Congress Abstracts - Society for Developmental Biology

investigate this issue, we used targeted mutagenesis of embryonic stem (ES) cells to generate mice with a polyalanine expansion
mutation in Sox3 (Sox3-26ala) that is associated with X-linked Hypopituitarism (XH) in humans. By investigating both ES cells and
chimeric mice we show that endogenous polyalanine expanded SOX3 does not form protein aggregates in vivo, but rather is present at
dramatically reduced levels within the nucleus of mutant cells. Importantly, the residual mutant protein of chimeric embryos is able to
rescue a block in gastrulation but is not sufficient for normal development of the hypothalamus, a region that is functionally
compromised in Sox3 null embryos and individuals with XH. Together, these data provide the first definitive example of a diseaserelevant
PA mutant protein that is both nuclear and functional, thereby manifesting as a partial loss-of-function allele.
Program/Abstract # 407
The epigenetic factor Reptin regulates zebrafish development through both cilia dependent and independent pathways
Sun, Zhaoxia (Yale U, USA)
The cilium, an antenna-like organelle, has emerged as a key center of sensation, signal transduction, growth and motility for the
vertebrate cell. Consistently, ciliary defects have been linked to a growing list of human diseases, collectively referred to as
“ciliopathies”. Although the importance of the cilium is now firmly established, how the cilium performs its vital motility and sensory
functions remains largely unclear. Addressing this question in a vertebrate model system will be critical for understanding and treating
ciliopathies. In zebrafish, a number of cilia mutants have been isolated and they provide valuable research tools for studying cilia and
ciliopathies. From a previous genetic screen for cystic kidney mutants, we isolated hi2394, an insertional mutant displaying kidney
cyst formation and ventral body curvature, phenotypes typically associated with ciliary defects in zebrafish. In this study, we show
that hi2394 is a loss of function allele of reptin. Interestingly, reptin encodes an AAA ATPase that is known to be involved in
epigenetic regulation. We further provide evidence that reptin genetically interacts with known ciliary genes and it is essential for the
normal function of cilia. In addition, since recently it was suggested that in some ciliopathies, DNA damage response (DDR) is
activated as shown by an increased level of phosphorylated H2AX, we examined DDR in fish mutant embryos using the same
approach. Results showed that while the signal in the classic cilia mutant ift172 hi2211 is comparable to wild type siblings, the level of
phosphorylated H2AX is greatly increased in both the eye and the brain of reptin hi2394 mutants. Consistently, compared to IFT
mutants, reptin hi2394 mutants are more neocrotic and die earlier. Taken together, these results suggest that reptin is involved in cilia
dependent and independent pathways during vertebrate development.
Program/Abstract # 408
Impaired Folate Uptake and Neural Tube Closure Defects in Lrp2 Deficient Mice
Mecklenburg, Nora; Kur, Esther (Max- Delbrück Centrum, Germany); Cabrera, Robert (University of Texas, USA); Willnow,
Thomas E.; Hammes, Annette (Max- Delbrück- Centrum, Germany)
The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2) is a multifunctional cell surface receptor highly expressed in the
embryonic neuroepithelium. Loss of receptor activity in the developing murine CNS causes holoprosencephaly (HPE) due to impaired
SHH signalling (Christ et al. 2012). Besides the HPE phenotype, we observe additional neural tube defects (NTDs) in LRP2 deficient
embryos, unrelated to SHH dependent forebrain development. Thus, the rostral neural tube is still open at somite stages 17-26
whereas in wild type controls closure of this neural tube region is completed at the 15 somites stage (E9.0). Similar NTDs have been
described previously to be caused by impaired folate metabolism in mice. A function for LRP2 in endocytosis of folate bound to
soluble folate receptors (FolR1) has been already suggested for the kidney (Birn, 2005). Therefore we analyzed whether LRP2
expressed in the neuroepithelium is required for delivery of folate to neuroepithelial cells during neurulation. Expression levels for
FolR1 and Slc19a1, a carrier that transports folate directly across the cell membrane (the second import route for folate) are
unchanged in LRP2 deficient mice. However, uptake assays in whole embryo cultures showed that LRP2 deficient neuroepithelial
cells are unable to mediate uptake of soluble FolR1. Consequently, folate concentrations are significantly reduced in Lrp2 -/- embryos
compared to control littermates. Moreover, expression of the folic acid dependent gene Alx3 is significantly down regulated in Lrp2
mutants. In conclusion, this study suggests that LRP2 is essential for cellular folate uptake in the developing neural tube, a step
crucial for proper closure of the neural tube.
Program/Abstract # 409
Wnt/catenin Signaling System Functions in Embryoid Bodies Aggregated from Human Embryonic Stem Cell
Xu, Xuehong (Shaanxi Normal Univ, China); Xu, MengMeng (Shaanxi Normal Univ/Duke University, USA); Zhou, Xin (Shaanxi
Normal Univ, China); Jones, Odell (Univ of Maryland School of Med, USA); Pan, Yuexin (Case Western Reserve Univ, USA); Bryant,
Joseph (Univ of Maryland School of Med, USA); Anthony, Donald (Case Western Reserve Univ, USA)
As an essential molecule in Wnt/β-catenin signaling, β-Catenin plays a crucial role in the decision making for tissue differentiation in
embryogenesis and pathogenesis. Associated with other proteins such as LEF/TCF family of transcription factors, the complex of β-
Catenin and LEF/TCF is accumulated in the cytoplasm and transported to the nucleus. In mouse skin, the deferential fate of the skin
stem cell depends on β-catenin, which organizes stem cells into follicular or epidermal lineages. These indicate that Wnt/β-catenin
signaling should also function in lineage differentiation of hESC. To study the function of β-catenin in early differentiation, we
cultured H9 stem cell and aggregated them into embryoid bodies (EB). Using confocal microscopy combined with immunostaining,
we revealed that in early EBs some guarding cells were first differentiated from EB stem cell aggregates. These early differentiated
cells for guarding epithelial cells have strongest expression of β-catenin within EB. These cells were flattened on the surface of EB,
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