Development 143: doi:10.1242/dev.128538: Supplementary information
Development • Supplementary information
Supplemental Figure 1 Targeting strategy for the RLFNG allele. Targeting
mechanism for RLFNG allele. The targeting vector (top) contains a novel first exon
fusing the N-terminus of Radical fringe (Rfng) to the mature domain of Lfng.
Homologous recombination replaces the endogenous exon 1 of Lfng with this novel
exon, and inserts a Neo/Testis Cre cassette in the first exon. This cassette silences the
targeted allele in the chimeric mice carrying the LfngRLFNGneo allele. The floxed cassette is
removed upon passage through the male germline, resulting in the active LfngRLFNG allele
being expressed in heterozygous, F1 offspring of the chimeras.
Development 143: doi:10.1242/dev.128538: Supplementary information
Development • Supplementary information
Supplemental Figure 2. Long exposures and qRT-PCR reveal low levels of Lfng and Rlfng transcripts in
LfngRLFNG/+ mutant embryos. A) At 9.0 d.p.c. a 48 hour revelation after in situ for Lfng transcripts reveals Lfng
expression in the caudal PSM of mutant embryos (c). Even after long exposure, Lfng expression in wild type embryos
still appears oscillatory (a,b). B) qRT-PCR was performed on individual posterior PSMs isolated from embryos of
indicated genotypes to measure total Lfng transcript levels from both alleles. Wild type embryos exhibit oscillatory
expression, with Lfng transcript levels varying from embryo to embryo. In contrast, LfngRLFNG/+ heterozygous embryos
exhibit little variability from embryo to embryo, and the overall levels are close to the lower limit of those observed
in wild type embryos. C) Allele specific semiquantitative PCR was performed on cDNA from individual PSMs to
measure expression from the two alleles. Top: The two alleles are shown, with approximate primer sites indicated as
arrows (the shared reverse primer is contained entirely within exon 2). Middle: By regular PCR of cDNA, only the
wild type Lfng allele is observed in wild type embryos, while in LfngRLFNG/+ embryos both the wild type and Rlfng
allele are observed, demonstrating that both alleles are expressed abd properly spliced in the heterozygous embryos.
Bottom: Quantitative RT-PCR was performed on cDNA from individual PSMs. Wild type embryos (the first two in
part B above) exhibit varying levels of wild type Lfng transcript, but no Rlfng transcript. LfngRLFNG/+ embryos (the
first two in part B above) express both alleles to similar levels.
Development 143: doi:10.1242/dev.128538: Supplementary information
Table S1. In situ probes and antibodies used
Gene/antigen
Citation or probe
Lfng
(1)
Mesp2
(2)
Uncx
(3)
Tbx18
(4)
Spry2
(5)
Axin2
(6)
Hes7
(7)
Hes7 intron
(7)
Nrarp
(7)
Notch1
(8)
Dll1
(9)
Snai1
(10)
Pax1
Base pairs 316-1062 of NM_008780.2
Myog
Base pairs 1041-1480 of NM_031189.2
NICD
Cell Signaling Technology Clone D3B8,
Catalog #4147, Lot 6
Gene names and the citations for the probes are shown.
1. Johnston SH, et al (1997) A family of mammalian fringe genes implicated in boundary
determination and the notch pathway. Development 124(11): 2245-2254.
2. Saga Y, Hata N, Koseki H & Taketo MM (1997) Mesp2: A novel mouse gene
expressed in the presegmented mesoderm and essential for segmentation initiation. Genes
Dev 11(14): 1827-1839.
3. Mansouri A, et al (1997) Paired-related murine homeobox gene expressed in the
developing sclerotome, kidney, and nervous system. Dev Dyn 210(1): 53-65.
5. Wahl MB, Deng C, Lewandoski M & Pourquié O (2007) FGF signaling acts upstream
of the NOTCH and WNT signaling pathways to control segmentation clock oscillations
in mouse somitogenesis. Development 134: 4033-4041.
6. Ishikawa A, et al (2004) Mouse Nkd1, a wnt antagonist, exhibits oscillatory gene
expression in the PSM under the control of notch signaling. Mech Dev 121(12): 14431453.
7. Shifley ET, et al (2008) Oscillatory lunatic fringe activity is crucial for segmentation
of the anterior but not posterior skeleton. Development 135(5): 899-908.
8. Del Amo FF, et al (1992) Expression pattern of motch, a mouse homolog of drosophila
notch, suggests an important role in early postimplantation mouse development.
Development 115(3): 737-744.
Development • Supplementary information
4. Kraus F, Haenig B & Kispert A (2001) Cloning and expression analysis of the mouse
T-box gene Tbx18. Mechanisms of Development 100(1): 83-6.
Development 143: doi:10.1242/dev.128538: Supplementary information
9. Bettenhausen B, Hrabe de Angelis M, Simon D, Guenet JL & Gossler A (1995)
Transient and restricted expression during mouse embryogenesis of Dll1, a murine gene
closely related to drosophila delta. Development 121(8): 2407-2418.
Development • Supplementary information
10. Smith DE, Franco del Amo F & Gridley T (1992) Isolation of sna, a mouse gene
homologous to the drosophila genes snail and escargot: Its expression pattern suggests
multiple roles during postimplantation development. Development 116(4): 1033-1039.