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. 2013 Jun 21;3(6):979-91.
doi: 10.1534/g3.113.006338.

Identification of Cilia Genes That Affect Cell-Cycle Progression Using Whole-Genome Transcriptome Analysis in Chlamydomonas Reinhardtti

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Free PMC article

Identification of Cilia Genes That Affect Cell-Cycle Progression Using Whole-Genome Transcriptome Analysis in Chlamydomonas Reinhardtti

Alison J Albee et al. G3 (Bethesda). .
Free PMC article

Abstract

Cilia are microtubule based organelles that project from cells. Cilia are found on almost every cell type of the human body and numerous diseases, collectively termed ciliopathies, are associated with defects in cilia, including respiratory infections, male infertility, situs inversus, polycystic kidney disease, retinal degeneration, and Bardet-Biedl Syndrome. Here we show that Illumina-based whole-genome transcriptome analysis in the biflagellate green alga Chlamydomonas reinhardtii identifies 1850 genes up-regulated during ciliogenesis, 4392 genes down-regulated, and 4548 genes with no change in expression during ciliogenesis. We examined four genes up-regulated and not previously known to be involved with cilia (ZMYND10, NXN, GLOD4, SPATA4) by knockdown of the human orthologs in human retinal pigment epithelial cells (hTERT-RPE1) cells to ask whether they are involved in cilia-related processes that include cilia assembly, cilia length control, basal body/centriole numbers, and the distance between basal bodies/centrioles. All of the genes have cilia-related phenotypes and, surprisingly, our data show that knockdown of GLOD4 and SPATA4 also affects the cell cycle. These results demonstrate that whole-genome transcriptome analysis during ciliogenesis is a powerful tool to gain insight into the molecular mechanism by which centrosomes and cilia are assembled.

Keywords: GLOD4; NXN; SPATA4; ZMYND10; deflagellation; flagella.

Figures

Figure 1
Figure 1
Expression profiles during ciliogenesis are similar between RNAseq and qRT-PCR. Genes with previous support for involvement in ciliogenesis (TUA1, RSP3, ODA6, KLP1, and FAP178) and new genes (ABCA, GLOD4, MOT8, KCN1, NXN, and ZMYND10) were examined for the up-regulation pattern by RNAseq and qRT-PCR. Error bars on qRTPCR data are the average of two independent experiments. Expression profiles generally agree between the two methods.
Figure 2
Figure 2
Profile clustering identifies 16 principal up-regulation expression profiles organized into five pattern groups. (A) The Arch pattern shows increased expression at 3 min, a peak expression at 10 min, and then decreasing expression at 30 and 60 min. This profile pattern group accounts for 37% of up-regulated gene profiles. The first and second most common principal expression profiles are found with this pattern: Arch1 (n = 336; 18%) and Arch 2 (n = 343; 19%), respectively. The Arch1 pattern shows expression that peaks at 10 or 30 min but is still up at 60 min. The Arch2 pattern is similar to Arch1 except that the genes are not up-regulated at the 60-min time point. (B) The Staggered (Stag) pattern shows genes with a burst of expression at the 3, 10, 30, or 60 min time points. The third, fourth, fifth, and tenth most common expression profiles are found with this pattern: Stag30 (n = 226; 12%), Stag10 (n = 189; 10%), Stag60 (n = 165; 9%), and Stag3 (n = 49; 3%). (C) The Pulse pattern shows up-regulation at a single time point and accounts for 11% (n = 196) of the up-regulated genes. (D) The Up-Tick pattern describes genes that show increased expression at one time point followed by down-regulation at another time point followed by up-regulation at a third time point. Up-Tick patterns comprise 3% (n = 51) of up-regulated genes and can be further subdivided by the time point of the up-tick. (E) Hump patterns comprise 16% (n = 290) and are profiles that are pulse-like, but significant up-regulation is sustained over two consecutive time points. Of the remaining fraction, 0.5% (n = 9) show profiles that are outliers in that their profiles are not adequately similar to any principal expression profile found in this analysis.
Figure 3
Figure 3
Knockdown by shRNA as a method to validate our dataset. (A) On Day 0, hTERT-RPE1 expressing centrin/GFP was infected with lentivirus containing shRNAs. The next day, puromycin was added to select for cells that incorporated the shRNAs. After selection (Day 3), cells were passed 1:5 and plated onto coverslips. On Day 4, one set of coverslips received EdU and the other set had their medium changed to serum-free to induce cilia growth. After 24 hr in EdU (Day 5), the coverslips were fixed and stained. After 3 d in serum-free medium (Day 7), the coverslips were fixed and stained for acetylated α-tubulin. (B) The amount of transcript remaining for each gene normalized to GAPDH. Although we used 4−5 shRNAs per gene, only the construct with the greatest degree of knockdown is shown here.
Figure 4
Figure 4
Depletion ZMYND10 causes short cilia. (A) Micrograph of control cells showing normal cilia (left) and a micrograph of a representative image from ZMYND10 knockdown. Scale bar is 10 μm. Blue, DNA; red, acetylated α-tubulin; green, centrin/GFP. (B) Graph showing cilia length after gene knockdown. Error bars represent SD from at least 100 cilia per gene knockdown. Significance was determined using a Student’s t-test with two tails and unequal variance. **P < 0.001. (C) Percent ciliated cells normalized to the control. (D) Percent of cells with 2, 3, 4, 5, or ≥6 basal bodies/centrioles. Bracket indicates gene knockdowns that have increased numbers of basal bodies/centrioles compared with the control. (E) Distance between the mother and daughter basal bodies/centrioles. Error bars represent SD from at least 100 cells per gene knockdown. Significance was determined using a Student’s t-test with two tails and unequal variance. (F) Percent of cells that up took EdU normalized to the control. (G) Examples of localization of ZMYND10 in cells with and without cilia. Green, gene-YFP; red, centrioles left panel, cilia right panel; blue, DNA.
Figure 5
Figure 5
NXN knockdown causes cells to have short cilia and basal bodies/centrioles to be far apart. (A) A representative image from NXN knockdown. Scale bar is 10 μm. Blue, DNA; red, acetylated α-tubulin; green, centrin/GFP. (B) Cilia length after gene knockdown. Error bars represent SD from at least 100 cilia per gene knockdown. Significance was determined using a Student’s t-test with two tails and unequal variance. **P < 0.001. (C) Percent ciliated cells normalized to the control. **Fewer cilia. (D) Percent of cells with 2, 3, 4, 5, or ≥6 basal bodies/centrioles. (E) Distance between the mother and daughter basal bodies/centrioles. Error bars represent SD from at least 100 cells per gene knockdown. Significance was determined using a Student’s t-test with two tails and unequal variance, **P < 0.001. (F) Percent of cells that incorporated EdU normalized to the control. (G) Examples of localization of NXN in cells with and without cilia. Green, gene-YFP; red, centrioles left panel, cilia right panel; blue, DNA.
Figure 6
Figure 6
GLOD4 causes short cilia and altered cell-cycle progression. (A) A representative image from GLOD4 knockdown. Scale bar is 10 μm. Blue, DNA; red, acetylated α-tubulin; green, centrin/GFP. (B) Cilia length after gene knockdown. Error bars represent SD from at least 100 cilia per gene knockdown. Significance was determined using a Student’s t-test with two tails and unequal variance. **P < 0.001 (C) Percent ciliated cells normalized to the control. (D) Percent of cells with 2, 3, 4, 5, or ≥6 basal bodies/centrioles. (E) Distance between the mother and daughter basal bodies/centrioles. Error bars represent SD from at least 100 cells per gene knockdown. (F) Percent of cells that incorporated EdU normalized to the control. (G) Examples of localization of GLOD4 in cells with and without cilia. Green, gene-YFP; red, centrioles left panel, cilia right panel; blue, DNA.
Figure 7
Figure 7
SPATA4 knockdown arrests cells in S-phase. (A) A representative image from SPATA4 and UPF1 knockdown. Scale bar is 10 mm. Blue, DNA; red, acetylated a-tubulin; green, centrin/GFP. (B) Graph showing the percent ciliated cells normalized to the control. **Because there were no cilia after UPF1 knockdown, cilia lengths could not be measured. (C) Percent ciliated cells normalized to the control. **Fewer cilia. (D) Percent of cells with 2, 3, 4, 5, or >6 basal bodies/centrioles. Bracket indicates gene knockdowns that have increased numbers of basal bodies/centrioles compared to the control. (E) Distance between the mother and daughter basal bodies/centrioles. Error bars represent SD from at least 100 cells per gene knockdown. Significance was determined using a Student's t-test with two tails and unequal variance. **P <0.001. (F) Percent of cells that incorporated EdU normalized to the control. (G) Localization of SPATA4 in cells with and without cilia. Green, SPATA4-YFP; red, centrioles left panel, cilia right panel; blue, DNA.

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