A Wisp3 Cre-knockin Allele Produces Efficient Recombination in Spermatocytes during Early Prophase of Meiosis I

Individuals with the autosomal recessive skeletal disorder Progressive Pseudorheumatoid Dysplasia have loss-of-function mutations in WISP3, and aberrant WISP3 expression has been detected in tumors from patients with colon and breast cancer. In mice however, neither absence nor over-expression of WISP3 was found to cause a phenotype, and endogenous Wisp3 expression has been difficult to detect. To confirm that Wisp3 knockout mice have no phenotype and to identify potential sites of endogenous Wisp3 expression, we generated mice with a knockin allele (Wisp3GFP-Cre) designed to express Green Fluorescent Protein (GFP) and Cre-recombinase instead of WISP3. Heterozygous and homozygous knockin mice were fertile and indistinguishable from their wild-type littermates, confirming that mice lacking Wisp3 have no phenotype. We could not detect GFP-expression from the knockin allele, but we could detect Cre-expression after crossing mice with the knockin allele to Cre-reporter mice; the double heterozygous offspring had evidence of Cre-mediated recombination in several tissues. The only tissue that had high levels of Cre-mediated recombination was the testis, where recombination in spermatocytes occurred by early prophase of meiosis I. As a consequence, males that were double heterozygous for a Wisp3GFP-Cre and a floxed allele only contributed a recombined allele to their offspring. We detected no evidence of Cre-mediated recombination in the female ovary, although when double heterozygous females contributed the reporter allele to their offspring it had recombined ~7% of the time. Wisp3GFP-Cre expression therefore occurs less frequently and most likely at a later stage of oocyte development in female mice compared to male mice. We conclude that although WISP3 is dispensable in mice, male mice with a Wisp3GFP-Cre allele (Jackson Laboratory stock # 017685) will be useful for studying early prophase of meiosis I and for efficiently recombining floxed alleles that are passed to offspring

<i>Wisp3</i>^GFP-Cre activity occurs in spermatocytes early during meiosis I.

<p>Fluorescence microscope images of seminiferous tubules from <i>Wisp3</i>^+/GFP-Cre;<i>ROSA26</i>^+/mTmG male mice at 5, 10, 14, 15, 17, 19 and 27 days-of-age (P5 – P27). Cell nuclei are imaged with DAPI dye. Spermatocytes expressing membrane bound Green Fluorescent Protein (GFP) instead of Tomato Fluorescent Protein (TFP), which indicates that Cre-mediated recombination has occurred, become visible by P15 and increase in abundance with age. Germ cells and spermatogonial cells, which reside near the periphery of seminiferous tubules, do not express GFP. The location of the GFP expressing cells in the seminiferous tubules, coupled with PCR evidence of recombination by P10 (data not shown), suggests that the <i>Wisp3</i>^GFP-Cre allele is expressed in spermatocytes between the leptotene and pachytene stages of male meiosis I.</p

Endogenous <i>Wisp3</i> expression and <i>Wisp3</i>^{<i>GFP-Cre</i>} expression are not identical.

<p>(Upper panel) RT-PCR amplimers indicating the presence of <i>Wisp3</i> transcript in total RNA from a several different mouse tissues. A schematic of <i>Wisp3</i> mRNA is indicated (not drawn to scale) along with the locations of the intron-spanning PCR primers and the expected amplimer size for correctly splice mRNA (Lower panel). A schematic of the <i>ROSA26</i>^mTmG allele before and after Cre-recombination (not drawn to scale) along with the locations of the PCR primers and the expected amplimer sizes for the non-recombined and recombined alleles. PCR amplimers indicating non-recombined <i>ROSA26</i>^mTmG DNA (upper gel) in all tissues and Cre-mediated recombination (lower gel arrowheads) in testis, heart, and brain recovered from <i>Wisp3</i>^{+/GFP- Cre};<i>ROSA26</i>^+/mTmG mice (floxed and recombined template DNA serves as controls for the two primer pairs). Note that there is poor correlation between endogenous <i>Wisp3</i> expression (upper panel) and <i>Wisp3</i>^GFP-Cre activity (lower panel).</p