Developmental Cell

Distal Visceral Endoderm (DVE) cells show a stereotypic unidirectional migration essential for correct orientation of the anterior-posterior axis. They migrate within a simple epithelium, the Visceral Endoderm (VE). It is unknown how DVE cells negotiate their way amongst the surrounding VE cells, what determines the bounds of DVE migration within the VE, and the relative contributions of different cell behaviours to this migration. To address these questions, we used lightsheet microscopy to generate a multi-embryo, singlecell resolution, longitudinal dataset of cell behaviour and morphology. We developed a machine learning based pipeline to segment cells and a data-informed systematic computational framework to extract and compare select morphological, behavioural and molecular parameters of all VE cells in a unified coordinate space. Unbiased clustering of this single-cell phenomic dataset reveals considerable patterned phenotypic heterogeneity within the VE and a previously unknown sub-grouping within the DVE. While migrating, DVE cells retain regular morphology, do not exchange neighbours and are crowded, all hallmarks of the jammed state. In contrast, VE cells immediately ahead of them deform and undergo neighbour exchange. We show that DVE cells are characterised by higher levels of apical F-actin and elevated tension relative to the VE cells immediately ahead of them through which they migrate, but stop migrating upon reaching a region of the VE with matching elevated tension. Lefty1 mutants, known to show abnormal over-migration of DVE cells, show disruption to this patterned tension in the VE. Our findings provide novel insights into the control of cell behaviour during the remodelling of curved epithelia, indicating that the collective migration of sub-sets of cells can be circumscribed by modulating the mechanical properties of surrounding cells and that migrating cells in this context remain as a jammed solid flock, with surrounding cells facilitating their movement by becoming unjammed. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/534937v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@10a7153org.highwire.dtl.DTLVardef@963a1forg.highwire.dtl.DTLVardef@c1fcd0org.highwire.dtl.DTLVardef@1bcdded_HPS_FORMAT_FIGEXP M_FIG C_FIG

Apical cell extrusion is a mechanical process that allows epithelia to eliminate apoptotic cells and prevent inflammation. Mechanosensitive ion channels are often invoked for their capacity to mediate rapid mechanical responses in dynamic morphogenetic processes. Here we report an unexpected strategy for PIEZO1 to support apoptotic extrusion. PIEZO1 inhibition blocks extrusion in cultured cells and zebrafish larvae. However, although PIEZO1 mediates calcium signals during the extrusion process, we show that extrusion is instead antagonized by increase in the preexisting mechanical tension of the epithelium when PIEZO1 is disrupted. Correcting enhanced pre-stress in PIEZO1-disrupted epithelia is sufficient to rescue apoptotic extrusion, even though it does not restore dynamic calcium signals. PIEZO1 supports mechanical homeostasis through a calcium/calcineurin-dependent pathway that protects MYPT1/myosin phosphatase from degradation to limit Myosin II activation. Therefore, PIEZO1 support the morphogenetic process of apoptotic extrusion through mechanical homeostasis.

Lumens or fluid-filled cavities are a ubiquitous feature of mammals and are often evolutionarily linked to the origin of body-plan complexity. Post-implantation, the pluripotent epiblast in a human embryo forms a central lumen, paving the way for gastrulation. While osmotic pressure gradients drive lumen formation in many developmental contexts, mechanisms of human epiblast lumenogenesis are unknown. Here, we study lumenogenesis in a pluripotent-stem-cell-based model of the epiblast using engineered hydrogels that model the confinement faced by the epiblast in the blastocyst. Actin polymerization into a dense mesh-like network at the apical surface generates forces to drive early lumen expansion, as leaky junctions prevent osmotic pressure gradients. Theoretical modeling reveals that apical actin polymerization into a stiff network drives lumen opening, but predicts that a switch to pressure driven lumen growth at larger lumen sizes is required to avoid buckling of the cell layer. Consistent with this prediction, once the lumen reaches a radius of around 12 m, tight junctions mature, and osmotic pressure gradients develop to drive further lumen growth. Human epiblasts show a transcriptional signature of actin polymerization during early lumenogenesis. Thus, actin polymerization drives lumen opening in the human epiblast, and may serve as a general mechanism of lumenogenesis.

Mammary glands contain branched networks of ducts and alveoli that function to produce milk for offspring. While the murine luminal epithelium is organized as a cellular monolayer, it originates from multilayered structures called terminal end buds (TEB). The TEBs generate ducts of monolayered epithelial cells as they invade the fat pad, but little is known about underlying mechanisms. While apoptosis provides a plausible mechanism for cavitation of the ductal lumen, it does not account for elongation of ducts behind the TEBs. Our spatial calculations suggest that most cells in TEBs need to intercalate into the outermost luminal layer and that this migration of cells is the primary driver of cavitation and ductal elongation. To study the progression of multilayered to monolayered epithelium, we developed a quantitative cell culture assay that determines the efficiency of intercalation into an epithelial monolayer. Using this tool, we verified that loss of adherens junctions prevents stable integration of cells into monolayers, consistent with previous data in cultured cells and in primary tissue. Interestingly, tight junction (TJ) proteins also play a key role in this integration process. Although loss of the ZO-1 TJ protein in intercalating cells suppresses intercalation, loss of ZO-1 in the monolayer has the reverse effect, promoting intercalation - even though ZO-1 is not necessary for establishment of TJs. ZO-1-positive puncta form between cells and the monolayer, which then resolves into a new intercellular boundary as intercalation proceeds. ZO-1 loss also reduces engraftment when cells are transplanted into the mammary gland via intraductal injection. We further show that intercalation is dependent on dynamic cytoskeletal rearrangements in both the existing monolayer and intercalating cells. These data identify luminal cell rearrangements necessary for mammary gland development and suggest a molecular mechanism for integration of cells into an existing monolayer.

Notch receptors control tissue morphogenic processes that involve coordinated changes in cell architecture and gene expression, but how a single receptor can produce these diverse biological outputs is unclear. Here we employ a 3D organotypic model of a ductal epithelium to reveal tissue morphogenic defects result from loss of Notch1, but not Notch1 transcriptional signaling. Instead, defects in duct morphogenesis are driven by dysregulated epithelial cell architecture and mitogenic signaling which result from loss of a transcription-independent Notch1 cortical signaling mechanism that ultimately functions to stabilize adherens junctions and cortical actin. We identify that Notch1 localization and cortical signaling are tied to apical-basal cell restructuring and discover a Notch1-FAM83H interaction underlies stabilization of adherens junctions and cortical actin. Together, these results offer new insights into Notch1 signaling and regulation, and advance a paradigm in which transcriptional and cell adhesive programs might be coordinated by a single receptor.

How complex three-dimensional (3D) organs coordinate cellular morphogenetic events to achieve the correct final form is a central question in development. The question is uniquely tractable in the late Drosophila pupal retina where cells maintain stereotyped contacts as they elaborate the specialized cytoskeletal structures that pattern the apical, basal and longitudinal planes of the epithelium. In this study, we combined cell type-specific genetic manipulation of the cytoskeletal regulator Abelson (Abl) with 3D imaging to explore how the distinct cellular morphogenetic programs of photoreceptors and interommatidial pigment cells coordinately organize tissue pattern to support retinal integrity. Our experiments revealed an unanticipated intercellular feedback mechanism whereby correct cellular differentiation of either cell type can non-autonomously induce cytoskeletal remodeling in the other Abl mutant cell type, restoring retinal pattern and integrity. We propose that genetic regulation of specialized cellular differentiation programs combined with inter-plane mechanical feedback confers spatial coordination to achieve robust 3D tissue morphogenesis.

1.Photosensitive organs are essential for most animals to perceive and respond to their environment. While the gene regulatory networks establishing retinal identity are deeply conserved across metazoans (reviewed in Gehring, 2012; Vopalensky & Kozmik, 2009; Hahn et al., 2023), the retinal architecture varies widely--from invertebrate compound eyes to vertebrate camera-type eyes (Lamb et al., 2007; Schwab, 2017; Arendt & Wittbrodt, 2001). Despite this morphological diversity, early eye anlagen in both, invertebrates and vertebrates, share an initial pseudo-stratified epithelial organization (Weasner & Kumar, 2022; Randlett et al., 2010; Das et al., 2003; Kitambi & Malicki, 2008), which is maintained and elaborated into multi-layered retinae in vertebrates. In contrast, the invertebrate neuroepithelium is re-organized as ommatidia develop. Laminar organisation of the vertebrate retina appears to be a consequence of initial polarisation of the retinal neuroepithelium. This is, however, challenging to test in the organismal context. To address the plasticity of retinal architecture and the impact of epithelial polarity on the structuring of retinal tissue, we take advantage of retinal organoids derived from medaka (Oryzias latipes) (Zilova et al., 2021) that allow to modulate polarity cues and test their impact on the level of epithelialization and structural organisation of the forming retina. We show that under specific culture conditions, medaka retinal organoids undergo a striking morphological switch depending on the level of apico-basal polarity imposed. When polarity cues are continuously provided, a laminated retinal epithelium is established in the organoid. The absence of polarity cues results in the formation of horizontal cellular clusters containing the retinal cell types, which form the vertical retinal column in the developing embryo. We demonstrate that the emergence of this alternative retinal architecture is associated with a loss of epithelial polarity, notably the absence of extracellular matrix (ECM) components, such as laminin, which efficiently rescues lamination. Our findings indicate that tissue-level polarization and lamination in vertebrate retinae require specific extrinsic cues, and that in their absence, differentiating retinal cell types self-organize into structurally distinct, retinal units. This reveals an unexpected plasticity in vertebrate retinal development and indicates a potential for alternative modes of retinal patterning. O_FIG O_LINKSMALLFIG WIDTH=155 HEIGHT=200 SRC="FIGDIR/small/688026v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@100bb1eorg.highwire.dtl.DTLVardef@fceb9borg.highwire.dtl.DTLVardef@d036a7org.highwire.dtl.DTLVardef@1b41483_HPS_FORMAT_FIGEXP M_FIG C_FIG Retinal cells in medaka organoids adopt either a continuous layered epithelium when supported by laminin or a unit-based, ommatidia-like organization when epithelial continuity is lost. This dual outcome suggests that epithelial integrity represents a branching point between vertebrate and invertebrate strategies of retinal patterning, providing an experimental system to replay alternative evolutionary trajectories of eye design.

The myoepithelial cell compartment of the murine postnatal mammary gland is generated from basal cap cells in the terminal end bud and maintained by self-renewal. Transdifferentiation to the luminal lineage does not normally occur but can be induced by DNA damage, luminal cell death or transplantation into a recipient mammary fat pad. Myoepithelial cells cultivated in vitro can also transdifferentiate towards the luminal lineage. Little is known about the molecular mechanisms and gene regulatory networks underlying this plasticity. Using a transgenic mouse (Tg11.5kb-GFP) that marks cap cells with GFP, we discovered that mature myoepithelial cells placed in culture begin to express GFP within [~]24 hrs and later express the Keratin 8 (K8) luminal marker. Cell tracking showed that most K8+ cells arose from GFP+ cells, suggesting that myoepithelial cells de-differentiate towards a progenitor state before changing lineage. Differential gene expression analysis, comparing pure GFP+ cap cells with mature myoepithelial cells, identified multiple transcription factors that iRegulon predicted might regulate the myoepithelial to cap cell transition. Knockout of one of these genes, Regulatory Factor 3 (Rfx3), significantly reduced the population of GFP+ cells and increased differentiation to the K8+ luminal lineage. Rfx3 knockout also reduced mammosphere growth and mammary gland regeneration efficiency in a transplantation assay, but had no effect on proliferation in vitro. Together, these data support a key role for Rfx3 in the stabilization of the mammary basal cell lineages.

Cell proliferation is central to epithelial tissue development, repair and homeostasis. During cell division, small RhoGTPases control both actomyosin dynamics and cell-cell junction remodelling to faithfully segregate the duplicated genome while maintaining tissue polarity and integrity. To decipher the mechanisms of RhoGTPases spatiotemporal regulation during epithelial cell division, we generated a transgenic fluorescently tagged library for Drosophila Rho Guanine exchange factors (GEF) and GTPase activating proteins (GAP), and systematically characterized their endogenous distributions by time- lapse microscopy. Thereby, we unveiled candidate regulators of the interplay between actomyosin and junctional dynamics during epithelial cell division. Building on these findings, we uncovered that during cytokinesis, Cysts and RhoGEF4 play sequential roles in mechanosensing and de novo junction formation, respectively. We foresee that the RhoGEF/GAP library will be a key resource to understand the broad range of biological processes regulated by RhoGTPases.

Gastrulation is considered the sine qua non of embryogenesis, establishing a multidimensional structure and the spatial coordinates upon which all later developmental events transpire. At this time, the embryo adopts a heavy reliance on glucose metabolism to support rapidly accelerating changes in morphology, proliferation, and differentiation. However, it is currently unknown how this conserved metabolic shift maps onto the three-dimensional landscape of the growing embryo and whether it is spatially linked to the orchestrated cellular and molecular processes necessary for gastrulation. Here we identify that glucose is utilised during mouse gastrulation via distinct metabolic pathways to instruct local and global embryonic morphogenesis, in a cell type and stage-specific manner. Through detailed mechanistic studies and quantitative live imaging of mouse embryos, in parallel with tractable in vitro stem cell differentiation models and embryo-derived tissue explants, we discover that cell fate acquisition and the epithelial-to-mesenchymal transition (EMT) relies on the Hexosamine Biosynthetic Pathway (HBP) branch of glucose metabolism, while newly-formed mesoderm requires glycolysis for correct migration and lateral expansion. This regional and tissue-specific difference in glucose metabolism is coordinated with Fibroblast Growth Factor (FGF) activity, demonstrating that reciprocal crosstalk between metabolism and growth factor signalling is a prerequisite for gastrulation progression. We expect these studies to provide important insights into the function of metabolism in other developmental contexts and may help uncover mechanisms that underpin embryonic lethality, cancer, and congenital disease.

After gastrulation, the tailbud of Xenopus laevis emerges as a morphogenetic engine. We find that the transition from late neural to early tailbud stages is characterized by large-scale, counter-rotational tissue flows flanking the blastopore, and spatially coupled to dorsal elongation and ventral compression. Live imaging and quantitative flow analysis reveal that these rotations are maintained over [~]200 {micro}m, suggesting long-range mechanical coupling between dorsal and ventral tissues. High-resolution confocal microscopy shows fibronectin and laminin fibrils radiating ventrally from the blastopore in spoke-like arrays between ectoderm and mesoderm. Perturbations of cell proliferation, radial intercalation, and mediolateral intercalation fail to abolish these flows, whereas targeted disruption of ventral extracellular matrix integrity severely impairs rotational movement. These results identify a previously unrecognized ventral ECM network as a critical mechanical scaffold in regulating posterior tissue rotation, highlighting the interplay between ECM organization, tissue mechanics, and morphogenetic flow during tailbud development.

Blood vessel formation generates unique vascular patterns in each individual. The principles governing the apparent stochasticity of this process remain to be elucidated. Using mathematical methods, we find that the transition between two fundamental vascular morphogenetic programs - sprouting angiogenesis and vascular remodeling - is established by a shift on collective front-rear polarity of endothelial cells. We demonstrate that the competition between biochemical (VEGFA) and mechanical (blood flow-induced shear stress) cues controls this collective polarity shift. Shear stress increases tension at focal adhesions overriding VEGFA-driven collective polarization, which relies on tension at adherens junctions. We propose that vascular morphogenetic cues compete to regulate individual cell polarity and migration through tension shifts that translates into tissue-level emergent behaviors, ultimately leading to uniquely organized vascular patterns.

Symmetry breaking and pattern formation are critical events that occur throughout embryonic development. In early mouse development, a mass of non-polarized epiblast (EPI) cells in the blastocyst forms the egg-cylinder, while cells become apico-basally polarized and build a radial configuration. Yet, what drives the formation of this tissue architecture remains unclear. Here, we demonstrate that orientational patterning of EPI cells is dictated by heterogeneous tissue boundaries, which then defines central lumen positioning. We show that EPI cells progressively orient perpendicular to the visceral endoderm (VE) boundary enriched with laminin and active integrin {beta}1, but parallel to the extraembryonic ectoderm interface. These orientation dynamics are consistent with general boundary-induced alignment effects in polar materials, with a topological defect predicting the position where the pro-amniotic cavity nucleates. Knockout of laminin {gamma}1 and integrin {beta}1 confirms the essential role of adhesion at the EPI-VE-boundary. The established EPI pattern, in turn, facilitates ERK activation to ensure proper EPI maturation. Together, these findings present the mechanistic basis and functional significance of EPI tissue patterning.

Mechanical forces shape cell fate decisions during development and regeneration in many systems. Epithelial lumen volume changes, for example, generate mechanical forces that can be perceived by the surrounding tissue and integrated into cell fate decisions. Similar behavior occurs in regenerating Hydra tissue spheroids, where periodic osmotically driven inflation and deflation cycles generate mechanical stimuli in the form of tissue stretching. Using this model, we investigate how such mechanical input guides the de novo formation of differentiated body parts. We show that the expression of the organizer-defining factor Wnt3 functions as a quantitative readout of cellular stretching and, when supplied externally, enables successful regeneration without mechanical stimulation. This finding represents a previously undescribed cellular mechanism for converting mechanical stimuli to a biochemical signaling readout and guiding cell fate transitions. It also elucidates the role of mechanical oscillations in Hydra regeneration, which long remained unclear. The presence the Wnt/mechanics interplay in Hydra and its relatives underscores the ancient evolutionary history of this crosstalk, possibly extending back to the first metazoans. Since Wnt signaling crosstalks with cellular mechanics in various developmental and disease contexts, it can also represent a conserved feature of this signaling pathway.

After birth, tissues grow continuously until reaching adult size, with each organ exhibiting unique cellular dynamics, growth patterns, and (stem or non-stem) cell sources. Using a suite of experimental and computational multiscale approaches, we found that aortic expansion is guided by specific biological principles and scales with the vertebral column rather than animal body weight. Expansion proceeds via two distinct waves of arterial cell proliferation along blood flow that are spatially stochastic, yet temporally coordinated. Each wave exhibits unique cell cycle kinetics and properties, with the first wave exhibiting cell cycle durations as fast as 6 hours. Single-cell RNA sequencing showed changes in fatty acid metabolism concomitant with an increase in cell size. Mathematical modeling and experiments indicated endothelial cell extrusion is essential for homeostatic aortic growth and balancing excess proliferation. In a genetic model of achondroplasia, the aorta achieves proper scaling through enhanced cell extrusion while maintaining normal proliferation dynamics. Collectively, these results provide a blueprint of the principles that orchestrate aortic growth which depends entirely on differentiated cell proliferation rather than resident stem cells.

Central nervous system (CNS) architecture is established early by the organization and proliferative behavior of neuroepithelial (NE) cells, which form a pseudostratified epithelium during neural tube (NT) formation. In neurogenesis, newborn neurons have to detach and exit the neuroepithelium, in a process that requires the coordinated disassembly of apical junctions and the centrosome-cilia module. Although Rootletin--the structural component of the ciliary rootlet and centriolar linker--is classically viewed as a static mechanical element, its behavior in NE cells has not been described in detail. Here, we uncover a conserved, dynamic form of Rootletin fiber organization that remodels in synchrony with NE cell morphogenesis. We show that, in NE cells in interphase, Rootletin fibers extend from the basal body through the apical process toward the nucleus, and that Rootletin maintains its fibrous conformation throughout mitosis. As NE cells initiate apical constriction, Rootletin fibers retract from the apical process and assemble into an anisotropic rim-like structure that aligns with the apical junctional complex. This remodeling is coordinated with microtubule stabilization in low-tension apical endfeet and occurs prior to Lzts1 expression and increased actomyosin contractility. Forced neuronal delamination via Neurogenin-2 or Lzts1 promotes Rootletin rim formation and Rootletin alignment with the apical endfoot cortex. Finally, we show that Zika virus NS5 protein can aberrantly associate with all conformational states of Rootletin fibers, providing a potential mechanical link between ZikaV infection and premature delamination. Together, our findings identify Rootletin as a dynamically regulated cytoskeletal scaffold that orchestrates apical surface remodeling in NE cells and identify a potential mechanism by which ZikaV disrupts neurodevelopment.

The mammalian eye develops in concert with coordinated growth and remodeling of three vascular networks: the hyaloid vasculature, the choroid and retinal plexus. While retinal and choroidal systems support visual function in the mature eye, the hyaloid network plays a vital yet temporary role supporting the developing lens and inner retina. Regression of the hyaloid network is essential for optical clarity, yet the mechanisms guiding the process remain incompletely understood. Using single-cell RNA sequencing, we show that postnatal mouse hyaloid cells are broadly senescent. Hyaloid vascular smooth muscle, endothelial and immune cells display cell-cycle arrest marked by Cdkn1a with the expression of SASP factors. Genetic ablation of Cdkn1a impedes normal hyaloid regression, demonstrating that developmental senescence is essential for vascular remodeling and functions alongside apoptosis and macrophage-mediated clearance. These findings identify an unrecognized senescence-driven mechanism orchestrating hyaloid involution during ocular development, broadening the understanding of vascular remodeling in the eye.

Future boundaries of skeletal muscle segments are determined in the presomitic mesoderm (PSM). Within the PSM, future somitic cells undergo significant changes in both morphology and position. How such large-scale cellular changes are coordinated and the effect on the future border formation is unknown. We find that cellular rearrangements differ between cell populations within the PSM. In contrast to lateral somitic cells, which display less organized rearrangement, the adaxial cell layer undergoes significant tissue shearing with dorsal and ventral cells sliding posteriorly. This shear is generated by orientated intercalations of dorsally and ventrally located adaxial cells, which induces a chevron-like pattern. We find Shh signaling is required for the tissue shear and morphogenesis of adaxial cells. In particular, we observe Shh-dependent polarized recruitment of non-muscle myosin IIA drives apical constrictions, and thus the intercalations and shear. This reveals a novel role for Shh in regulating cell mechanics in the PSM.

During collective cell invasion, epithelial-to-mesenchymal transition (EMT) and morphogen signalling-mediated cell fate specification are traditionally viewed as a linear cascade: morphogens drive cell fates that activate EMT programs. Here, we uncover reciprocal coupling between EMT initiation and BMP signalling mediated by SNAI2 and SMAD1. Using substrate-induced EMT in ex vivo explants, we demonstrate that EMT initiation upregulates SMAD1 expression, priming cells for BMP signalling competence across germ layers. Single-cell RNA sequencing reveals SNAI2 and SMAD1 co-expression in EMT initiation regions, and SNAI2 overexpression is sufficient to induce ectopic SMAD1 expression in vivo. While BMP signalling is dispensable for EMT initiation, it regulates cell fate proportions, dispersal dynamics, precursor region depletion rates, and migration directionality. This coupling provides a mechanism for synchronising cell fate specification with invasion progression during axis elongation, positioning EMT as a process that actively modulates morphogen competence to coordinate tissue-level cell behaviours during collective cell invasion. HighlightsO_LIEMT initiation directly primes BMP pathway competence. C_LIO_LISNAI2 overexpression is sufficient to drive ectopic SMAD1 expression. C_LIO_LIBMP signalling is spatiotemporally restricted to EMT initiation zones. C_LIO_LIBMP signalling tunes cell fate proportions and invasion dynamics. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/691808v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@1ae1e78org.highwire.dtl.DTLVardef@526260org.highwire.dtl.DTLVardef@b892f1org.highwire.dtl.DTLVardef@1b74815_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mucociliary epithelia are found across different organs in animals, where they release bioactive substances and generate extracellular fluid flows. One key function of mucociliary epithelia is the clearance of pathogens, e.g. in the vertebrate lung and the epidermis of amphibian tadpoles. Mucociliary clearance relies on the correct balance between secretory cells that release mucus, ciliated cells that generate fluid flow as well as specialized cell types, including pH-regulating ionocytes and basal stem cells. Notch signaling, Hes repressors and cell type-inducing transcription factors (e.g. Foxi1, Mcidas, Spdef and Tp63) regulate cell fates and cell type proportions across mucociliary systems. Lateral inhibition was proposed to control mucociliary cell fates, but current models cannot explain how more than two cell types are generated and how Hes genes are employed as mediators during patterning. Using the Xenopus tadpole epidermis, we addressed these open questions in mucociliary biology through a combination of in vivo and organoid experiments, time-resolved transcriptomic studies and mathematical modeling. This revealed that ionocytes, ciliated cells, secretory cells and basal cells are preferentially specified at different time points and Notch signaling levels via sequentially expressed Hes factors. We termed this mode of patterning "competitive de-repression", because cell fates are selected by suppression of alternative fate choices, and demonstrate that this relies on differential active repression of cell fate transcription factors. Mathematical modeling further indicated the need for a positively Notch-regulated patterning factor, and we provide evidence that Spdef mediates Notch input for secretory and basal cell specification. Collectively, this work presents a coherent model for Notch- and Hes-mediated mucociliary cell fate specification in a vertebrate tissue, which allows for the specification of more than two cell fates within the Notch lateral-inhibition paradigm.