The segregation of Mib-GFP into the apical daughter was apparent at the time of birth (Figure 7F; Movie S3; ∼24 min). However, in the par-3 morphant, Mib-GFP selleck chemical was present in both the apical and basal daughter at the time of their birth ( Figure 7G; Movie S4; ∼18 min). Together, these results suggest that Mib is unequally segregated into the apical daughter upon asymmetric division in a Par-3-dependent manner, and such asymmetry is maintained in the daughter cells. In agreement with the disrupted Mib localization in the par-3 morphant,

we found that the asymmetry of both her4.1 ( Figures 8A–8C) and dla ( Figures 8D–8F) expression was lost in par-3-deficient embryos, demonstrating that Par-3 is essential for establishing Notch asymmetry in paired siblings. Mib mislocalization and lost asymmetry of Notch signaling components in par-3 morphants could result in either increased or diminished Notch activity in both daughter cells, which would in turn impact progenitor GW786034 supplier fate choice differently. To determine how Notch activity and cell fate might be affected in par-3-deficient embryos, we first analyzed the overall expression level of her4.1, dla, and the pan-neuronal marker Hu. These analyses showed that her4.1 expression ( Figures 8G and 8H; 88%, n = 16) was

increased, whereas neuronal numbers ( Figures 8I and 8J; 83%, n = 18) were Oxymatrine decreased in the par-3 morphant. In contrast the expression of dla was not changed significantly (

Figures 8K and 8L; 100%, n = 15). This is surprising, given the increase of her4.1 expression and the known negative feedback regulation of Notch ligands by hes/her genes. Quantitative reverse-transcription PCR analysis further confirmed the significant upregulation of her4.1 (and her6) mRNA expression, whereas the mRNA levels of dla, dld, notch1a, and notch1b were unchanged ( Figure 8M) ( Table S1). Thus, par-3 function is essential to restrict Notch activity, and is somehow also required for the feedback repression of Notch ligand expression. To understand the nature of these par-3 functions, we asked whether they are dependent on mib. In the mib−/− mutant, consistent with the disruption of Notch signaling, her4.1 expression was significantly reduced ( Figures S7J and S7K). The par-3 and mib double-deficient embryos also showed reduced her4.1 expression ( Figure S7M) that was indistinguishable from the mib−/− single mutant ( Figure S7K). This result indicates that Par-3 restricts Notch activity through Mib. Although the diminished Notch activation in the mib−/− mutant is expected to upregulate Notch ligand expression via the negative feedback loop, this was not what we observed. Instead, the dla mRNA level was significantly reduced in the mib−/− mutant ( Figures S7N and S7O) as well as in the par-3 and mib double-deficient embryos ( Figure S7Q).