The heterotaxy gene GALNT11 glycosylates Notch to orchestrate cilia type and laterality

Abstract

Heterotaxy is a disorder of left-right body patterning, or laterality, that is associated with major congenital heart disease. The aetiology and mechanisms underlying most cases of human heterotaxy are poorly understood. In vertebrates, laterality is initiated at the embryonic left-right organizer, where motile cilia generate leftward flow that is detected by immotile sensory cilia, which transduce flow into downstream asymmetric signals. The mechanism that specifies these two cilia types remains unknown. Here we show that the N-acetylgalactosamine-type O-glycosylation enzyme GALNT11 is crucial to such determination. We previously identified GALNT11 as a candidate disease gene in a patient with heterotaxy, and now demonstrate, in Xenopus tropicalis, that galnt11 activates Notch signalling. GALNT11 O-glycosylates human NOTCH1 peptides in vitro, thereby supporting a mechanism of Notch activation either by increasing ADAM17-mediated ectodomain shedding of the Notch receptor or by modification of specific EGF repeats. We further developed a quantitative live imaging technique for Xenopus left-right organizer cilia and show that Galnt11-mediated Notch1 signalling modulates the spatial distribution and ratio of motile and immotile cilia at the left-right organizer. galnt11 or notch1 depletion increases the ratio of motile cilia at the expense of immotile cilia and produces a laterality defect reminiscent of loss of the ciliary sensor Pkd2. By contrast, Notch overexpression decreases this ratio, mimicking the ciliopathy primary ciliary dyskinesia. Together our data demonstrate that Galnt11 modifies Notch, establishing an essential balance between motile and immotile cilia at the left-right organizer to determine laterality, and reveal a novel mechanism for human heterotaxy.

Figure 1. Galnt11 alters LR patterning and Notch signaling

Percent of embryos that have abnormal (a) cardiac looping (either A loops or L loops) or (b) abnormal coco or pitx2c expression L: left, R: right. c–j) Xenopus epidermal multiciliated cells marked by anti-acetylated α-tubulin (red). Embryos were injected at the two cell stage targeting the epidermis on either the right or left side with either galnt11 MO (d), GALNT11 RNA (f), notch1 MO (h), or nicd RNA (j) and compared to the uninjected side (c, e, g, i). GFP (insets) trace the injected side. All views are lateral views with dorsal to the top. k) Schematic of the Notch pathway l) Percent of embryos with abnormal pitx2c in galnt11 morphants or galnt11 morphants co-injected with members of the Notch pathway. Panel (a) UC n=130, galnt11 start MO n=145, galnt11 splice MO n=105, galnt11 splice MO + galnt11 RNA n=83, galnt11 RNA n=96. Panel (b) coco expression UC n=21, galnt11 MO n=24, galnt11 RNA n=23; pitx2c expression UC n=45, galnt11 MO n=54, galnt11 RNA n=34. Panel (l) UC n=96, galnt11 MO n=78, galnt11 MO + delta n=65, galnt11 MO + nicd n=86, galnt11 MO + su(h)ank n=62. Bars depict means. Additional details are in Methods.

Figure 2. GALNT11 glycosylation of NOTCH1 derived peptides and effects on ADAM-mediated cleavage

a) Depiction of NOTCH1 with MALDI-TOF spectra demonstrating O-GalNAc glycosylation of peptides derived from EGF6 (blue), EGF36 (magenta) and the juxtamembrane region (green). Yellow squares: O-GalNAc, Red triangles: O-Fuc. TM: transmembrane domain; NICD: Notch intracellular domain; # deammoniated peptide; & sodium adducts. b) MALDI-TOF time-course analysis of in vitro cleavage of the juxtamembrane peptide and glycopeptide by ADAM17. Scissors: Cleavage site. *nonspecific N-terminal degradation of two amino acids. c–d) HPLC analysis of the cleavage reactions with c) naked peptide and d) glycopeptide after 2 hrs (percentages above the peaks) with p<0.05 (two-tailed t-test).

Figure 3. Galnt11/Notch signaling switches cilia between motile and immotile types

a) Wildtype LRO explants expressing arl13b-mCherry reveal distinct populations of immotile and motile cilia. Cyan box highlights a single motile cilium, with a corresponding kymograph analyzing beating frequency. Magenta box highlights immotile cilium and kymograph. b) Analysis of multiple vehicle LROs reveals a specific geography for cilia types: immotile cilia are enriched along the fringes, while motile cilia are enriched in the center. c) Schematic of a wildtype LRO A: anterior, P: posterior, L: left, R: right. d) nicd overexpressants exhibit a decrease in motile cilia in the center of the LRO. notch1 (e) and galnt11 (f) morphants display a decrease in immotile cilia along the LRO periphery. (g–h) Quantification of motile cilia distribution in total LROs (g), and in the center and along the periphery of the LRO (h). Bars depict means and error bars are standard error. b,d,e,f) n=8. g,h) vehicle n=17, nicd RNA n=12, notch1 MO n=12, galnt11 MO n=15. Additional details are in Methods.