XB-ART-47888Development. October 1, 2013; 140 (20): 4277-86.
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. MYB 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.
PubMed ID: 24048590
PMC ID: PMC3787764
Article link: Development.
Grant support: 5T32 GM07276 NIGMS NIH HHS , 5U01HL099995 NHLBI NIH HHS , R01 GM096021 NIGMS NIH HHS , Howard Hughes Medical Institute , R01 GM098582 NIGMS NIH HHS
Genes referenced: cdh1 cdkn1a epha8 foxj1.2 gmnn lamtor2 mcc myb myc nkx2-1 notch1 npat odc1 pcnt shh sox2
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|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. 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. 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.|