Ectopic Expression of Ptf1a Induces Spinal Defects, Urogenital Defects, and Anorectal Malformations in Danforth's Short Tail Mice

Abstract

Danforth's short tail (Sd) is a semidominant mutation on mouse chromosome 2, characterized by spinal defects, urogenital defects, and anorectal malformations. However, the gene responsible for the Sd phenotype was unknown. In this study, we identified the molecular basis of the Sd mutation. By positional cloning, we identified the insertion of an early transposon in the Sd candidate locus approximately 12-kb upstream of Ptf1a. We found that insertion of the transposon caused overexpression of three neighboring genes, Gm13344, Gm13336, and Ptf1a, in Sd mutant embryos and that the Sd phenotype was not caused by disruption of an as-yet-unknown gene in the candidate locus. Using multiple knockout and knock-in mouse models, we demonstrated that misexpression of Ptf1a, but not of Gm13344 or Gm13336, in the notochord, hindgut, cloaca, and mesonephros was sufficient to replicate the Sd phenotype. The ectopic expression of Ptf1a in the caudal embryo resulted in attenuated expression of Cdx2 and its downstream target genes T, Wnt3a, and Cyp26a1; we conclude that this is the molecular basis of the Sd phenotype. Analysis of Sd mutant mice will provide insight into the development of the spinal column, anus, and kidney.

Underlying genetic factors of the VATER/VACTERL association with special emphasis on the “Renal” phenotype

“…As a consequence and contrary to wildtype littermates, Sd mice showed ectopic Ptf1a expression in the notochord and hindgut at E8.5 to E9.5, which extended to the cloaca and mesonephros at E10.5 and to the pancreatic bud at E10.5 and E11.5 [46]. The resultant phenotype of this Sd mutation not only causes reduction or absence of kidneys, but mirrors the complete phenotype of the human VATER/VACTERL association.…”

Non-essential function of KRAB zinc finger gene clusters in retrotransposon suppression

“…Furthermore, recent reports have suggested that KRAB-ZFP gene content is distinct in different strains of laboratory mice (37,38), and reduced KRAB-ZFP gene content could contribute to increased activity in individual mice. Although we have yet to find obvious phenotypes in the mice carrying new insertions, novel ETn germ line insertions have been shown to cause phenotypes from short tails (39)(40)(41) to limb malformation (3) and severe morphogenetic defects including polypodia (42) depending upon their insertion site.…”

Retrotransposon Activates Ectopic Ptf1 Expression: A Short Tail

“…Exploded view shows the completely sequenced region, the ETn (red triangle) as the only variant in Sd . (F) Semba et al [7] demonstrate both the functional significance of the ETn and the requirement for a Ptf1a open reading frame using serial gene targeting of wild type–and Sd /+-derived ES cells and conventional transgenic mice. Blue triangles, variant loxP sites; red triangles, ETn insertion; colored boxes, neo (green), Ptf1a (deep purple), and Gm13336 (light purple) replacement cassettes.…”

“…In this issue of PLOS Genetics , three laboratories independently identify the Sd mutation as an 8.5 kb ETn retrotransposon insertion 12.3 kb upstream from the Ptf1a gene [5]–[7]. Ptf1a encodes a cell type–restricted basic helix-loop-helix transcription factor required for development of the pancreas and cerebellum [8]–[10].…”

“…By physical mapping with an Sd / Sd cosmid library constructed for that purpose, they found an unexpectedly large fragment containing the 8.5 kb ETn element as a candidate mutation [7]. To test its functional relevance to Sd , the group performed a true tour-de-force of mouse genetics, producing a series of targeted and transgenic alleles to determine which gene products were functionally important to the Sd phenotype in the context of the ETn (Figure 1F).…”