014). A role for Arl3 in cilia function was first identified in the protozoan Leishmania donovani (Cuvillier et al., 2000). Subsequent work found that Arl3 directly binds with RP2 (retinitis pigmentosa protein 2), which functions as the GAP (GTPase activating protein) for Arl3 (Veltel et al., 2008). Both RP2 and Arl3 localize to the connecting cilium of photoreceptors (Grayson et al., 2002). Arl3 has been linked to the trafficking of lipidated membrane-associated proteins (Hanke-Gogokhia et al., 2016a; Hanke-Gogokhia et al., 2016b). Defects Arl3-mediated trafficking of prenylated proteins in rod photoreceptors led to cell death and retinal degeneration (Wright et al., 2016) Although human mutations in ARL3 have not yet been identified, the Arl3^−/− knockout mice exhibit phenotypes consistent with ciliary defects (Schrick et al., 2006). Unlike Arl6 and Arl3, which function both inside and outside the cilium, localization of Arl13b is limited to primary cilia. Arl13b was first linked to cilia in a forward genetic screen for kidney cysts in zebrafish (Sun et al., 2004). Shortly thereafter, the mouse Arl13b^hnn mutant was described as having defects in neural tube patterning, shortened cilia, and polydactyly, which were phenotypes consistent with cilia defects (Caspary et al., 2007). Mutations in ARL13B were subsequently identified in families with Joubert Syndrome (Cantagrel et al., 2008). Arl13b has been proposed to function at the guanine nucleotide exchange factor (GEF) for Arl3, thereby spatially restricting Arl3 activation and cargo release to the cilium (Gotthardt et al., 2015).

Mutations disrupting the structure or function of cilia in zebrafish lead to a common set of phenotypes that include rapid photoreceptor cell death, kidney cysts, left-right asymmetry, and a curly body axis (Sun et al., 2004; Tsujikawa and Malicki, 2004; Pathak et al., 2007; Omori et al., 2008). Zebrafish lacking arl13b exhibit many of these phenotypes (Sun et al., 2004). We recently reported, however, that zebrafish arl13b^−/− mutants undergo a slow, progressive photoreceptor degeneration (Song et al., 2016). While the arl13b^−/− mutants develop kidney cysts during larval stages and exhibit phenotypes seen in other zebrafish cilia mutants, the mild retinal phenotype was unusual. One possible explanation for these results is functional redundancy with a closely related protein. Here, we explored the hypothesis that Arl13a may compensate for Arl13b in zebrafish photoreceptor function. Cellular localization studies revealed that Arl13a colocalized to microtubules in both ciliated and dividing cells of early zebrafish embryos. We report, however, that while arl13a expression overlaps with arl13b during early zebrafish development, the expression patterns differed at later stages during neural differentiation. We conclude that Arl13a likely does not compensate for Arl13b in the zebrafish retina.

METHODS

Zebrafish care and maintenance

Zebrafish were maintained in a 14:10 hr light-dark cycle on Aquatic Habitats recirculating water systems (Pentair; Apopka, FL). Animals were maintained in accordance with protocols approved by the Cleveland Clinic Institutional Animal Care and Use Committee (IACUC). To image and localize basal bodies, we used the transgenic line Tg(5actb2:cetn2-GFP) ^cu6, which expresses a centrin2-GFP fusion protein from the β-actin promoter (Randlett et al., 2011). The arl13b (sco)^hi459 line was previously described (Sun et al., 2004; Song et al., 2016).

In situ hybridization and immunocytochemistry

Localization of mRNA by in situ hybridization was done using digoxigenin-and fluorescein-labeled riboprobes as described (Jowett, 2001). The full -length zebrafish arl13a cDNA clone (NCBI Reference Sequence: NM_200818.1) was purchased from ATCC (ID 10167628). The full-length zebrafish hmx4 cDNA clone was purchased from GE Dharmacon via Thermo Scientific. The pax2a clone was previously described (Perkins et al., 2005). Images were obtained on a Zeiss AxioZoom.V16 fluorescence stereomicroscope using an AxioCam digital camera (Carl Zeiss Microscopy, Thornberg, NY).

Whole-mount immunostaining was performed as described previously (Lunt et al., 2009). A monoclonal antibody against acetylated α-tubulin (Clone 6–11B-1, Sigma, St Louis, MO) was used at 1:100 dilution to identify cilia. Centrosomes were labeled with a monoclonal anti-γ-tubulin antibody (Sigma, GTU-88) at 1:5000 dilution. Spindle microtubules were labeled with a monoclonal antibody against anti-alpha-tubulin (Clone T9026, Sigma) used at 1:500 dilution. Rabbit polyclonal antibodies against mCherry (Catalog number 5993; BioVision, Milpitas, CA) were used at 1:400 dilution to detect the Arl13a-mCherry fusion protein. Chicken polyclonal antibodies against GFP (Catalog number 13970; Abcam; Cambridge, MA) were used at 1:500 dilution to detect GFP. Alexa secondary antibodies (Invitrogen) were diluted 1:500 and used for fluorescent detection. Embryos for whole-mount immunohistochemistry were mounted on depression slides in PBST. Serial optical sections were obtained with a Zeiss AxioImager.Z2 fluorescent microscope fitted with a 63x PlanApo objective and the Apotome.2 (Carl Zeiss Microscopy, Thornwood, NY). Images were prepared using Adobe Photoshop software.

Phylogenetics

All amino acid sequences for Arl13a were obtained from NCBI using the following access numbers: human (Hs; NP_001155963.1), mouse (Mm; NP_083223.1), rat (Rn; NP_001019537.1), chicken (Gg; XP_015134146.1), zebrafish (Dr; NP_957112.1), Xenopus tropicalus (Xt; XP_004916818.1), Xenopus laevis (Xl; AAH99310.1). Amino acid sequences of Arl13b from NCBI used the following accession numbers: human (NP_001167621.1), mouse (NP_080853.3), rat (NP_001100571.1), chicken (XP_004938370.1), zebrafish (NP_775379.1), Xenopus tropicalus (NP_001184084.1). Accession numbers for Arl13 amino acid sequences were: worm (Ce; NP_001032986.1), and algae (Cr; XP_001691430.1). Sequence alignment was conducted using the online Multiple Sequence Comparison by Log-Expectation (MUSCLE) (McWilliam et al., 2013). Phylogenetic tree construction was performed using Phylodendron (http://iubio.bio.indiana.edu/treeapp/treeprint-form.html) with default parameters.

Imaging

In situ Images were obtained on a Zeiss AxioZoom.V16 fluorescence stereomacroscope using an AxioCam digital camera. Serial optical sections were obtained with a Zeiss AxioImager.Z2 fluorescent microscope fitted with a 63x PlanApo objective and the Apotome.2. Images were prepared using Adobe Photoshop.

RESULTS

To identify additional zebrafish homologs of arl13b, we searched the Ensembl Genome database (http://useast.ensembl.org/Danio_rerio/Info/Index) for paralogs and identified arl13a as a likely candidate. The arl13a gene is predicted to encode a 434 amino acid protein that is 28% identical to Arl13b. Both proteins are part of the Arl family of GTPases. Sequence alignment of Arl13a and Arl13b proteins from human, mouse, rat, chicken, frog, zebrafish, worm (Arl13) and Chlamydomonas (ARL13) found that sequence similarities were highest in the N-terminal region of the proteins, which contained the GTPase domain (Fig. 1). A highly conserved P loop (GLDNAGKT) required for nucleotide binding was located near amino acids 28–35 in most Arl13a and Arl13b homologs (Fig. 1, black bar). Phylogenetic comparisons revealed that Arl13a orthologs are more similar to each other and cluster separately from the Arl13b orthologs (Fig. 2). Furthermore, the C. elegans Arl13 gene and the Chlamydomonas Arl13 gene cluster with the tetrapod Arl13b genes, suggesting that Arl13b is more closely related functionally to the ancestral form.

Figure 1. Sequence alignment of Arl13a and Arl13b shows strong similarity in the N-terminal region of the protein.

Using ClustalW, amino acid sequences for Arl13a homologs from human (Hs; NP_001155963.1), mouse (Mm; NP_083223.1), rat (Rn; NP_001019537.1), chicken (Gg; XP_015134146.1), zebrafish (Dr; NP_957112.1), Xenopus tropicalus (Xt; XP_004916818.1), Xenopus laevis (Xl; AAH99310.1), were aligned against amino acid sequences of Arl13b homologs from human (NP_001167621.1), mouse (NP 080853.3), rat (NP_001100571.1), chicken (XP_004938370.1), zebrafish (NP_775379.1), Xenopus tropicalus (NP_001184084.1), and Arl13 amino acid sequences from worm (Ce; NP 001032986.1), and algae (Cr; XP_001691430.1). Identical residues are noted in black and similar residues (minimum 50%) highlighted in gray. The nucleotide binding site (P loop) is underlined.

Figure 2. Phylogenetic relationship between Arl13 family members.

Phylogenetic tree was constructed using Phylodendron from an alignment of Arl13a and Arl13b amino acid sequences from Chlamydomonas (Cr), C. elegans (Ce), zebrafish (Dr), Xenopus tropicalus (Xt), chicken Gg), rat (Rn), mouse (Mm), and human (Hs).

The expression of arl13a and arl13b was examined by whole-mount in situ hybridization beginning at the 1-cell stage and progressing through 5 days post fertilization (dpf). Both arl13a and arl13b mRNAs were maternally expressed, although expression of arl13b was significantly higher (Fig. 3A, 1-cell). By the 1k-cell stage and through 50% epiboly, both genes were highly expressed throughout the blastula (Fig. 3A, 1k -cell, 50%). During somitogenesis, both arl13a and arl13b remained widely expressed throughout neural tissues. Beginning at 26 hours post fertilization (hpf), arl13a expression could be observed in the ventricular zone extending from a boundary in the midbrain, which was demarcated by pax2a expression (Fig. 3B, arrowhead), to the forebrain. We found that expression of arl13a was excluded from otic vesicles and the optic stalk, as no overlap was observed with pax2a expression (Fig. 3B, open arrow). A similar expression pattern was observed for arl13b, although faint expression was observed in the otic vesicle (Fig. 3F; open arrow). At 32–40 hpf, both arl13a and arl13b exhibited diffuse expression throughout the central nervous system (Figs. 3C and G). At 2 and 3 days post fertilization, the expression of arl13a remained diffusely expressed in neural tissues and appeared in the pronephros (Fig. 3D). At 4 dpf, expression of arl13a was no longer detected in the pronephros but was robust expression was observed in the olfactory pit and liver (Fig. 3E). By 5 dpf, expression had expanded into lower cranial structures. It is interesting to note that the expression of arl13a in the olfactory pit, liver, and cranial structures was considerably more robust than the neural expression at 2–3 dpf. The time required to detect in situ probe signal at 4–5 dpf was much shorter than the time needed to observe signal in neural tissues at earlier time points. Sense control probes did not result in detectable signal. In contrast, expression of arl13b remained elevated in neural tissues and the pronephros in 2 dpf, 3 dpf, and 5 dpf animals. In the retina, arl13b expression was observed through all cellular layers (Figs. 3H, I).

Figure 3. Developmental expression of zebrafish arl13a and arl13b from 1-cell to 5 dpf by in situ hybridization.

(A) Expression patterns of arl13a (top row) and arl13b (bottom row) from 1-cell stage to 21 somite stage (ss) using antisense RNA probes. Control (sense) RNA probes for arl13a are shown for 1-cell stage and for the 1k-cell stage embryos (inset). Anterior is to the left. For images of 2ss and 11ss embryos, dorsal views (upper inset) and front views (lower inset) are also shown. Views of 21ss specimens are lateral and dorsal views, respectively. (B) Expression of arl13a at 26 hpf. The upper panels show views of an embryo following whole-mount in situ hybridization noting expression the CNS. Upper left shows lateral view and upper right image is a dorsal view. Lower images show two-color in situ hybridization with arl13a (blue) and pax2a or hmx4 (orange). Note that arl13a and pax2a expression domains do not overlap except in a small patch at the midbrain-hindbrain boundary (arrowhead). arl13a expression is excluded from the otic vesicle (open arrow) and in the lens, as noted by hmx4 expression. (D) Expression of arl13a by WISH at 2 dpf (top) and 3 dpf (bottom). (E) arl13a is highly expressed at upper lip (insets from the black rectangle) and lower lip (right image, ventral view) at 4 dpf, and at jaw at 5dpf (lower images, inset is the higher magnification from the black rectangle). F, forebrain; M, midbrain; L, lens; Vent, ventral view. (F-I) Expression patterns of arl13b from 26 hpf to 5 dpf. In F, the arrow indicates the otic vesicles; G, the arrow (upper images) indicates kidney and the arrow (lower right) indicates otic vesicle; H and I, higher expression of arl13b at photoreceptor layer at 3 dpf and 5 dpf (sections, lower left images, respectively). I, arrow indicates olfactory bulbs at 5 dpf (lower right). Scale bar: 100 µm.

To determine the subcellular localization of Arl13a, mRNA encoding an Arl13a-mCherry fusion protein was injected into 1-cell zebrafish embryos. Embryos were fixed at the 6 somite stage and ciliated cells in presomitic mesoderm were analyzed by whole-mount immunocytochemistry. We first compared the subcellular localization of Arl13a to Arl13b by injected Arl13a-mCherry mRNA into the Tg(−3.2actnb:arl13b-GFP) transgenic line, which expresses an Arl13b-GFP fusion protein localizing to cilia (Borovina et al., 2010). The fluorescence signal from Arl13a-mCherry was observed in small puncta that showed considerable overlap with the signal from Arl13b-GFP, which is consistent with ciliary localization (Fig. 4A; arrow and arrowhead). To confirm that Arl13a localized to cilia, wild-type embryos were injected with Arl13a-mCherry mRNA and stained with acetylated tubulin. The Arl13a-mCherry fluorescence puncta partially overlapped with acetylated tubulin (Fig. 4B), indicating that Arl13a localized to cilia. Out of 150 cilia observed across 7 embryos, 79 cilia (53%) showed colocalization between Arl13a and acetylated tubulin and 71 (47%) were stained only with acetyled tubulin. To further define the spatial distribution of Arl13a, we used the Tg(−3.2actnb:cetn4-GFP) transgenic line that localizes to the basal bodies of cilia (Randlett et al., 2011; Ramsey and Perkins, 2013). The Arl13a-mCherry signal was immediately distal of signal from the centrin-GFP fusion protein in 3 of 4 cilia observed (Fig. 4C). The localization of Arl13a was also assessed in cells undergoing cell division in the presomitic mesoderm. Embryos were injected with Arl13a-mCherry mRNA and immunolabeled for α-tubulin to label microtubules of the mitotic spindle (Fig. 4D) or γ-tubulin to mark the centrosomes as the spindle poles (Fig. 4E). At each stage of the cell cycle, Arl13a-mCherry colocalized with α-tubulin to label microtubules of the mitotic spindle and the midbody (Fig. 4D). Arl13a was excluded from the spindle pole and did not colocalize with γ-tubulin.

Figure 4. Arl13a colocalizes with microtubules in zebrafish embryos.

(A) Tg(−3.2actnb:arl13b-GFP) transgenic larvae injected with mRNA encoding Arl13a-mCherry were fixed at 6 somite stage (ss) and stained with antibodies against mCherry (red) or GFP (green). Cilia on two cells (arrow and arrowhead). Inset panels show enlarged images of individual channels and the merged image. (B) Wild-type larvae (6 ss) were fixed and stained with antibodies against mCherry (red) or acetylated-tubulin (green) to label cilia. (C) Tg(−3.2actnb:cetn4-GFP) transgenic larvae injected with mRNA encoding Arl13a-mCherry were fixed at 6 somite stage (ss) and stained with antibodies against mCherry (red) or GFP (green). (D)Mitotic cells in presomitic mesoderm of wild-type larvae (6 ss) were fixed and stained with antibodies against mCherry (red) or acetylated-tubulin (green) to label microtubules or (E) γ-tubulin to label centrosomes. All larvae were counterstained with DAPI to label nuclei. Scale bar: 10 μm (A-C); 20 μm (D, E).

We previously reported that arl13b mutants had mild retinal phenotypes during larval stages (Song et al., 2016). To determine if mutations in arl13b results in compensatory elevated expression of arl13a, we examined arl13a in arl13b mutants (Fig. 5). At both 48 hpf and 3 dpf, expression of arl13a was not significantly different between arl13b mutants and wild-type siblings (Figs 5A and 5B).

Figure 5. Expression of zebrafish arl13a in 48 hpf and 3dpf arl13b^−/−mutants by in situ hybridization.

(A) Expression of arl13a by WISH at 48 hpf and (B) 3 dpf in wild-type (top) and arl13b^−/−homozygous mutants (bottom). Lateral views are shown in left and middle panels. Right panels show the ventral views of the head. Insets of left panels show higher magnification of the trunk.

Discussion

Members of the Arf-like (Arl) subfamily of small GTPases are implicated in diseases known as ciliopathies (Zhang et al., 2013). While biochemical and functional studies have revealed important details about GTP-binding and the role of Arl2/3 and Arl13b proteins in protein trafficking, the Arl subfamily is large and questions about functional redundancy remain largely unanswered.

We previously reported that ciliary extension and long-term photoreceptor survival requires Arl13b (Song et al., 2016). Mutations in ARL13B cause Joubert Syndrome in humans (Cantagrel et al., 2008) and mice lacking Arl13b die in utero (Caspary et al., 2007). The Arl13b paralog, Arl13a, is a protein of unknown function and we questioned whether arl13a was expressed in similar domains as arl13b and could potentially provide functional compensation. While arl13a was expressed in a similar manner as arl13b during early zebrafish development, we did not observe expression in the retina or other neural tissues following somitogenesis. These results suggest that arl13a does not compensate for the loss of arl13b in the zebrafish retina. It is interesting to note, however, that Arl13a and Arl13b both localize to microtubules in ciliated and dividing cells of the developing zebrafish embryo. Arl13b directly binds tubulin and this interaction requires the N-terminal G-domain (Revenkova et al., 2018). As a small GTPase, Arl13a shares this G-domain and may also directly bind tubulin through this domain. As both genes exhibit overlapping expression patterns during early development, some functional redundancy may exist.

To date, this is the first study of Arl13a expression and cellular localization and no other functional studies have been conducted. The expression of arl13a in cranial structures is intriguing as craniofacial abnormalities are can be a symptom of Joubert Syndrome (Damerla et al., 2015). Loss of function studies will be required to identify the precise role of Arl13a and any functional redundancy in early development with Arl13b.