RESUMO
Anterior definitive endoderm (ADE) is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other visceral organs. Here we describe a method for the differentiation of mouse embryonic stem (ES) cells to endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (aggregation or embryoid body) culture and in a serum-free adherent monolayer culture. Purified ES cell-derived ADE cells appear committed to endodermal fates and can undergo further differentiation in vitro towards liver and pancreas with enhanced efficiency.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Animais , Adesão Celular , Agregação Celular , Embrião de Mamíferos/citologia , CamundongosRESUMO
The mammalian acrosome reaction is a specialised exocytotic event. Although molecular motors are known to be involved in exocytosis in many cell types, their potential involvement in the acrosome reaction has remained unknown. Here, it has been shown that actin is localised within the equatorial segment and in the marginal acrosomal ridge of the heads of unreacted bull spermatozoa. Myosins IIA and IIB are found within the anterior acrosomal margins of virtually all sperm cells and, less prominently, within the equatorial segment. Tubulin was detected in the equatorial segment and around the periphery of the acrosome while kinesin was prominent in the equatorial segment. After induction of the acrosome reaction by means of the calcium ionophore A23187, the number of cells exhibiting actin fluorescence intensity in the anterior acrosomal margin decreased four-fold and those displaying equatorial segment fluorescence decreased 3.5-fold; myosin IIA immunofluorescence decreased in intensity with most spermatozoa losing equatorial staining, whereas there was little change in the distribution or intensity of myosin IIB immunofluorescence, except for approximately 20% decrease in the number of cells exhibiting acrosomal staining. Tubulin became largely undetectable within the head and kinesin staining spread rostrally over the main acrosome region. A possible sequence of events that ties in these observations of molecular motor involvement with the known participation of SNARE proteins is provided.
Assuntos
Reação Acrossômica , Actinas/metabolismo , Cinesinas/metabolismo , Miosinas/metabolismo , Espermatozoides/metabolismo , Tubulina (Proteína)/metabolismo , Reação Acrossômica/efeitos dos fármacos , Animais , Calcimicina/farmacologia , Cálcio/metabolismo , Bovinos , Exocitose , Ionóforos/farmacologia , Masculino , Microscopia Confocal , Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Transporte Proteico , Espermatozoides/efeitos dos fármacosRESUMO
The use of embryonic stem cell (ESC) differentiation to generate functional hepatic or pancreatic progenitors and as a tool for developmental biology is limited by an inability to isolate in vitro equivalents of regionally specified anterior definitive endoderm (ADE). To address this, we devised a strategy using a fluorescent reporter gene under the transcriptional control of the anterior endoderm marker Hex alongside the definitive mesendoderm marker Cxcr4. Isolation of Hex(+)Cxcr4(+) differentiating ESCs yielded a population expressing ADE markers that both can be expanded and is competent to undergo differentiation toward liver and pancreatic fates. Hex reporter ESCs were also used to define conditions for ADE specification in serum-free adherent culture and revealed an unexpected role for FGF signaling in the generation of ADE. Our findings in defined monolayer differentiation suggest FGF signaling is an important regulator of early anterior mesendoderm differentiation rather than merely a mediator of morphogenetic movement.
