Congress Abstracts - Society for Developmental Biology

downstream asymmetric signals. The mechanism that specifies these two cilia types remains unknown. We now show that the O-
glycosylation enzyme GALNT11 is crucial to such determination. We previously identified GALNT11 in a patient with Htx, and now
demonstrate, in Xenopus, that galnt11 activates Notch signaling. By mass spectrometry, GALNT11 glycosylates NOTCH1 peptides
with GalNAc, a previously undescribed form of Notch glycosylation. Surprisingly, this glycosylation can increase Notch1 peptide
cleavage by ADAM17, suggesting a novel mechanism for Notch activation. We further developed a quantitative live imaging
technique for Xenopus LRO cilia and show that knockdown of galnt11 or notch1 converted immotile LRO cilia into motile cilia and
produced a laterality defect reminiscent of loss of the ciliary sensor Pkd2. Strikingly, paralyzing a subset of these converted motile
cilia by knockdown of axonemal dynein dnah9 rescued laterality, thereby suggesting that motility masks the sensory function of
motile cilia. Together, our data demonstrates that Galnt11 modifies Notch, establishing an essential balance between motile and
immotile cilia at the LRO to determine laterality and identifies a novel mechanism for human Htx.
Program/Abstract # 254
N-cadherin locks left-right asymmetry by ending the leftward movement of Hensen’s node cells
Saude, Leonor; Mendes, Raquel V. (Instituto de Medicina Molecular, Portugal); Martins, Gabriel G. (Centro de Biologia Ambiental,
Portugal)
The stereotypic left-right (LR) asymmetric distribution of internal organs is due to an asymmetric molecular cascade in the lateral
plate mesoderm (LPM) that has its origin at the embryonic node. In chicken embryos, molecular asymmetries at Hensen’s node are
created by leftward cell movements that occur transiently. What terminates these movements, and moreover what is the impact of
prolonging them on the LR asymmetry cascade, was entirely unknown. We show that leftward movements last longer when N-
cadherin function is blocked and cease prematurely when N-cadherin is overexpressed on the right side of the node. The prolonged
leftward movements lead to loss of asymmetric expression of wnt3a, fgf8 and nodal at the node region. This originates an abnormal
expression of the asymmetric genes cer1 and snai1 in the LPM, resulting in a mispositioned heart. We conclude that N-cadherin stops
the leftward cell movements, and that this termination is an essential step in the establishment of LR asymmetry.
Program/Abstract # 255
Novel complementary asymmetric gene expression of linked genes at the Pitx2 locus establishes a role for chromatin
regulation of L-R patterning
Welsh, Ian Christophe; Chen, Frances; Kurpios, Natasza (Cornell University, USA)
The transcription factor Pitx2 is required for the asymmetric development of multiple organs including that of the dorsal mesentery
(DM), a structure whose dynamic changes in cell behavior are critical for directing the chiral rotation of the midgut. To identify the
downstream cellular effectors that mediate Pitx2 influence on cell behavior, our lab performed a laser capture microdissection and
microarray analysis of the left and right DM at the onset of gut rotation. Unexpectedly, we found that the most differentially expressed
gene in the right DM is located immediately adjacent to Pitx2 on chicken chromosome 4. Furthermore, additional genes from this
locus, both proximal and distal to Pitx2, also exhibit mirrored right-specific DM expression. Significantly, we discovered that this
novel complementary expression occurs early during L-R patterning at both the node and in the lateral plate mesoderm well prior to
formation of the DM. These data suggest this phenomenon is a fundamental characteristic of regulatory mechanisms directing
expression of Pitx2. To our knowledge, this is the first report of such binary expression of linked genes across the L-R axis.
Evolutionarily, the organization and content of this locus, including the presence of a large (~600kb) gene desert proximally flanking
Pitx2, is highly conserved. The gene desert harbors numerous conserved noncoding elements (CNEs) with established transcriptional
or structural regulatory function. We propose a model where long range interactions amongst these CNEs differentially organizes
chromatin at the locus in nuclei on the left or right to drive asymmetric expression of Pitx2 or its neighbors during L-R organogenesis.
Program/Abstract # 256
RhoA GTPase Signaling During Development of the Left-Right Body Axis
Amack, Jeffrey D.; Wang, Guangliang (State University of NY Upstate Med Univ, USA)
Evidence from patients and animal models implicates Rho GTPase signaling pathways in establishing left-right (LR) asymmetry in
vertebrate embryos, but the underlying mechanisms remain unclear. We are using the zebrafish embryo to identify and characterize
the role(s) of RhoA signaling during development of LR asymmetry. The small GTPase RhoA is a molecular switch that can activate
downstream effectors including Rho kinase (Rock) proteins to modulate cytoskeletal dynamics and control several cell behaviors.
Previously, we found that the Rho kinase Rock2b mediates cell shape changes that establish an anteroposterior (AP) asymmetric
distribution of motile cilia in Kupffer’s vesicle, which is necessary for these cilia to generate asymmetric fluid flow and direct normal
LR patterning. Partial depletion of RhoA protein levels altered AP asymmetry in Kupffer’s vesicle, but also revealed defects in cilia
formation. While cilia phenotypes were not observed in Rock2b deficient embryos, antisense depletion of another Rho kinase,
Rock2a, or brief treatments with a Rho kinase inhibitor disrupted cilia and LR development. Videomicroscopy of beads injected into
Kupffer’s vesicle demonstrated that interfering with RhoA or Rock2a function disrupted asymmetric fluid flow. Finally, LR defects in
RhoA deficient embryos were partially rescued by ectopic expression of constitutively active Rock proteins, indicating RhoA indeed
signals through Rho kinases to control LR asymmetry. These results uncover new roles for RhoA signaling via different downstream
effectors (Rock2a and Rock2b) that control multiple steps of LR development.
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