October 1, 2013;
Myb promotes centriole amplification and later steps of the multiciliogenesis program.
The transcriptional control of primary cilium
formation and ciliary motility are beginning to be understood, but little is known about the transcriptional programs that control cilium
number and other structural and functional specializations. One of the most intriguing ciliary specializations occurs in multiciliated cells (MCCs), which amplify their centrioles to nucleate hundreds of cilia
per cell, instead of the usual monocilium. Here we report that the transcription factor MYB
, which promotes S phase and drives cycling of a variety of progenitor cells, is expressed in postmitotic epithelial cells of the mouse airways and ependyma destined to become MCCs. MYB
is expressed early in multiciliogenesis, as progenitors exit the cell cycle and amplify their centrioles, then switches off as MCCs mature. Conditional inactivation of Myb
in the developing airways blocks or delays centriole
amplification and expression of FOXJ1
, a transcription factor that controls centriole
docking and ciliary motility, and airways fail to become fully ciliated. We provide evidence that MYB
acts in a conserved pathway downstream of Notch
signaling and multicilin, a protein related to the S-phase regulator geminin
, and upstream of FOXJ1
can activate endogenous Foxj1
expression and stimulate a cotransfected Foxj1
reporter in heterologous cells, and it can drive the complete multiciliogenesis program in Xenopus embryonic epidermis
. We conclude that MYB
has an early, crucial and conserved role in multiciliogenesis, and propose that it promotes a novel S-like phase in which centriole
amplification occurs uncoupled from DNA synthesis, and then drives later steps of multiciliogenesis through induction of Foxj1
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References [+] :
Fig. 1. Myb expression in the developing bronchial epithelium. (A) Schematic of multiciliogenesis in developing airway epithelial cells. FOXJ1 expression is indicated by green nuclei. Stage I: the presumptive multiciliated cell (MCC) has exited the cell cycle and pericentriolar material components begin to accumulate near the centrosome. Stage II: new centrioles begin to appear. Stage III: centrioles migrate and dock at the apical cell surface. Stage IV: each docked centriole (now called a basal body) nucleates a motile 9+2 ciliary axoneme in a mature MCC (Vladar and Stearns, 2007). (B) In situ hybridization of Myb on a section of E15.5 mouse lung. Myb mRNA is detected only in the large proximal airways (bronchus, Br), in a ‘salt and pepper’ pattern, and not in smaller, more distal airways (outlined) that have not yet begun to differentiate or in surrounding mesenchyme or blood vessels (V). (C-E) Immunostaining of E15.5 lung section for MYB and Ki67, a marker of cycling cells. MYB is not expressed in cells that express Ki67. Scale bars: 50 μm in B; 10 μm in C-E.
Fig. 2. MYB is expressed early and transiently during multiciliogenesis in the lung and brain. (A,B) Immunostaining of E17.5 mouse lung sections for MYB and either SCGB1A1 (Clara cells) or FOXJ1 (MCCs). MYB is not detected in SCGB1A1-expressing cells (boxed region, insets in A) but many MYB-positive cells also express FOXJ1 (boxed region, insets in B). (C) Sections of E14.5 through E17.5 lungs were stained for MYB, FOXJ1 and SOX2 and the number of epithelial cells (marked by SOX2) expressing MYB only, FOXJ1 only, or both MYB and FOXJ1 were counted. Total SOX2-positive cells scored was >1000 per age. (D) Mouse tracheal epithelial cell (MTEC) cultures were immunostained for MYB and FOXJ1 at the times indicated after exposure to an air-liquid interface (ALI) to promote differentiation. The number of cells that were MYB+, FOXJ1+ or double-positive were counted. Similar to in vivo, the FOXJ1+ population continually increased during culturing, whereas the percentage of MYB+ and double-positive cells initially increased and then declined. Total cells scored was >100 per time point. (E) Immunostaining of E17.5 lung section for MYB, FOXJ1 and acetylated tubulin (ACT, cilia marker). MYB is seen in developing (upper inset) but not mature (lower inset) MCCs. (F) Immunostaining of ALI +8 days MTEC cultures for MYB and ACT. MYB is not detected in mature MCCs. Arrowheads (E,F), ciliary tufts of mature MCCs. (G,H) Whole-mount ventricles of P2 mouse brain stained for FOXJ1 and MYB (G) or Ki67 and MYB (H). (I) Stage-specific expression of MYB during multiciliogenesis in MTECs. ALI +6 days MTECs were stained for MYB and pericentrin (PCNT), a centriole marker, and cells were staged as described in Fig. 1A. MYB is detected at stages I and II, before and during centriole amplification, but not during stages III or IV. Dotted lines show cell outline. Scale bars: 10 μm in A,B,E-H; 5 μm in I.
Fig. 3. Effect of Myb deletion on initiation of FOXJ1 expression in the lung. (A-F) Mice carrying a conditional allele of Myb (Mybfl) and a Cre recombination reporter (mTmG) were crossed to mice carrying Shh-Cre or Nkx2.1-Cre transgenes to selectively delete Myb from the developing airway epithelium (Myb CKO), and lungs from E15.5 control (Myb+/fl) and Myb CKO mice were immunostained for the proteins indicated. (A,B) MYB immunostaining confirms loss of MYB expression in Myb CKO tissue. (C,D) FOXJ1 expression is not detected in Myb CKO tissue. (E,F) Large concentrations of PCNT, indicative of ciliating cells with amplified centrioles (inset in E), are not observed in Myb CKO tissue (inset in F). PCNT staining of centrosomes (arrowheads) is seen in both control and Myb CKO non-MCCs. (G,H) Lungs from E15.5 Foxj1 control heterozygous and homozygous knockout (Foxj1-/-) mice immunostained for MYB. MYB is expressed normally in the absence of Foxj1. Scale bars: 10 μm.
Fig. 4. Effect of MYB on FOXJ1 expression in MTECs and a HEK293T cell cotransfection assay. (A,B) MTECs infected with a lentivirus expressing either a tdTomato-MYB fusion protein (tdT-MYB) or RFP alone were harvested at ALI +4 days and stained for RFP (A) or tdTomato (B) and FOXJ1. Scale bar: 20 μm. (C) Quantification of the percentage of infected cells that express FOXJ1. *P<0.0001 by chi-square test. (D) Luciferase activity driven by the Foxj1 reporter illustrated (nucleotides -876 to +162 with respect to the Foxj1 transcription initiation site) cotransfected into human HEK293T cells with pCDH empty vector (-) or pCDH constructs expressing the indicated proteins. Reporter activity is normalized to the vector-only (pCDH) control. *P<0.05, **P<0.003; n.s., not significant (Student’s t-test). Error bars indicate s.e.m.
Fig. 5. Effect of Myb conditional knockout on multiciliogenesis in the lung. Lungs from Myb CKO mice (E17.5 in B,D,F and P14 or P21 in H,J,L) or littermate controls (E17.5 in A,C,E and P14 or P21 in G,I) were immunostained for the indicated markers. Cadherin 1 (CDH1) was used to visualize the epithelium in control tissues without mTmG reporter. (A,B) Immunostaining for ACT shows that the large apical tufts of ACT of mature MCCs (inset in A) are not present in Myb CKO tissue (inset in B) at E17.5. Immunostaining for FOXJ1 (C,D) and PCNT (E,F) shows that the multiciliogenesis program has begun to recover (compare with E15.5 in Fig. 3C-F). (G-L) ACT staining in postnatal lungs shows that multiciliogenesis in the large proximal airways recovers by P14 (G,H) (note that white indicates overlap of green and purple). However, small distal airways of Myb CKO lungs lack mature cilia (J) or are only sparsely ciliated (L) even at P21, as compared with control distal airways (I), as quantified in K. n, number of airways scored in three mice for each condition. Samples were stained simultaneously and scanned with the same confocal laser settings. Scale bars: 20 μm in A-F,I,J,L; 50 μm in G,H.
Fig. 6. Effect of the multiciliogenesis regulator multicilin (MCIN) on MYB expression in MTECs. (A-C) MTECs were infected with a control lentivirus expressing GFP (A), a myc-tagged wild-type MCIN (B) or dominant-negative MCIN (DN-MCIN) (C), and stained for MYB and GFP or myc at ALI +4 days. (D) Quantification of the percentage of infected cells that express MYB. *P<0.001 by chi-square test. For examples of infected cells, see supplementary material Fig. S6. Scale bar: 10 μm.
Fig. 7. Myb acts downstream of Mcin and can promote multiciliogenesis in Xenopus laevis embryonic epidermis. (A) Two-cell stage X. laevis embryos were injected with Notch intracellular domain (ICD) mRNA alone to repress multiciliogenesis or together with MCIN (ICD/MCIN-HGR) to induce it, and Foxj1 and Myb mRNA levels were measured by qRT-PCR, normalized to Odc mRNA levels as an internal control. Values are mean ± s.d. (B-D) Two-cell stage embryos were injected with control (B) or Myb mRNA (C) and Hysl-GFP mRNA to visualize centrioles and membrane RFP mRNA to visualize cell boundaries and stained for acetylated tubulin (ACT) to label cilia. Scale bar: 10 μm. (D) Quantification of B and C. Note that the percentage of MCCs is increased in Myb mRNA-injected embryos, whereas the percentage of outer cells (OCs) and proton-secreting cells (PSCs) declines slightly. P<0.05 for all three cell types by Student’s t-test. Error bars indicate s.d.
Fig. 8. Proposed molecular pathway governing multiciliogenesis. MYB and other transcriptional regulators associated with each step in the program are shown along with the regulatory relationships among them, together with the Notch signaling pathway that controls initiation of the program. The dashed line indicates that there is an auxiliary pathway that can partially bypass the requirement for MYB, at least in some MCCs.
Bender, Critical functions for c-Myb at three checkpoints during thymocyte development. 2004, Pubmed