Date of Award
Open Access Thesis
Medical Doctor (MD)
Medicine, Developmental biology
Heterotaxy is a disease of abnormal left-right (LR) body patterning associated with congenital heart disease that has very poor outcomes. Despite advances in surgical management, the two most severe forms of heterotaxy, right and left atrial isomerism, have a 29% and 64% 5-year survival rate, respectively. Through copy number variant analysis of heterotaxy patients, GALNT11 was recently identified as a novel gene important in human LR development. However, the mechanism by which Galnt11 causes heterotaxy has not been elucidated. In order to discover the mechanism of GALNT11 in patterning the LR axis, I performed loss of function and gain of function studies in Xenopus tropicalis and expression analysis in Mus musculus. In Xenopus, knockdown of galnt11 = induced heart looping defects that were successfully rescued with human GALNT11 mRNA indicating that the phenotype was specific to Galnt11. Via immunohistochemistry, Galnt11 protein strongly localizes to the crown cells surrounding the LRO. Manipulations of Galnt11 altered the density of ciliated epidermal cells, but based on gliding assays and ultrastructural analysis did not alter the cilia. Galnt11 and Notch effects on epidermal ciliated epidermal cell density, heart looping, as well as PitX2 and Coco expression were very similar, and Galnt11 morphants were rescued with Notch ICD and Su(H)-Ank, but not Delta suggesting that
galnt11 acts in the notch pathway downstream of the ligand. GALNT11 RNA no longer had any effect on heart looping or PitX2 expression following a conservative point mutation of its catalytic glycosylation domain. Galnt11 morphants had significantly narrower LROs, and much stronger expression of motile ciliary markers FoxJ1 and RFX2, while GALNT11 RNA injected embryos had almost no detectable FoxJ1 and RFX2. Taken together, these results indicate that Galnt11 is a GalNAc-transferase that is necessary for proper left-right axis establishment and heart looping. Its function is to specify between motile and sensory cell fates at the Left-Right Organizer by glycosylating Notch receptor and modifying Notch signaling.
Boskovski, Marko, "Galnt11 Is A Novel Galnac-Transferase That Glycosylates Notch1 Receptor To Specify Between Motor And Sensory Ciliary Fates In The Vertebrate Left-Right Organizer" (2012). Yale Medicine Thesis Digital Library. 1696.