RESUMEN
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.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Animales , Proteínas de Ciclo Celular/metabolismo , Proteínas Portadoras/metabolismo , División Celular , Epidermis/metabolismo , Diferenciación Celular/genética , Huso Acromático/metabolismo , Polaridad Celular , Mamíferos/metabolismoRESUMEN
Whilst tissues form during development, some cells are extruded from epithelial monolayers. Rather than dying or differentiating, a new study shows that displaced cells can reintegrate after dividing. Surprisingly, this 'intrusion' pathway shares common features with axon guidance.
Asunto(s)
Orientación del Axón , Axones , EpitelioRESUMEN
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.
Asunto(s)
Labio Leporino/genética , Fisura del Paladar/genética , Proteínas de Microfilamentos/genética , Mutación/genética , Nectinas/genética , Animales , Células Epiteliales/metabolismo , Humanos , Integrasas/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Organogénesis , Hueso Paladar/embriología , Penetrancia , SíndromeRESUMEN
Stem cells in stratified epithelia are generally believed to adhere to a non-hierarchical single-progenitor model. Using lineage tracing and genetic label-retention assays, we show that the hard palatal epithelium of the oral cavity is unique in displaying marked proliferative heterogeneity. We identify a previously uncharacterized, infrequently-dividing stem cell population that resides within a candidate niche, the junctional zone (JZ). JZ stem cells tend to self-renew by planar symmetric divisions, respond to masticatory stresses, and promote wound healing, whereas frequently-dividing cells reside outside the JZ, preferentially renew through perpendicular asymmetric divisions, and are less responsive to injury. LRIG1 is enriched in the infrequently-dividing population in homeostasis, dynamically changes expression in response to tissue stresses, and promotes quiescence, whereas Igfbp5 preferentially labels a rapidly-growing, differentiation-prone population. These studies establish the oral mucosa as an important model system to study epithelial stem cell populations and how they respond to tissue stresses.
Asunto(s)
Mucosa Bucal/citología , Mucosa Bucal/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , División Celular/fisiología , Linaje de la Célula/fisiología , Células Cultivadas , Femenino , Citometría de Flujo , Fluorescencia , Inmunohistoquímica , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
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.
Asunto(s)
Células Epidérmicas/citología , Células Epiteliales/citología , Telofase/fisiología , Actomiosina/fisiología , Anafase , Animales , Autorrenovación de las Células , Forma de la Célula , Citoesqueleto/ultraestructura , Epidermis/embriología , Femenino , Genes Reporteros , Microscopía Intravital , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/fisiología , Conformación Proteica , Interferencia de ARN , Huso Acromático/ultraestructura , Vinculina/genética , Vinculina/fisiología , alfa Catenina/genética , alfa Catenina/fisiologíaRESUMEN
Oral epithelia protect against constant challenges by bacteria, viruses, toxins and injury while also contributing to the formation of ectodermal appendages such as teeth, salivary glands and lingual papillae. Despite increasing evidence that differentiation pathway genes are frequently mutated in oral cancers, comparatively little is known about the mechanisms that regulate normal oral epithelial development. Here, we characterize oral epithelial stratification and describe multiple distinct functions for the mitotic spindle orientation gene LGN (Gpsm2) in promoting differentiation and tissue patterning in the mouse oral cavity. Similar to its function in epidermis, apically localized LGN directs perpendicular divisions that promote stratification of the palatal, buccogingival and ventral tongue epithelia. Surprisingly, however, in dorsal tongue LGN is predominantly localized basally, circumferentially or bilaterally and promotes planar divisions. Loss of LGN disrupts the organization and morphogenesis of filiform papillae but appears to be dispensable for embryonic hair follicle development. Thus, LGN has crucial tissue-specific functions in patterning surface ectoderm and its appendages by controlling division orientation.
Asunto(s)
Proteínas Portadoras/metabolismo , Epitelio/metabolismo , Folículo Piloso/embriología , Folículo Piloso/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Inmunohistoquímica , Ratones , Microscopía Electrónica de Rastreo , Morfogénesis/genética , Morfogénesis/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Papilas Gustativas/embriología , Papilas Gustativas/metabolismo , Lengua/embriología , Lengua/metabolismoRESUMEN
Alterations in genes that regulate brain size may contribute to both microcephaly and brain tumor formation. Here, we report that Aspm, a gene that is mutated in familial microcephaly, regulates postnatal neurogenesis in the cerebellum and supports the growth of medulloblastoma, the most common malignant pediatric brain tumor. Cerebellar granule neuron progenitors (CGNPs) express Aspm when maintained in a proliferative state by sonic hedgehog (Shh) signaling, and Aspm is expressed in Shh-driven medulloblastoma in mice. Genetic deletion of Aspm reduces cerebellar growth, while paradoxically increasing the mitotic rate of CGNPs. Aspm-deficient CGNPs show impaired mitotic progression, altered patterns of division orientation and differentiation, and increased DNA damage, which causes progenitor attrition through apoptosis. Deletion of Aspm in mice with Smo-induced medulloblastoma reduces tumor growth and increases DNA damage. Co-deletion of Aspm and either of the apoptosis regulators Bax or Trp53 (also known as p53) rescues the survival of neural progenitors and reduces the growth restriction imposed by Aspm deletion. Our data show that Aspm functions to regulate mitosis and to mitigate DNA damage during CGNP cell division, causes microcephaly through progenitor apoptosis when mutated, and sustains tumor growth in medulloblastoma.