Generation of EGFP-Rab5c, EGFP-Rab11a, and EGFP-Rab7 Transgenic Lines
To mark and study early, recycling, and late endosomes, we generated constructs encoding N-terminal fluorescent fusions of Rab5c, Rab11a, and Rab7. We chose Rab5c (early), Rab11a (recycling), and Rab7 (late) for analysis based on their established localization and functions in these endosome subtypes and conservation of protein function/localization across evolution (Zhang et al., 2007). Most vertebrates have three paralogs of Drosophila Rab5 (Rab5a–c). However, in zebrafish there are additionally two rab5a genes (rab5aa and rab5ab) (see Supp. Fig. S1a, which is available online). Transcripts of rab5aa, rab5ab, and rab5c are all ubiquitously expressed in zebrafish embryos, unlike rab5b, where expression is limited to the yolk syncytial layer, pronephric duct, and telencephalon (Thisse and Thisse, 2004). Thus, rab5c was chosen for examination in zebrafish due to its ubiquitous expression and to avoid potential complications due to gene duplication (rab5aa and rab5ab). Whereas many vertebrates have a single rab11 gene, zebrafish possess four paralogs: rab11a, rab11a-like, rab11ba, and rab11bb. We selected rab11a as a representative recycling endosome marker because rab11a shows ubiquitous expression, unlike the other rab11 paralogs that show some degree of tissue specificity (Thisse and Thisse, 2004). Additionally, the zebrafish Rab11a protein is the most similar zebrafish paralog to the well-studied Drosophila RAB11 (Supp. Fig. S1a). For rab7, only one homolog has been curated in zebrafish, and this gene was chosen for characterizing late endosomes. Interestingly, other species such as frog, mouse, rat, and human possess two rab7 genes: rab7a and rab7b (Supp. Fig. S1). Protein alignments against the most recent build of the zebrafish genome (Zv8), however, indicated the presence of two hypothetical proteins (LOC 449549 and LOC 431725) with significant homology to Rab7 protein, suggesting that other rab7 paralogs are present in zebrafish (Supp. Fig. S1b).
Transgenic zebrafish lines expressing EGFP-tagged versions of the Rab proteins from h2ax regulatory sequence were generated using standard protocols (Fig. 1a) (Kawakami, 2004, 2005; Kwan et al., 2007). N-terminal fusions were generated as they have been shown to not interfere with protein function in other systems (Zhang et al., 2007). These lines were designated Tg(h2afx:EGFP-Rab5c)mw5, Tg(h2afx:EGFP-Rab11a)mw6, and Tg(h2afx:EGFP-Rab7)mw7 and will subsequently be referred to as EGFP-Rab5c, EGFP-Rab11, and EGFP-Rab7 (Fig. 1b). For each line, transgenic fish showed punctate EGFP expression throughout the embryos. Single-copy, functional transgenes were maintained through outcrosses to wild-type strains. No abnormal phenotypes were noted in transgenic embryos and the fish developed and reproduced normally. To determine if the expression of the EGFP-Rab fusions resulted in compensatory down-regulation of the endogenous transcripts, quantitative RT-PCR was performed on both transgenic and non-transgenic larvae. No differences in endogenous rab5c, rab11a, or rab7 transcript abundance were observed between wild-type and transgenic fish (Supp. Fig. S2). We next attempted to investigate whether EGFP-Rab puncta co-localized with endogenous Rab5c, Rab11, and Rab7 proteins. Unfortunately, we were unable to obtain antibodies specific to the zebrafish Rab proteins, precluding us from performing definitive co-localization studies through fluorescent microscopy or immuno-electron microscopy. We, therefore, used alternative strategies to address whether the EGFP-Rab labeling marked the expected endosome compartments.
Figure 1. Transgenic construct models and Rab protein alignments. A: Schematic of constructs used to generate the transgenic lines. N-Terminal GFP fusions of wild-type Rab proteins are expressed under control of the ubiquitous h2afx promoter. N-terminal mCherry fusions of mutated Rab proteins are expressed under control of the conditional UAS promoter for tissue-specific and temporal regulation of mutant protein expression using the GAL4/UAS system. Flanking Tol2 sequences faciliate genomic integration. B: Allele designation and common names of EGFP-Rab and mCherry-Rab Mutant transgenic lines. C: Sequence alignments of portions of the GTP-binding pockets of the Rab proteins, indicating high homology between zebrafish, fly, and human. Amino acids highlighted in yellow are mutated to generate either dominant-negative or constitutively active Rab proteins by affecting GTP/GDP affinity (dominant-negative mutations) or GTPase activity (constitutively active mutations). Amino acid sequence is conserved for each set of orthologs except when marked by an asterisk (*). Numbers give amino acid position for each protein.
Download figure to PowerPoint
Early, recycling, and late endosomes can be characterized by their distribution in polarized cells and the time-dependent accumulation of cargo in uptake assays. To assist in quantifying the number, size, degree of polarized localization, and dynamic features of the EGFP-Rab-marked vesicles, we developed an automated tracking program. The automated tracking program runs on the Windows operating system as a stand-alone program or from within the MATLAB environment on any operating system. The program segments (delineates) and then tracks each fluorescently labeled endosome throughout the image sequence. After segmentation and tracking, the program generates an Excel spreadsheet with organelle size and position information for each endosome. The whole process takes a few minutes to analyze a 60-frame image sequence. Initial results using the automated tracker were confirmed through manual measurements. The tracking software is available free of charge and can be downloaded from https://pantherfile.uwm.edu/cohena/www/rabtools.html.
To begin characterization and investigate the specificity of the three EGFP-Rab lines, we first analyzed the size distribution of puncta marked by each fusion protein. Published electron microscopic studies from various cell types have shown that endosomes range in cross-sectional areas from 0.01–1.3 μm2 (Gorvel et al., 1991; Bucci et al., 1992, 1994; Cataldo et al., 1997). Confocal time-lapse microscopy revealed EGFP-Rab-labeled puncta of various sizes, some actively associating/dissociating with vesicles and others remaining stably complexed throughout much of the time-course (Supp. Movies S1–3). Analysis of stable fluorescent puncta (present in five consecutive frames) within retinal neuroepithelial cells indicated that the cross-sectional size distribution fell within the expected range for endosomal compartments (Fig. 2). We then characterized the degree of polarization of EGFP-Rab puncta for each transgenic line in a variety of polarized cells. Previous studies of endosome subtypes in polarized cells, either cultured MDCK cells or epithelia in vivo, have shown non-uniform distribution along the apical-basal axis. Specifically, polarized cells exhibit enriched localization of early endosomes at the apical surface, as indicated by RAB5 expression (Bucci et al., 1994; Leung et al., 2000). Similarly, RAB11-positive recycling endosomes are known to reside in a pericentriolar region at the apical surface (Ullrich et al., 1995). As RAB7 replaces RAB5 protein with late endosome maturation, localization shifts more centrally (Marois et al., 2006). Examination of transgenic embryos showed differences in the polarization of the EGFP-Rab puncta in neuroepithelial cells from the retina, hindbrain, and otic vesicle (Fig. 3). Ensuing characterization of the bright vesicular structures showed that both Rab5c- and Rab11a-positive endosomes localized towards the apical surface, while EGFP-Rab7-positive vesicles were located more centrally in polarized cells of the developing retina, hindbrain, and ear (Fig. 3A–R). Quantization confirmed the polarized distribution, where EGFP-Rab5c and EGFP-Rab11a were most apical (Fig. 3S–U).
Figure 2. Tracking and cross-sectional area. Determination of EGFP-Rab-positive endosomes. A: Example of the tracking software over five time-series of an EGFP-Rab5c embryo. Tracked endosomes are automatically circled in red and are given an identification number to correlate numerical data in generated spreadsheets with visual images. Arrows point to endosomes recognized by the program (outlined in green) that were not followed for a minimum of five consecutive frames, and, therefore, no data were obtained from these endosomes and they were not given identification numbers. Insert in panel T=0 is a higher magnification of the boxed region showing numbering and outlining of tracked endosomes. T represents progression of time in seconds between frames. B: Distribution of cross-sectional area measurements determined for each of the EGFP-Rab lines. Most of the tracked endosomes have cross-sectional areas between 0.2 and 0.6 μm2, with very few endosomes larger than 1 μm2. P value is calculated from a one-way ANOVA.
Download figure to PowerPoint
Figure 3. Expression and polarization of EGFP-Rab fusion proteins in neuroepithelial cells. Representative single-plane, confocal images of 28hpf embryos expressing (A–F) EGFP-Rab5c, (G–L) EGFP-Rab11a, or (M–R) EGFP-Rab7. Distinct fluorescent endosomes can be visualized as bright structures above a general membrane background in B, D, F, H, J, L, N, P, and R. Bright-field images (A, C, E, G, I, K, M, O, and Q) illustrate the structure of the developing epithelium with apical and basal surfaces indicated. Scale bars = 25 μm;. Asterisk in E, K, and Q indicates the presence of the anterior otolith. S–U: Quantification of endosome distance from the apical surface in (S) retinal neuroepithelial cells, and (T) hindbrain epithelial cells, (U) otic vesicle epithelial cells. In each cell type, EGFP-Rab5c, and EGFP-Rab11a, polarization is more towards the apical surface than EGFP-Rab7-positive endosomes. N>200 endosomes from images of 10 embryos across multiple clutches for each genotype and cell type. Mean distance from the apical surface is indicated with error bars indicating SEM. One-way ANOVA results indicate P < 0. 0001 for each cell type, followed by the Tukey's post-test with ***P < 0.001.
Download figure to PowerPoint
To verify that the EGFP-Rab proteins mark distinct structures indicative of vesicle progression from early (Rab5c-positive) to recycling (Rab11a-positive) or late (Rab7-positive) endosomes, we performed an in vivo lipophilic dye uptake assay (Fig. 4 and Supp. Fig. S3). Otic vesicle epithelial cells were imaged to examine the time-course of dye co-localization within endosome subtypes. The lumen of otic vesicles of 28-hr post-fertilization (hpf) EGFP-Rab transgenic larvae were injected with FM4-64 lipophilic dye to continuously label membranes of adjacent neuroepithelia. Internalization of the dye in otic neuroepithelia was apparent within minutes, indicating a high degree of endocytosis in the otic vesicle epithelial cells, a phenomenon maintained as the cells differentiate into sensory hair cells (Seiler and Nicolson, 1999). At early time points (two minutes post injection), the FM4-64 dye co-localized to a high degree with vesicles marked by EGFP-Rab5c and EGFP-Rab11a, consistent with early and recycling endosomes (Fig. 4E). For this time-point, very few FM4-64-positive structures overlapped with EGFP-Rab7 expression, suggesting EGFP-Rab7 labeled a “late” endosome subtype. Conversely, 10 min after injection of the lipophilic dye, there was a high degree of co-localization of FM4-64-positive structures with EGFP-Rab11a and EGFP-Rab7, but not EGFP-Rab5c (Fig. 4E). Together, these experiments confirmed that the transgenic lines mark the expected endosome subtypes. Interestingly, there was a high degree of co-localization of the FM4-64 dye with EGFP-Rab11a at both the 2- and 10-min time-points. Consistent with this observation, it was previously noted that RAB11 partially co-localizes with RAB5 (Ullrich et al., 1996) and these markers of early and recycling endosomes can exist on sub-domains of the same vesicular structure (Sonnichsen et al., 2000). Therefore, the high degree of EGFP-Rab11a co-localization with FM4-64 dye at both time-points is suggestive of (1) vesicles transitioning from early to recycling compartments and (2) active recycling of material throughout the duration of the experiment. Overall, these experiments coupled with a wealth of published data suggest that the EGFP-Rab5c, EGFP-Rab11a, and EGFP-Rab7 transgenic lines label early, recycling, and late endosomes, respectively.
Figure 4. FM 4-64 injections into the developing otic vesicle indicate progression of endosome sub-types. A–C: 50-μm images thresholded for high fluorescence providing an example of FM 4-64 dye injection into the otic vesicle of a 28hpf EGFP-Rab5c embryo, 2 min post-injection. Single-channel images show intracellular puncta positive for the FM 4-64 dye (A), EGFP-Rab5c (B), and merged in C. D: Boxed image in C is magnified. Circles in A–D indicate non-membrane-associated, intracellular FM 4-64-positive vesicles. Arrows indicate examples of vesicles positive for both EGFP-Rab5c and the FM 4-64 dye, while the asterisk (*) indicates an FM 4-64-positive vesicle that is EGFP-Rab5c negative. E: Quantification of the percentage of FM 4-64-positive vesicles that co-localize with EGFP-Rab-positive vesicles at 2 and 10 min post-FM 4-64 dye injection. Numerical values below the x-axis indicate total number of FM 4-64 puncta analyzed from a minimum of 10 embryos. n-values indicate total number of FM 4-64 vesicles positive for EGFP-Rab at each time-point. Statistical Analysis: Fisher's Exact Test. ***P < 0.001; ns, not significant.
Download figure to PowerPoint
Generation of Dominant-Negative and Constitutively Active UAS-Driven Rab Transgenic Lines
To establish tools for examining Rab protein function in developing tissues, constructs encoding dominant-negative (DN) and constitutively active (CA) versions of Rab5c, Rab11a and Rab7 proteins were generated (Fig. 1). For each rab cDNA, mutations were introduced to codons corresponding to critical amino acids previously characterized to confer dominant-negative or constitutively active functions when altered in human and Drosophila Rab proteins (Stenmark et al., 1994a, 1994b; Feng et al., 1995; Meresse et al., 1995; Chen et al., 1998). These amino acid substitutions result in proteins with either reduced GTP affinity (dominant-negative) or reduced GTPase activity (constitutively active). Protein alignments revealed the location of the conserved, critical amino acids in the zebrafish homologs (Fig. 1c). The mutant Rab proteins were generated as N-terminal mCherry fusions and expressed from a UAS promoter, thus providing temporal and tissue-specific expression through use of Gal4 transactivating lines (Asakawa and Kawakami, 2008). Using these constructs, the following transgenic lines were established: Tg(UAS:mCherry-Rab5c S34N)mw33, Tg(UAS:mCherry-Rab5c Q81L)mw34, Tg(UAS:mCherry-Rab11a S25N)mw35, Tg(UAS:mCherry-Rab11a Q70L)mw36, Tg(UAS:mCherry-Rab7 T22N)mw37, and Tg(UAS:mCherry-Rab7 Q67L)mw38, and will subsequently be referred to as Rab5cDN, Rab5cCA, Rab11aDN, Rab11aCA, Rab7DN, and Rab7CA, respectively.
To begin to verify the functional consequences of the zebrafish mutant Rab variants, mCherry-rabDN or mCherry-rabCA mRNA was synthesized for each. The DN and CA versions are predicted to disrupt endogenous Rab functions in a dosage-dependent manner. Indeed, increasing concentrations of injected mRNA resulted in augmented expression of each mutant Rab protein and in larger percentages of embryos displaying defects such as altered body curvature, vascular anomalies, and increased cell death (Fig. 5, data not shown). Embryos injected with higher concentrations for the mCherry-rabDN mRNAs phenocopied body curvature defects described in previous reports of morpholino-based protein knockdown for Rab5c and Rab11a (see Fig. 7B,C, data not shown) (Kalen et al., 2009). Global defects in embryogenesis may be due to disruptions in cell polarity, migration, signaling, or survival as previous reports have defined roles of either Rab5c or Rab11a in these processes (Ulrich et al., 2005; Kalen et al., 2009; Yu et al., 2009; Tay et al., 2010; Nowak et al., 2011). Importantly, morphogenic defects induced by high levels of mCherry-rab5cDN expression were diminished in an EGFP-Rab5c background, but not in EGFP-Rab7 transgenic fish (Fig. 5B,C). Similar results were observed for all transgenic lines with mRNA injection. As a control, injection of mCherry-CAAX mRNA resulted in >83% of embryos displaying WT morphology (115/137) with no embryos displaying moderate or severe phenotypes. These experiments suggest that wild-type EGFP-Rab transgenes are functional and the mutant versions are specific in that each wild-type Rab can abrogate the deleterious effects caused by the corresponding dominant-negative version.
Figure 5. Dosage-dependent defects of mCherry-Rab5cDN expression are rescued by expression of the EGFP-Rab5c transgene. A: Representative montaged confocal images of 24hpf embryos showing phenotypic designations of Wild-type (WT), Mild, Moderate, or Severe after injection with mCherry-Rab5cDN RNA. Arrow indicates region of edema and blood pooling posterior to the yolk extension, characteristic of embryos with the “Mild” phenotype label. B: Quantification of relative percentage of embryos displaying phenotypes shown in A after injection of mCherry-Rab5cDN mRNA. C: Table indicating the total number of embryos examined for each category represented in B. Statistical Analysis: Chi-Squared compared to 300 pg RNA injected into WT embryo category, ***P < 0. 0001. Three or more clutches were used for each category.
Download figure to PowerPoint
From a sub-cellular perspective, over-expression of constitutively active Rab proteins has been found to enlarge the endosome in which the Rab protein associates (Marois et al., 2006; Zhang et al., 2007). To examine alterations in size distribution as a consequence of RabCA expression, we injected mCherry-rabCA mRNA into EGFP-Rab transgenic embryos. The automated tracker was again used to determine cross-sectional areas of individual endosomes. The sizes of both EGFP-Rab-positive and mCherry-RabCA-positive structures were analyzed. As expected, expression of the mCherry-RabCA proteins resulted in mCherry-RabCA- or EGFP-Rab-labeled endosomes that were much larger than the EGFP-Rab vesicles in uninjected transgenic embryos (Fig. 6). For example, on average, the size of mCherry-Rab5cCA-labeled endosomes was approximately two-fold larger than EGFP-Rab5c-labeled endosomes from uninjected embryos. Similar increases were observed with Rab11a and Rab7. Interestingly, mCherry-Rab mutant protein expression only partially overlapped with wild-type transgenes (Supp. Fig. S4), consistent with previous observations for localization with atypical GTP/GDP exchange dynamics (Zhang et al., 2007). Together, these experiments are consistent with a gain-of-function for the mCherry-RabCA transgenic lines and loss-of-function for the mCherry-RabDN transgenic lines.
Figure 6. mCherry-RabCA expression increases the size of corresponding EGFP-Rab-labeled endosomes. Expression of constitutively active mutants in the EGFP-Rab lines increases the size of tracked (A) EGFP-Rab5c, (B) EGFP-Rab11a, and (C) EGFP-Rab7 endosomes from >10 embryos for each genotype. RNAs encoding mCherry-RabCA proteins were injected into corresponding EGFP-Rab embryos (for example, mCherry-Rab5cCA injected into EGFP-Rab5c embryos). Time-lapse images were generated, detecting either green or red fluorescence, and subsequently processed through the tracking software. Endosomes were scored as described under each scatter plot. Individual points represent mean cross-sectional area with average area and SEM indicated. Results of one-way ANOVA, P < 0.0001 for A–C followed by Tukey's post-test. *P < 0.05, ***P < 0.001.
Download figure to PowerPoint
Apical Crumbs2a Localization in Neuroepithelia Depends on Endocytic Recycling
The utility of the UAS transgenic lines expressing mutant Rab proteins was confirmed by crossing with several Gal4 driver lines. The dose-dependence of function disruption was tested by crossing UAS:mCherry-RabDN/CA transgenic fish with carriers of an hsp70:Gal4 transgene. The hsp70 promoter can be induced to varying degrees by altering the timing of mild heat-shocks (Scheer et al., 2002). The resultant double transgenic embryos were, therefore, subjected to varying pulses of heat-shocks. Similar to embryos injected with mRNA encoding Rab-disrupting proteins, graded induction of the RabDN and RabCA transgenes displayed dose-dependent embryonic defects that mimic phenotypes resulting from mRNA injection (Rab11a DN shown in Fig. 7, data not shown). In general, the defects caused by heat-shock induction of the transgenes were mild compared to embryos injected with mRNA, likely owing to the developmental timing and transient nature of protein expression. The consequences of altering particular Rab activities in a tissue-specific manner was evaluated by observing Crumbs protein localization following expression of mutant Rabs specifically in neuroepithelia. Crumbs (Crb) is a single-pass transmembrane protein and is a component of an apical protein complex that regulates cellular polarity (Bulgakova and Knust, 2009). In Drosophila, cell surface levels of CRB protein are increased in larval discs mutant for RAB5, owing to a reduction in the internalization of CRB and other apical membrane proteins (Lu and Bilder, 2005). Expression of Rab5CA results in CRB localization within large internal vesicles (Lu and Bilder, 2005). RAB11 mutant flies or those expressing a Rab11DN, however, have more severely disrupted apical cell junctions and show significant levels of internalized CRB protein, indicating a role of endocytic recycling in proper CRB localization (Roeth et al., 2009). To investigate the specificity of the zebrafish mutant Rab transgenic lines, we assessed the localization of Crumbs2a (Crb2a) protein following expression of mutant Rabs in hindbrain neuroepithelia. Expression of mutant Rabs in hindbrain neuroepithelia was accomplished using regulatory sequence associated with the vsx2 gene. The Tg(vsx2:Gal4vp16)mw39 transgenic line (referred to as vsx2:Gal4) activates UAS transgenes strongly in neuroepithelia of the developing retina and hindbrain (Liu et al., 1994; Rowan and Cepko, 2005; Kimura et al., 2006) and to a lesser extent in other regions of the embryo (Supp. Fig. S5). Using the zs4 monoclonal antibody, which recognizes Crb2a protein (Hsu and Jensen, 2010), immunoreactivity was restricted to the apical surface of neuroepithelial cells. In cells expressing Rab5cCA, an increased amount of Crb2a protein localized to large internal vesicles, consistent with augmented internalization of the apical membrane protein (Fig. 8D–H,X). Like previous reports in Drosophila, Rab11aDN expression resulted in severe disorganization of Crb2a localization (Fig. 8I–M,Y). Hindbrain morphogenesis was also severely disrupted in vsx2:Gal4/UAS:Rab11DN double transgenic embryos (Fig. 8I–M). In these embryos, however, neuroepithelia not expressing the transgenes showed normal morphology and appropriate polarized distribution of Crbs2a (Fig. 8A–C,N–R). Rab7DN expression did not affect Crb2a localization, consistent with the lack of a functional link between late endosomes and Crumbs localization to the apical region of polarized cells (Fig. 8S–W).
Figure 7. mCherry-Rab11aDN expression mimics morpholino-induced Rab11a loss-of-function. Confocal montaged images of 24–28hpf embryos examining the effect of Rab11a loss of function. A: Control embryo. B: Rab11a morphant (1.6 ng injected). C: Embryo injected with mCherry-Rab11aDN mRNA. D: Transgenic hsp70:Gal4;UAS:mCherry-Rab11aDN embryos after high-dose (30 min) heat-shock at 70% epiboly. E: Transgenic hsp70:Gal4;UAS:mCherry-Rab11aDN embryos after low-dose (10 min) heat-shock at 70% epiboly.
Download figure to PowerPoint
Figure 8. Crumbs2a localization is altered when either Rab11aDN or Rab5cCA proteins are expressed. Immunolocalization of Crumbs2a to the apical membrane in the hindbrain epithelium of (A–C) 28hpf vsx2:Gal4 control; (D–H) vsx2:Gal4;mCherry-Rab5cCA; (I–R) vsx2:Gal4/mCherry-Rab11aDN; and (S–W) vsx2:Gal4/mCherry-Rab7DN transgenic embryos. Images B,C, E–H, J–M, O–R, and U–W represent higher magnification of the boxed regions of the brightfield images in A, D, I, N, and S, respectively. Boxed regions in H and M are shown as higher magnifications in X and Y, respectively. Arrows in F–H and X indicate Crumbs2a immunostaining localized to large intracellular mCherry-Rab5cCA puncta. Hindbrain morphogenesis (asterisk in J) and Crumbs2a localization is disrupted in mCherry-Rab11aDN-expressing embryo (I–M,Y). Crumbs2a immunostaining is preserved, however, in the forebrain neuroepithelia (N–R), and in regions devoid of Rab11aDN expression as indicated by the arrowheads. S–W: Expression of mCherry-Rab7DN does not appear to affect Crumbs2a localization and expression. Scale bars in A, B, D, E, I, J, N, O, S, and T = 50 μm; scale bars in X and Y = 10 μm.
Download figure to PowerPoint