Wnt members act as morphogens essential for embryonic patterning and adult homeostasis. Currently, it is still unclear how Wnt secretion and its gradient formation are regulated. In this study, we examined the roles of N-glycosylation and lipidation/acylation in regulating the activities of Wingless (Wg), the main Drosophila Wnt member. We show that Wg mutant devoid of all the N-glycosylations exhibits no major defects in either secretion or signaling, indicating that N-glycosylation is dispensable for Wg activities. We demonstrate that lipid modifications at Serine 239 (S239) rather than that at Cysteine 93 (C93) plays a more important role in regulating Wg signaling in multiple developmental contexts. Wg S239 mutant exhibits a reduced ability to bind its receptor, Drosophila Frizzled 2 (dFz2), suggesting that S239 is involved in the formation of a Wg/receptor complex. Importantly, while single Wg C93 or Wg S239 mutants can be secreted, removal of both acyl groups at C93 and S239 renders Wg incapable of reaching the plasma membrane for secretion. These data argue that lipid modifications at C93 and S239 play major roles in Wg secretion. Further experiments demonstrate that two acyl attachment sites in the Wg protein are required for the interaction of Wg with Wntless (Wls, also known as Evi or Srt), the key cargo protein involved in Wg secretion. Together, our data demonstrate the in vivo roles of N-glycosylation and lipid modification in Wg secretion and signaling.

The canonical Wnt signaling pathway plays critical roles during development and homeostasis. Dysregulation of this pathway can lead to many human diseases, including cancers. A key process in this pathway consists of regulation of β-catenin concentration through an Axin-recruited destruction complex. Previous studies have demonstrated a role for Tankyrase (TNKS), a protein with poly (ADP-ribose) polymerase, in the regulation of Axin levels in human cancers. However, the role of TNKS in development is still unclear. Here, we have generated a Drosophila tankyrase (DTNKS) mutant and provided compelling evidence that DTNKS is involved in the degradation of Drosophila Axin (Daxin). We show that Daxin physically interacts with DTNKS, and its protein levels are elevated in the absence of DTNKS in eye discs. In S2 cells, DTNKS suppressed the levels of Daxin. Surprisingly, we found that Daxin in turn down-regulated DTNKS protein level. In vivo study showed that DTNKS regulated Wg signaling and wing patterning at a high Daxin protein level, but not at a normal level. Taken together, our findings identified a conserved role of DTNKS in regulating Daxin levels, and thereby Wg/Wnt signaling during development.

Lgr5+ stem cells are crucial to gut epithelium homeostasis; however, how these cells are maintained is not fully understood. Zinc finger HIT-type containing 1 (Znhit1) is an evolutionarily conserved subunit of the SRCAP chromosome remodeling complex. Currently, the function of Znhit1 in vivo and its working mechanism in the SRCAP complex are unknown. Here we show that deletion of Znhit1 in intestinal epithelium depletes Lgr5+ stem cells thus disrupts intestinal homeostasis postnatal establishment and maintenance. Mechanistically, Znhit1 incorporates histone variant H2A.Z into TSS region of genes involved in Lgr5+ stem cell fate determination, including Lgr5 , Tgfb1 and Tgfbr2 , for subsequent transcriptional regulation. Importantly, Znhit1 promotes the interaction between H2A.Z and YL1 (H2A.Z chaperone) by controlling YL1 phosphorylation. These results demonstrate that Znhit1/H2A.Z is essential for Lgr5+ stem cell maintenance and intestinal homeostasis. Our findings identified a dominant role of Znhit1/H2A.Z in controlling mammalian organ development and tissue homeostasis in vivo.