Directed Differentiation of Human Embryonic Stem Cells Reveals Novels Insights into the 'Master Regulators' of Multiciliogenesis

Multiciliated cells (MCCs) drive fluid flow over specialised epithelia, and in the respiratory
tract, they constitute an essential part of mucociliary clearance, whereby pathogens are
eliminated by motile cilia decorating their apical surface. If compromised, this causes a
susceptibility towards respiratory diseases such as primary ciliary dyskinesia (PCD). Central
to the development of diagnostic and treatment approaches of diseases, is the understanding
of their genetic underpinnings. While causal loci for PCD have typically been linked with
structural defects in cilia, recently our attention has moved towards the transcriptional
regulation of MCC differentiation. Although extensively investigated in animal models, this
process has not been as elaborately established in the context of the human airway.
Here, we have developed a directed differentiation approach to generate MCCs from
human embryonic stem cells (hESCs) to investigate the function and localisation of master
regulators of the MCC transcription cascade. Using genome editing we show that MCIDAS
(MCI), a master transcriptional regulator of MCC biogenesis, is essential for the development
and function of multiple cilia. Although the absence of MCI does not compromise the
specification of MCC precursors, we have demonstrated that in differentiating MCCs it
triggers a de novo centriolar amplification pathway by accumulating in the cytoplasm and
progressively associating with centriolar proteins. MCIDAS loss blocks cytoplasmic
translocation of E2F4, a co-transcriptional regulator recently shown to also have a cytoplasmic
role in centriole formation, and centriole biogenesis is completely inhibited. Additionally, in
the nucleus, MCIDAS induces genes that participate in centriole biogenesis. In concert we
infer that GMNC, a paralogous protein, previously demonstrated to trigger MCC
differentiation in animal models, may not be as important in humans. In GMNC-/- hESCs, we
show that MCC formation appears unhindered and motile cilia are formed normally. We
investigate the role of the downstream effectors E2F4, E2F5 and CCNO in multiciliogenesis.
Demonstrating the separate, dispensable, and integral functions E2F4 and E2F5, respectively,
and confirming the importance of CCNO in centriole amplification.
For the first time in a human airway model our findings confirm the ability of the
nodal MCC transcription factor MCIDAS and its downstream effector CCNO to induce
explosive centriole biogenesis; clarify that the paralogous protein GMNC and cell cycle
regulator E2F4 may be dispensable for MCC formation and suggest that E2F5 acts
independently to facilitate multiciliogenesis.