Taking a different pathway: Nle1 plays with Wnt during embryogenesis Amy C. Lossie 1,2 , Chiao-Ling Lo 1,3 and Jeremy B. Sherrill 1,4 1 Department of Animal Science, Purdue University, West Lafayette, IN; 2 Dept of Medicine, Indiana University School of Medicine, Indianapolis, IN; 3 PULSe Interdisciplinary Graduate Program, Purdue University, West Lafayette, IN; 4 Department of Biological Sciences, Purdue University, West Lafayette, IN. • Nle1 mutants more severe than single Notch mutants or disruption of Notch signaling • Mice undergo gastrulation in the absence of Notch signaling • ICM from Nle1 KO mice fails to grow • Mutants undergo apoptosis at E3.5 plus 1 day in culture (Cormier et al., 2006) • Downstream targets will be downregulated • Notch receptors & ligands will be upregulated • No misregulation of downstream target genes or members of the Notch pathway • Cdkn1a is upregulated (p=1.9 X 10 - 8 ) • Fzd4, Fzd7 and Wnt11 are downregulated • NLE1 is not a positive regulator of Notch signaling during pre- implantaiton development • NLE1 acts via multiple pathways during embryonic development Conclusions • Trp53 is not upregulated in mutants at the morula, blastocyst or hatched blastocyst stage Is Trp53 upregulated in mutants? • Examine acetylated form of p53 • Examine expression of miR-34a • Examine other genes involved in cell- cycle arrest or apoptosis (Bcl2) • Investigate whether Nle1 acts in the Notch pathway at later stages Future Studies Our interests lie in understanding the genetic pathways and epigenetic factors that are important for establishing and maintaining maternal-fetal interactions during pregnancy. L1 & L4 are excellent models of miscarriage Nle1 encodes a WD40 Repeat Protein Notch Figure modiﬁed from Haltiwanger R.S. and Lowe J.B. 2004. Annu. Rev. Biochem The Notch Pathway • NLE1 binds NICD in Drosophila and Xenopus (Royet et al., 1998; Cormier et al., 2006) • Disruption of the Notch pathway in lower vertebrates does not affect mesoderm induction Miscarriage • 50% of mammalian pregnancies result in fetal loss by peri-implantation (Rai and Regan, 2006) • 15% chance in women under 35 • 25% chance in women under 40 • Recurrent miscarriage (3 consecutive) occurs in ~1% of the population Notch mutants are less severe than Nle1 mutants Notch Wnt Summary • L1 and L4 have mutations in Nle1 • L1 and L4 phenocopy the Nle1 -/- knockout • Cdkn1a is upregulated in mutants at all stages • Wnt pathway genes are downregulated Notch PCR Array at E3.5 Katherine Baumgarner

Nle1 mutants more severe than single Notch mutants or disruption of Notch signaling

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Page 1: Nle1 mutants more severe than single Notch mutants or disruption of Notch signaling

Taking a different pathway: Nle1 plays with Wnt during embryogenesis

Amy C. Lossie1,2, Chiao-Ling Lo1,3 and Jeremy B. Sherrill1,4

1Department of Animal Science, Purdue University, West Lafayette, IN; 2Dept of Medicine, Indiana University School of Medicine, Indianapolis, IN; 3PULSe

Interdisciplinary Graduate Program, Purdue University, West Lafayette, IN; 4Department of Biological Sciences, Purdue University, West Lafayette, IN.

• Nle1 mutants more severe than single Notch mutants or disruption of Notch signaling

• Mice undergo gastrulation in the absence of Notch signaling

• ICM from Nle1 KO mice fails to grow

• Mutants undergo apoptosis at E3.5 plus 1 day in culture (Cormier et al., 2006)

• Downstream targets will be downregulated• Notch receptors & ligands will be upregulated

• No misregulation of downstream target genes or members of the Notch pathway

• Cdkn1a is upregulated (p=1.9 X 10-8)• Fzd4, Fzd7 and Wnt11 are downregulated

• NLE1 is not a positive regulator of Notch signaling during pre-implantaiton development

• NLE1 acts via multiple pathways during embryonic development

Conclusions

• Trp53 is not upregulated in mutants at the morula, blastocyst or hatched blastocyst stage

Is Trp53 upregulated in mutants?

• Examine acetylated form of p53• Examine expression of miR-34a• Examine other genes involved in cell-cycle arrest

or apoptosis (Bcl2)• Investigate whether Nle1 acts in the Notch

pathway at later stages

Future Studies

Our interests lie in understanding the genetic pathways and epigenetic factors that are important for establishing and maintaining maternal-fetal interactions during pregnancy.

L1 & L4 are excellent models of miscarriage

Nle1 encodes a WD40 Repeat Protein

Notch Figure modified from Haltiwanger R.S. and Lowe J.B. 2004. Annu. Rev. Biochem

The Notch Pathway

• NLE1 binds NICD in Drosophila and Xenopus (Royet et al., 1998; Cormier et al., 2006)

• Disruption of the Notch pathway in lower vertebrates does not affect mesoderm induction

Miscarriage• 50% of mammalian pregnancies result in fetal loss

by peri-implantation (Rai and Regan, 2006)

• 15% chance in women under 35• 25% chance in women under 40• Recurrent miscarriage (3 consecutive) occurs in

~1% of the population

Notch mutants are less severe than Nle1 mutants

Notch Wnt

Summary• L1 and L4 have mutations in Nle1• L1 and L4 phenocopy the Nle1-/- knockout• Cdkn1a is upregulated in mutants at all stages• Wnt pathway genes are downregulated

Notch PCR Array at E3.5

Katherine Baumgarner