Disruption of the nectin-afadin complex recapitulates features of the human cleft lip/palate syndrome CLPED1.

Cleft palate (CP), one of the most common congenital conditions, arises from failures in secondary palatogenesis during embryonic development. Several human genetic syndromes featuring CP and ectodermal dysplasia have been linked to mutations in genes regulating cell-cell adhesion, yet mouse models have largely failed to recapitulate these findings. Here, we use in utero lentiviral-mediated genetic approaches in mice to provide the first direct evidence that the nectin-afadin axis is essential for proper palate shelf elevation and fusion. Using this technique, we demonstrate that palatal epithelial conditional loss of afadin (Afdn) - an obligate nectin- and actin-binding protein - induces a high penetrance of CP, not observed when Afdn is targeted later using Krt14-Cre We implicate Nectin1 and Nectin4 as being crucially involved, as loss of either induces a low penetrance of mild palate closure defects, while loss of both causes severe CP with a frequency similar to Afdn loss. Finally, expression of the human disease mutant NECTIN1W185X causes CP with greater penetrance than Nectin1 loss, suggesting this alteration may drive CP via a dominant interfering mechanism.

The cell adhesion molecule Echinoid promotes tissue survival and separately restricts tissue overgrowth in Drosophila imaginal discs.

The interactions that cells in Drosophila imaginal discs have with their neighbors are known to regulate their ability to survive. In a screen of genes encoding cell surface proteins for gene knockdowns that affect the size or shape of mutant clones, we found that clones of cells with reduced levels of echinoid (ed) are fewer, smaller, and can be eliminated during development. In contrast, discs composed mostly of ed mutant tissue are overgrown. We find that ed mutant tissue has lower levels of the anti-apoptotic protein Diap1 and has increased levels of apoptosis which is consistent with the observed underrepresentation of ed mutant clones and the slow growth of ed mutant tissue. The eventual overgrowth of ed mutant tissue results not from accelerated growth, but from prolonged growth resulting from a failure to arrest growth at the appropriate final size. Ed has previously been shown to physically interact with multiple Hippo-pathway components and it has been proposed to promote Hippo pathway signaling, to exclude Yorkie (Yki) from the nucleus, and restrain the expression of Yki-target genes. We did not observe changes in Yki localization in ed mutant tissue and found decreased levels of expression of several Yorkie-target genes, findings inconsistent with the proposed effect of Ed on Yki. We did, however, observe increased expression of several Yki-target genes in wild-type cells neighboring ed mutant cells, which may contribute to elimination of ed mutant clones. Thus, ed has two distinct functions: an anti-apoptotic function by maintaining Diap1 levels, and a function to arrest growth at the appropriate final size. Both of these are unlikely to be explained by a simple effect on the Hippo pathway.

AGS3 antagonizes LGN to balance oriented cell divisions and cell fate choices in mammalian epidermis.

Oriented cell divisions balance self-renewal and differentiation in stratified epithelia such as the skin epidermis. During peak epidermal stratification, the distribution of division angles among basal keratinocyte progenitors is bimodal, with planar and perpendicular divisions driving symmetric and asymmetric daughter cell fates, respectively. An apically restricted, evolutionarily conserved spindle orientation complex that includes the scaffolding protein LGN/Pins/Gpsm2 plays a central role in promoting perpendicular divisions and stratification, but why only a subset of cell polarize LGN is not known. Here, we demonstrate that the LGN paralog, AGS3/Gpsm1, is a novel negative regulator of LGN and inhibits perpendicular divisions. Static and ex vivo live imaging reveal that AGS3 overexpression displaces LGN from the apical cortex and increases planar orientations, while AGS3 loss prolongs cortical LGN localization and leads to a perpendicular orientation bias. Genetic epistasis experiments in double mutants confirm that AGS3 operates through LGN. Finally, clonal lineage tracing shows that LGN and AGS3 promote asymmetric and symmetric fates, respectively, while also influencing differentiation through delamination. Collectively, these studies shed new light on how spindle orientation influences epidermal stratification.

A flagellate-to-amoeboid switch in the closest living relatives of animals.

Amoeboid cell types are fundamental to animal biology and broadly distributed across animal diversity, but their evolutionary origin is unclear. The closest living relatives of animals, the choanoflagellates, display a polarized cell architecture (with an apical flagellum encircled by microvilli) that resembles that of epithelial cells and suggests homology, but this architecture differs strikingly from the deformable phenotype of animal amoeboid cells, which instead evoke more distantly related eukaryotes, such as diverse amoebae. Here, we show that choanoflagellates subjected to confinement become amoeboid by retracting their flagella and activating myosin-based motility. This switch allows escape from confinement and is conserved across choanoflagellate diversity. The conservation of the amoeboid cell phenotype across animals and choanoflagellates, together with the conserved role of myosin, is consistent with homology of amoeboid motility in both lineages. We hypothesize that the differentiation between animal epithelial and crawling cells might have evolved from a stress-induced switch between flagellate and amoeboid forms in their single-celled ancestors.

Telophase correction refines division orientation in stratified epithelia.

During organogenesis, precise control of spindle orientation balances proliferation and differentiation. In the developing murine epidermis, planar and perpendicular divisions yield symmetric and asymmetric fate outcomes, respectively. Classically, division axis specification involves centrosome migration and spindle rotation, events occurring early in mitosis. Here, we identify a novel orientation mechanism which corrects erroneous anaphase orientations during telophase. The directionality of reorientation correlates with the maintenance or loss of basal contact by the apical daughter. While the scaffolding protein LGN is known to determine initial spindle positioning, we show that LGN also functions during telophase to reorient oblique divisions toward perpendicular. The fidelity of telophase correction also relies on the tension-sensitive adherens junction proteins vinculin, α-E-catenin, and afadin. Failure of this corrective mechanism impacts tissue architecture, as persistent oblique divisions induce precocious, sustained differentiation. The division orientation plasticity provided by telophase correction may enable progenitors to adapt to local tissue needs.

RARα and RARγ reciprocally control K5+ progenitor cell expansion in developing salivary glands.

Understanding the mechanisms of controlled expansion and differentiation of basal progenitor cell populations during organogenesis is essential for developing targeted regenerative therapies. Since the cytokeratin 5-positive (K5+) basal epithelial cell population in the salivary gland is regulated by retinoic acid signaling, we interrogated how isoform-specific retinoic acid receptor (RAR) signaling impacts the K5+ cell population during salivary gland organogenesis to identify RAR isoform-specific mechanisms that could be exploited in future regenerative therapies. In this study, we utilized RAR isoform-specific inhibitors and agonists with murine submandibular salivary gland organ explants. We determined that RARα and RARγ have opposing effects on K5+ cell cycle progression and cell distribution. RARα negatively regulates K5+ cells in both whole organ explants and in isolated epithelial rudiments. In contrast, RARγ is necessary but not sufficient to positively maintain K5+ cells, as agonism of RARγ alone failed to significantly expand the population. Although retinoids are known to stimulate differentiation, K5 levels were not inversely correlated with differentiated ductal cytokeratins. Instead, RARα agonism and RARγ inhibition, corresponding with reduced K5, resulted in premature lumenization, as marked by prominin-1. With lineage tracing, we demonstrated that K5+ cells have the capacity to become prominin-1+ cells. We conclude that RARα and RARγ reciprocally control K5+ progenitor cells endogenously in the developing submandibular salivary epithelium, in a cell cycle-dependent manner, controlling lumenization independently of keratinizing differentiation. Based on these data, isoform-specific targeting RARα may be more effective than pan-RAR inhibitors for regenerative therapies that seek to expand the K5+ progenitor cell pool. SUMMARY STATEMENT: RARα and RARγ reciprocally control K5+ progenitor cell proliferation and distribution in the developing submandibular salivary epithelium in a cell cycle-dependent manner while regulating lumenization independently of keratinizing differentiation.