Aim 1: Analyzing the formation of the epithelium of visceral endoderm surrounding the internal cavity in locally-instructed embryoids
Sub-aim 1.1: Investigating the formation of an endodermal epithelium around the cavity
Rationale: VE is derived from PrE, an epithelial tissue that separates the ICM from the blastocoel. PrE and Epiblast (Epi) cells exist in a ‘salt and pepper’ manner in the ICM by E3.5, which can be visualized by the complementary expression patterns of Gata6 (PrE) and Nanog (Epi) (Chazaud et al., 2006). The PrE cells are sorted from the ICM through differential adhesion (Gerbe et al., 2008) and cortical tension (Moore et al., 2009) and become a morphologically distinct tissue by E4.5. PrE/VE epithelial tissue surrounds in the …show more content…
Analysis of DBA+ cells in relation to the position of the cavity will be completed using compound and confocal microscopy with chromatic and fluorescent resolution of DBA labeling. Expected outcomes: Hypothesis 1: All epithelial cells surrounding the cavity are DBA lectin+. No DBA lectin+ cells in the mass of the EB. Hypothesis 2: ‘Salt and pepper’ DBA lectin + cells in the EB progressively invade the pre-existing epithelium lining the cavity. The resulting epithelium has patchy DBA lectin staining due to some pre-existing epiblast cells contributing to the epithelium. Hypothesis 3: Initially, the cavity is not lined with an epithelium. As PrE cells are sorted from the Epi cells, a DBA lectin+ epithelium forms around the …show more content…
Manipulation of Wnt signaling through alterations in embryoid instruction followed by analysis with markers of AVE (Dkk1) and primitive streak (Wnt3a) in embryoids fixed 1, 2, and 3 days following instruction will reveal the effects of Wnt signaling on AVE behavior and AP axis development. Expected results: Lefty1 should be expressed earlier than the other inhibitors and will identify a DVE-like cell population within the PrE that may be asymmetrically localized to the future anterior side well before expression of EmVE or PS markers. Dkk1 and Cer1 expression patterns will reveal an AVE-like cell population. Wnt signaling and localized activity of its inhibitor Dkk1 are expected to be essential for orienting the AP axis. Despite initial patterning differences between our embryoids and mouse embryos, these data may prove a useful model to study the requirements of AVE behavior during the AP axis formation in
Sub-aim 1.1: Investigating the formation of an endodermal epithelium around the cavity
Rationale: VE is derived from PrE, an epithelial tissue that separates the ICM from the blastocoel. PrE and Epiblast (Epi) cells exist in a ‘salt and pepper’ manner in the ICM by E3.5, which can be visualized by the complementary expression patterns of Gata6 (PrE) and Nanog (Epi) (Chazaud et al., 2006). The PrE cells are sorted from the ICM through differential adhesion (Gerbe et al., 2008) and cortical tension (Moore et al., 2009) and become a morphologically distinct tissue by E4.5. PrE/VE epithelial tissue surrounds in the …show more content…
Analysis of DBA+ cells in relation to the position of the cavity will be completed using compound and confocal microscopy with chromatic and fluorescent resolution of DBA labeling. Expected outcomes: Hypothesis 1: All epithelial cells surrounding the cavity are DBA lectin+. No DBA lectin+ cells in the mass of the EB. Hypothesis 2: ‘Salt and pepper’ DBA lectin + cells in the EB progressively invade the pre-existing epithelium lining the cavity. The resulting epithelium has patchy DBA lectin staining due to some pre-existing epiblast cells contributing to the epithelium. Hypothesis 3: Initially, the cavity is not lined with an epithelium. As PrE cells are sorted from the Epi cells, a DBA lectin+ epithelium forms around the …show more content…
Manipulation of Wnt signaling through alterations in embryoid instruction followed by analysis with markers of AVE (Dkk1) and primitive streak (Wnt3a) in embryoids fixed 1, 2, and 3 days following instruction will reveal the effects of Wnt signaling on AVE behavior and AP axis development. Expected results: Lefty1 should be expressed earlier than the other inhibitors and will identify a DVE-like cell population within the PrE that may be asymmetrically localized to the future anterior side well before expression of EmVE or PS markers. Dkk1 and Cer1 expression patterns will reveal an AVE-like cell population. Wnt signaling and localized activity of its inhibitor Dkk1 are expected to be essential for orienting the AP axis. Despite initial patterning differences between our embryoids and mouse embryos, these data may prove a useful model to study the requirements of AVE behavior during the AP axis formation in