_:b5405898 "MUPP1 is a paralogue of Patj, which regulates apico-basal polarity and apical membrane organization in cultured cells as well as in Drosophila (Bachmann et al., >>2001<<; Shin et al., 2005; Richard et al., 2006; Sugihara-Mizuno et al., 2007)." . _:b406456719 "3"^^ . _:b406456812 . _:b406456718 "3"^^ . _:b5405894 . . _:b406456762 . _:b406456809 . . _:b406456792 . . _:b406456743 . _:b406456808 . _:b5405889 . _:b406456715 "4"^^ . _:b406456726 . . _:b5405890 . _:b406456811 . _:b5405900 "MUPP1 is a paralogue of Patj, which regulates apico-basal polarity and apical membrane organization in cultured cells as well as in Drosophila (Bachmann et al., 2001; Shin et al., 2005; Richard et al., >>2006<<; Sugihara-Mizuno et al., 2007)." . _:b406456714 "5"^^ . _:b406456725 "3"^^ . _:b406456810 . _:b406456766 . _:b406456724 "3"^^ . _:b406456727 "3"^^ . . . _:b406456761 . _:b406456726 "3"^^ . _:b406456724 . _:b406456764 . . _:b406456721 "3"^^ . _:b5405903 . . . _:b406456720 "3"^^ . _:b5405889 "Cells were cultured as described previously (Tanoue and Takeichi, >>2004<<). For isolating stable cDNA transfectants, 400 \u00B5g/ml G418 (Invitrogen) or 250 \u00B5g/ml hygromycin B (Invitrogen) were used." . . _:b406456723 "3"^^ . _:b5405901 . _:b5405873 _:b5405874 . _:b5405873 _:b5405875 . . _:b5405873 _:b5405876 . _:b406456722 "3"^^ . . _:b5405873 _:b5405877 . _:b5405873 _:b5405878 . _:b5405873 _:b5405879 . _:b406456733 "2"^^ . _:b5405873 "introduction" . _:b5405873 _:b5405880 . _:b5405873 _:b5405881 . _:b5405873 _:b5405882 . _:b5405873 _:b5405883 . _:b406456732 "2"^^ . _:b5405873 _:b5405884 . _:b5405873 _:b5405885 . _:b5405873 _:b5405886 . _:b5405899 . _:b5405873 _:b5405887 . _:b5405890 "Immunostaining was performed as described previously (Tanoue and Takeichi, >>2004<<). Cell aggregation assays were performed as described previously (Nagafuchi et al., 1987)." . _:b406456735 "2"^^ . . _:b406456734 "2"^^ . _:b5405899 "MUPP1 is a paralogue of Patj, which regulates apico-basal polarity and apical membrane organization in cultured cells as well as in Drosophila (Bachmann et al., 2001; Shin et al., >>2005<<; Richard et al., 2006; Sugihara-Mizuno et al., 2007)." . . . . _:b5405896 "The discontinuous distribution of Fat and Dachsous was also observed for the Drosophila homologues (Ma et al., >>2003<<), suggesting that this unique localization pattern reflects certain conserved nature of these molecules." . _:b406456729 "2"^^ . _:b406456795 . . _:b5405885 "In mammals, four types of Fats have been identified: Fat1, Fat2, Fat3, and Fat4 (Rock et al., 2005; Tanoue and Takeichi, >>2005<<). Among them, Fat4 is thought to be the vertebrate counterpart of Drosophila Fat based on their sequence similarity." . _:b406456728 "3"^^ . . . _:b406456720 . _:b406456775 . _:b406456731 "2"^^ . . _:b5405901 "MUPP1 is a paralogue of Patj, which regulates apico-basal polarity and apical membrane organization in cultured cells as well as in Drosophila (Bachmann et al., 2001; Shin et al., 2005; Richard et al., 2006; Sugihara-Mizuno et al., >>2007<<). The interaction between Fat4 and MUPP1 was confirmed by detecting MUPP1 from the pulled-down materials with anti-MUPP1 antibodies (Fig." . _:b406456730 "2"^^ . _:b406456796 . . _:b406456741 "2"^^ . . _:b5405888 "materials and methods" . _:b406456740 "2"^^ . _:b5405885 . _:b5405897 "In Drosophila, in the absence of fat, the level of Dachsous protein is down-regulated; conversely, without dachsous, the Fat protein level is up-regulated (Ma et al., >>2003<<). To test whether such a relationship also exists for the mammalian homologues, we depleted these molecules in the embryonic cortices by electroporating siRNAs targeting their transcripts." . . _:b406456743 "2"^^ . _:b406456785 . _:b406456742 "2"^^ . _:b406456800 . _:b406456733 . . _:b406456737 "2"^^ . . _:b406456736 "2"^^ . _:b406456739 "2"^^ . . _:b406456752 . _:b5405904 "Because the Stardust complex has been reported to regulate the apical membrane organization in Drosophila (Pellikka et al., >>2002<<; Hong et al., 2003; Richard et al., 2006), we asked whether Fat4 and Dachsous1 had similar functions." . _:b406456738 "2"^^ . _:b5405882 . _:b406456749 "2"^^ . . _:b406456748 "2"^^ . _:b5405908 . . . _:b406456748 . _:b406456751 "2"^^ . _:b406456750 "2"^^ . . _:b5405898 . _:b5405905 "Because the Stardust complex has been reported to regulate the apical membrane organization in Drosophila (Pellikka et al., 2002; Hong et al., >>2003<<; Richard et al., 2006), we asked whether Fat4 and Dachsous1 had similar functions." . _:b406456745 "2"^^ . _:b406456801 . . _:b406456744 "2"^^ . _:b406456747 "2"^^ . _:b406456750 . . _:b406456746 "2"^^ . _:b5405875 . _:b406456774 . _:b406456790 . _:b406456757 "2"^^ . _:b406456810 . _:b406456756 "2"^^ . _:b406456715 . _:b406456728 . _:b406456759 "2"^^ . _:b406456758 "2"^^ . _:b5405908 "We did not examine the effect of MUPP1 removal because of the presence of its paralogue, Patj, in the cortex (Srinivasan et al., >>2008<<)." . _:b5405878 . . _:b406456772 . _:b5405895 "results and discussion" . _:b406456753 "2"^^ . . . _:b406456752 "2"^^ . _:b406456727 . _:b406456791 . _:b406456755 "2"^^ . _:b406456780 . _:b406456739 . _:b406456754 "2"^^ . . _:b406456765 "2"^^ . _:b5405879 "Fat is well known as a regulator of cell proliferation and planar cell polarity (PCP; Bryant et al., 1988; Mahoney et al., 1991; Yang et al., 2002; Ma et al., >>2003<<; Casal et al., 2006)." . _:b406456764 "2"^^ . _:b406456735 . _:b5405882 "dachsous mutants show several morphogenetic defects (Clark et al., 1995), and both fat and dachsous mutants commonly exhibit defects in PCP (Yang et al., >>2002<<; Ma et al., 2003)." . . . _:b406456805 . _:b406456767 "2"^^ . _:b5405888 _:b5405892 . _:b5405895 _:b5405912 . . _:b5405895 _:b5405913 . _:b5405888 _:b5405893 . . _:b5405888 _:b5405894 . . _:b5405895 _:b5405914 . _:b406456766 "2"^^ . _:b5405888 _:b5405889 . . _:b5405888 _:b5405890 . _:b406456758 . "PMC0" . _:b406456809 . _:b406456761 "2"^^ . _:b406456717 . _:b5405888 _:b5405891 . _:b406456734 . _:b5405895 _:b5405904 . _:b5405895 _:b5405905 . _:b406456745 . . _:b5405873 . _:b5405895 _:b5405906 . . _:b406456760 "2"^^ . _:b406456716 . _:b5405895 _:b5405907 . _:b5405895 _:b5405908 . _:b5405895 _:b5405909 . _:b5405895 _:b5405910 . _:b5405895 _:b5405911 . _:b5405893 "Detailed procedures were described previously (Saito, >>2006<<). The following Stealth siRNAs were used for the RNAi experiments:" . _:b406456763 "2"^^ . . _:b406456719 . _:b5405895 _:b5405896 . . _:b5405895 _:b5405897 . _:b5405875 "In Drosophila melanogaster, there are two Fats, Fat and Fat-like (Tanoue and Takeichi, >>2005<<), which are different in their functions." . _:b5405895 _:b5405898 . _:b5405895 _:b5405899 . _:b406456762 "2"^^ . . _:b406456718 . . _:b5405895 _:b5405900 . . _:b5405895 _:b5405901 . _:b5405895 _:b5405902 . _:b406456760 . _:b5405895 _:b5405903 . _:b406456773 "2"^^ . . _:b406456751 . . _:b5405891 . _:b406456772 "2"^^ . _:b406456765 . _:b5405911 "The AJ is known as the major cell junctional structure observed in the apical portions of neural progenitor cells (Ho et al., 2000; Lien et al., 2006; Kadowaki et al., >>2007<<). We found that Fat4 and Dachsous1 were located more apical to the AJ and that the plasma membranes at the corresponding region showed a simple apposition, which we defined as the subapical membrane apposition. Such apically extended" . _:b406456715 . _:b406456775 "2"^^ . . . . _:b406456774 "2"^^ . _:b406456714 . . _:b406456725 . _:b406456769 "2"^^ . _:b5405902 . _:b5405894 "Detailed procedures were described previously (Tanoue and Takeichi, >>2004<<)." . _:b406456776 . _:b406456724 . _:b406456768 "2"^^ . _:b5405912 . _:b406456771 . _:b406456727 . _:b406456771 "2"^^ . _:b406456799 . _:b406456811 . _:b406456726 . _:b406456770 "2"^^ . _:b406456778 . . _:b406456812 . _:b406456721 . _:b406456781 "2"^^ . . _:b406456768 . _:b5405910 . _:b406456720 . _:b406456780 "2"^^ . . . _:b406456757 . _:b406456723 . _:b406456783 "2"^^ . _:b406456770 . . . _:b5405884 "In mammals, four types of Fats have been identified: Fat1, Fat2, Fat3, and Fat4 (Rock et al., >>2005<<; Tanoue and Takeichi, 2005). Among them, Fat4 is thought to be the vertebrate counterpart of Drosophila Fat based on their sequence similarity." . _:b406456722 . _:b406456803 . _:b406456782 "2"^^ . _:b406456722 . _:b406456777 "2"^^ . _:b406456733 . _:b5405893 . _:b406456732 . _:b406456776 "2"^^ . . _:b406456735 . _:b406456779 "2"^^ . . . _:b406456734 . _:b406456778 "2"^^ . . _:b5405911 . _:b406456755 . _:b406456729 . _:b406456789 "2"^^ . _:b5405914 . _:b5405888 . _:b406456728 . _:b406456788 "2"^^ . _:b406456784 . _:b406456731 . _:b406456791 "2"^^ . . _:b5405895 . _:b406456730 . _:b406456790 "2"^^ . . _:b5405900 . . _:b406456729 . _:b406456785 "2"^^ . _:b406456741 . _:b5405897 . _:b406456741 . _:b406456740 . _:b406456784 "2"^^ . _:b5405896 . . _:b406456746 . . _:b406456743 . _:b406456787 "2"^^ . . _:b406456773 . _:b406456742 . _:b406456786 "2"^^ . _:b406456732 . _:b5405880 . _:b406456737 . _:b406456797 "2"^^ . _:b406456736 . _:b406456796 "2"^^ . _:b406456739 . _:b406456799 "2"^^ . . _:b406456738 . _:b406456798 "2"^^ . . _:b406456749 . _:b406456793 "2"^^ . _:b406456763 . . . _:b406456748 . _:b406456792 "2"^^ . _:b406456808 . _:b5405914 "Because Crumbs also forms a complex with MUPP1/PatJ and Pals1 (van de Pavert et al., >>2004<<), it is important to determine how the Fat and Crumbs systems share these binding partners for their potential cooperation in future studies." . _:b5405886 . _:b406456751 . _:b5405876 "Fat is well known as a regulator of cell proliferation and planar cell polarity (PCP; Bryant et al., >>1988<<; Mahoney et al., 1991; Yang et al., 2002; Ma et al., 2003; Casal et al., 2006)." . _:b406456795 "2"^^ . _:b5405887 "At the mRNA level, the former is broadly expressed in mouse embryos, whereas the latter exhibits a very restricted expression (Rock et al., >>2005<<). Their functions also remain to be explored." . _:b406456750 . _:b406456794 "2"^^ . . . _:b406456745 . _:b406456805 "2"^^ . . _:b406456744 . _:b406456804 "2"^^ . . . _:b406456747 . _:b406456807 "2"^^ . _:b406456719 . _:b406456746 . _:b5405913 . _:b406456806 "2"^^ . . _:b406456756 . _:b406456744 . . . . _:b406456801 "2"^^ . _:b406456757 . . _:b406456802 . _:b406456797 . _:b406456756 . _:b406456800 "2"^^ . _:b5405906 . _:b406456759 . _:b406456803 "2"^^ . _:b5405906 "Because the Stardust complex has been reported to regulate the apical membrane organization in Drosophila (Pellikka et al., 2002; Hong et al., 2003; Richard et al., >>2006<<), we asked whether Fat4 and Dachsous1 had similar functions. We prepared ultrathin sections for electron microscopy of E15.5 cortices, which had been electroporated with siRNAs targeting Fat4, Dachsous1, or Pals1 (Fig." . _:b406456758 . _:b406456802 "2"^^ . . _:b406456753 . _:b406456752 . _:b406456812 "2"^^ . _:b406456807 . _:b406456779 . . . _:b406456755 . _:b406456793 . _:b406456754 . _:b5405878 "Fat is well known as a regulator of cell proliferation and planar cell polarity (PCP; Bryant et al., 1988; Mahoney et al., 1991; Yang et al., >>2002<<; Ma et al., 2003; Casal et al., 2006)." . _:b406456765 . _:b5405904 . _:b5405876 . _:b406456809 "2"^^ . . . _:b406456753 . _:b5405877 . _:b406456808 "2"^^ . _:b406456764 . _:b406456767 . _:b5405878 . _:b406456811 "2"^^ . _:b406456766 . _:b5405879 . _:b406456810 "2"^^ . "10.1083%2Fjcb.200811030" . _:b406456761 . . _:b406456788 . . . . _:b406456760 . _:b5405879 . _:b5405873 . . . _:b5405877 . _:b406456763 . _:b5405874 . _:b5405892 "N terminus\u2013truncated forms of Fat4 and Dachsous1 were constructed by using pCA-Sig-IRES vector (Tanoue and Takeichi, >>2004<<). For production of a GST-fused cytoplasmic region and deletion mutants of Fat4 in Escherichia coli, the pGEX-2T vector (GE Healthcare) was used." . . . _:b5405875 . _:b406456762 . . . _:b406456723 . _:b406456767 . _:b5405884 . _:b406456773 . . . . _:b406456772 . _:b5405885 . _:b5405883 . . _:b406456775 . _:b5405886 . . _:b5405887 . _:b406456781 . _:b406456774 . _:b406456754 . _:b406456769 . _:b5405880 . _:b406456806 . _:b5405874 . _:b406456742 . _:b406456768 . _:b5405881 . _:b406456714 . _:b406456737 . . _:b406456777 . _:b406456716 . _:b406456717 . _:b5405882 . _:b406456771 . _:b406456730 . _:b5405912 "We found that Fat4 and Dachsous1 could interact in a heterophilic fashion and regulated the protein level of the partner in a way similar to that found in Drosophila (Ma et al., >>2003<<). Then, the question arises as to how the Fat4\u2013Dachsous1 system regulates the apical membrane architecture." . _:b406456770 . _:b5405913 "Stardust, the Drosophila counterpart of Pals1, and Crumbs are well known to regulate the apical membrane organization in Drosophila retinal cells (Pellikka et al., >>2002<<). It is attractive to hypothesize that Fat may cooperate with these apical regulators and in turn takes part in the apical membrane organization." . _:b5405883 . . _:b406456781 . _:b5405892 . . _:b5405891 "Cell aggregation assays were performed as described previously (Nagafuchi et al., >>1987<<)." . _:b406456780 . _:b5405893 . . _:b406456783 . _:b5405894 . . _:b406456782 . _:b406456808 . _:b5405895 . _:b406456809 . _:b406456810 . _:b406456811 . _:b5405877 "Fat is well known as a regulator of cell proliferation and planar cell polarity (PCP; Bryant et al., 1988; Mahoney et al., >>1991<<; Yang et al., 2002; Ma et al., 2003; Casal et al., 2006)." . _:b406456812 . _:b406456777 . . _:b5405888 . _:b406456725 . _:b406456800 . _:b5405909 "The AJ is known as the major cell junctional structure observed in the apical portions of neural progenitor cells (Ho et al., >>2000<<; Lien et al., 2006; Kadowaki et al., 2007)." . _:b5405889 . _:b406456776 . _:b406456801 . . _:b406456802 . _:b406456803 . _:b406456804 . _:b5405890 . _:b406456779 . _:b406456805 . _:b406456806 . _:b406456807 . _:b5405887 . _:b406456792 . _:b5405891 . _:b406456778 . _:b406456793 . _:b406456794 . _:b406456795 . _:b5405884 . _:b406456796 . _:b5405900 . _:b406456789 . _:b406456797 . _:b406456798 . _:b406456799 . _:b406456784 . _:b5405901 . _:b406456788 . . _:b406456785 . _:b406456786 . _:b406456787 . _:b406456759 . . _:b406456788 . _:b5405902 . _:b406456791 . _:b406456789 . _:b406456790 . _:b406456791 . _:b406456776 . _:b5405903 . _:b406456790 . _:b406456777 . _:b406456778 . . _:b406456779 . _:b406456780 . _:b5405896 . _:b406456785 . . _:b406456781 . _:b406456782 . _:b5405883 "dachsous mutants show several morphogenetic defects (Clark et al., 1995), and both fat and dachsous mutants commonly exhibit defects in PCP (Yang et al., 2002; Ma et al., >>2003<<)." . _:b406456783 . _:b406456768 . _:b5405897 . _:b406456784 . _:b406456769 . . _:b406456770 . . _:b406456771 . _:b406456772 . _:b5405898 . _:b5405876 . _:b406456787 . _:b406456773 . _:b406456736 . _:b406456774 . _:b406456769 . _:b406456775 . _:b406456760 . _:b5405899 . _:b406456786 . _:b406456761 . _:b5405886 "phenotypes of Fat4-null mice were reported; they exhibit complicated morphological abnormalities, including abnormal orientation of inner ear hair cells, cystic dilation of kidney tubules, and defects in organ shapes (Saburi et al., >>2008<<). However, the molecular and cellular functions of Fat4 still remain to be analyzed." . _:b406456762 . _:b5405903 "As previous studies showed that MUPP1 forms a complex with Pals1, a vertebrate counterpart of Drosophila Stardust (van de Pavert et al., 2004; Sugihara-Mizuno et al., >>2007<<), we examined whether this interaction also occurred in the cortex, and as a result, we indeed detected Pals1 in the MUPP1 immunoprecipitates prepared from cortical lysates (Fig." . _:b406456804 . _:b406456794 . . _:b406456763 . _:b406456764 . _:b5405908 . _:b406456797 . _:b406456740 . _:b406456765 . _:b406456766 . _:b406456767 . _:b5405909 . . _:b406456752 . _:b5405909 . _:b406456796 . _:b406456753 . _:b406456754 . _:b406456755 . _:b5405881 . _:b406456749 . _:b406456756 . _:b5405910 . _:b406456799 . . _:b406456757 . . _:b406456758 . _:b406456759 . _:b5405907 . _:b406456738 . _:b406456744 . _:b5405911 . _:b406456798 . _:b406456745 . _:b406456746 . _:b406456747 . _:b406456748 . _:b5405904 . _:b406456793 . _:b406456749 . _:b406456750 . . _:b406456751 . _:b406456736 . _:b5405905 . _:b406456792 . . _:b406456737 . _:b5405905 . _:b406456731 . _:b406456738 . _:b5405880 "Fat is well known as a regulator of cell proliferation and planar cell polarity (PCP; Bryant et al., 1988; Mahoney et al., 1991; Yang et al., 2002; Ma et al., 2003; Casal et al., >>2006<<). In the PCP regulation, Fat interacts with Dachsous, another huge cadherin, through the extracellular region of each. dachsous mutants show several morphogenetic defects (Clark et al., 1995), and both fat and dachsous mutants commonly" . _:b406456739 . _:b406456740 . _:b5405906 . _:b406456795 . _:b406456741 . _:b406456798 . _:b406456742 . _:b406456743 . _:b406456728 . _:b5405907 . _:b406456794 . _:b406456729 . . _:b406456730 . . _:b5405910 "The AJ is known as the major cell junctional structure observed in the apical portions of neural progenitor cells (Ho et al., 2000; Lien et al., >>2006<<; Kadowaki et al., 2007)." . _:b406456731 . _:b406456732 . _:b406456805 . _:b406456733 . _:b406456734 . _:b406456735 . _:b406456718 . _:b406456720 . _:b406456804 . . _:b406456721 . _:b406456722 . _:b5405881 "dachsous mutants show several morphogenetic defects (Clark et al., >>1995<<), and both fat and dachsous mutants commonly exhibit defects in PCP (Yang et al., 2002; Ma et al., 2003)." . _:b406456723 . _:b406456724 . _:b406456807 . _:b406456747 . _:b406456725 . _:b406456726 . _:b406456727 . _:b406456721 . _:b406456806 . . _:b406456786 . _:b406456714 . _:b5405907 "5 A, red), as observed by others (Ho et al., >>2000<<). This gap appeared to contain amorphous electron-dense materials, but the cytoplasmic sides exhibited no specific structures." . _:b406456715 . _:b406456716 . _:b5405912 . _:b406456801 . _:b406456717 . _:b406456718 . _:b406456719 . _:b5405902 "As previous studies showed that MUPP1 forms a complex with Pals1, a vertebrate counterpart of Drosophila Stardust (van de Pavert et al., >>2004<<; Sugihara-Mizuno et al., 2007), we examined whether this interaction also occurred in the cortex, and as a result, we indeed detected Pals1 in the MUPP1 immunoprecipitates prepared from cortical lysates (Fig." . _:b406456800 . _:b5405913 . . _:b406456783 . _:b406456787 . _:b406456803 . _:b5405914 . _:b406456782 . _:b406456717 "3"^^ . _:b406456802 . _:b406456789 . . _:b5405892 . _:b406456716 "4"^^ . _:b5405874 "Although several molecules have been identified as regulators of epithelial apico-basal polarity (Shin et al., >>2006<<), precise mechanisms to regulate the apical domain organization in the cortical progenitor cells remain unknown." .