Early Prophase Research Articles

Meiotic Chromosome Structure and Function in Plants

Chromosome structure is important for many meiotic processes. Here, we outline 3 main determinants of chromosome structure and their effects on meiotic processes in plants. Cohesins are necessary to hold sister chromatids together until the first meiotic division, ensuring that homologous chromosomes and not sister chromatids separate during anaphase I. During meiosis in maize, Arabidopsis, and rice, cohesins are needed for establishing early prophase chromosome structure and recombination and for aligning bivalents at the metaphase plate. Condensin complexes play pivotal roles in controlling the packaging of chromatin into chromosomes through chromatin compaction and chromosome individualization. In animals and fungi, these complexes establish a meiotic chromosome structure that allows for proper recombination, pairing, and synapsis of homologous chromosomes. In plants, information on the role of condensins in meiosis is limited, but they are known to be required for successful completion of reproductive development. Therefore, we speculate that they play roles similar to animal and fungal condensins during meiosis. Plants generally have large and complex genomes due to frequent polyploidy events, and likely, condensins and cohesins organize chromosomes in such a way as to ensure genome stability. Hexaploid wheat has evolved a unique mechanism using a Ph1 locus-controlled chromosome organization to ensure proper chromosome pairing in meiosis. Altogether, studies on meiotic chromosome structure indicate that chromosome organization is not only important for chromatin packaging but also fulfills specific functions in facilitating chromosome interactions during meiosis, including pairing and recombination.

Unravelling the proteomic profile of rice meiocytes during early meiosis.

Transfer of genetic traits from wild or related species into cultivated rice is nowadays an important aim in rice breeding. Breeders use genetic crosses to introduce desirable genes from exotic germplasms into cultivated rice varieties. However, in many hybrids there is only a low level of pairing (if existing) and recombination at early meiosis between cultivated rice and wild relative chromosomes. With the objective of getting deeper into the knowledge of the proteins involved in early meiosis, when chromosomes associate correctly in pairs and recombine, the proteome of isolated rice meiocytes has been characterized by nLC-MS/MS at every stage of early meiosis (prophase I). Up to 1316 different proteins have been identified in rice isolated meiocytes in early meiosis, being 422 exclusively identified in early prophase I (leptotene, zygotene, or pachytene). The classification of proteins in functional groups showed that 167 were related to chromatin structure and remodeling, nucleic acid binding, cell-cycle regulation, and cytoskeleton. Moreover, the putative roles of 16 proteins which have not been previously associated to meiosis or were not identified in rice before, are also discussed namely: seven proteins involved in chromosome structure and remodeling, five regulatory proteins [such as SKP1 (OSK), a putative CDK2 like effector], a protein with RNA recognition motifs, a neddylation-related protein, and two microtubule-related proteins. Revealing the proteins involved in early meiotic processes could provide a valuable tool kit to manipulate chromosome associations during meiosis in rice breeding programs. The data have been deposited to the ProteomeXchange with the PXD001058 identifier.

Meiosis-Specific Cohesin Component, Stag3 Is Essential for Maintaining Centromere Chromatid Cohesion, and Required for DNA Repair and Synapsis between Homologous Chromosomes

Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin complexes. Furthermore, our data suggests that STAG3 is required for structural changes of chromosomes that mediate chromosome pairing and synapsis, DNA repair and progression of meiosis.

H2A.Z-Dependent Regulation of Cohesin Dynamics on Chromosome Arms

Structural maintenance of chromosomes (SMC) complexes and DNA topoisomerases are major determinants of chromosome structure and dynamics. The cohesin complex embraces sister chromatids throughout interphase, but during mitosis most cohesin is stripped from chromosome arms by early prophase, while the remaining cohesin at kinetochores is cleaved at anaphase. This two-step removal of cohesin is required for sister chromatids to separate. The cohesin-related Smc5/6 complex has been studied mostly as a determinant of DNA repair via homologous recombination. However, chromosome segregation fails in Smc5/6 null mutants or cells treated with small interfering RNAs. This also occurs in Smc5/6 hypomorphs in the fission yeast Schizosaccharomyces pombe following genotoxic and replication stress, or topoisomerase II dysfunction, and these mitotic defects are due to the postanaphase retention of cohesin on chromosome arms. Here we show that mitotic and repair roles for Smc5/6 are genetically separable in S. pombe. Further, we identified the histone variant H2A.Z as a critical factor to modulate cohesin dynamics, and cells lacking H2A.Z suppress the mitotic defects conferred by Smc5/6 dysfunction. Together, H2A.Z and the SMC complexes ensure genome integrity through accurate chromosome segregation.

Loading of the centromeric histone H3 variant during meiosis-how does it differ from mitosis?

In eukaryotic phyla studied so far, the essential centromeric histone H3 variant (CENH3) is loaded to centromeric nucleosomes after S-phase (except for yeast) but before mitotic segregation (except for metazoan). While the C-terminal part of CENH3 seems to be sufficient for mitotic centromere function in plants, meiotic centromeres neither load nor tolerate impaired CENH3 molecules. However, details about CENH3 deposition in meiocytes are unknown (except for Drosophila). Therefore, we quantified fluorescence signals after the immunostaining of CENH3 along meiotic and mitotic nuclear division cycles of rye, a monocotyledonous plant. One peak of fluorescence intensity appeared in the early meiotic prophase of pollen mother cells and a second one during interkinesis, both followed by a decrease of CENH3. Then, the next loading occurred in the male gametophyte before its first mitotic division. These data indicate that CENH3 loading differs between mitotic and meiotic nuclei. Contrary to the situation in mitotic cycles, CENH3 deposition is biphasic during meiosis and apparently linked with a quality check, a removal of impaired CENH3 molecules, and a general loss of CENH3 after each loading phase. These steps ensure an endowment of centromeres with a sufficient amount of correct CENH3 molecules as a prerequisite for centromere maintenance during mitotic cycles of the microgametophyte and the progeny. From a comparison with data available for Drosophila, we hypothesise that the post-divisional mitotic CENH3 loading in metazoans is evolutionarily derived from the post-divisional meiotic loading phase, while the pre-divisional first meiotic loading has been conserved among eukaryotes.

The transcriptome landscape of early maize meiosis.

BackgroundA major step in the higher plant life cycle is the decision to leave the mitotic cell cycle and begin the progression through the meiotic cell cycle that leads to the formation of gametes. The molecular mechanisms that regulate this transition and early meiosis remain largely unknown. To gain insight into gene expression features during the initiation of meiotic recombination, we profiled early prophase I meiocytes from maize (Zea mays) using capillary collection to isolate meiocytes, followed by RNA-seq.ResultsWe detected ~2,000 genes as preferentially expressed during early meiotic prophase, most of them uncharacterized. Functional analysis uncovered the importance of several cellular processes in early meiosis. Processes significantly enriched in isolated meiocytes included proteolysis, protein targeting, chromatin modification and the regulation of redox homeostasis. The most significantly up-regulated processes in meiocytes were processes involved in carbohydrate metabolism. Consistent with this, many mitochondrial genes were up-regulated in meiocytes, including nuclear- and mitochondrial-encoded genes. The data were validated with real-time PCR and in situ hybridization and also used to generate a candidate maize homologue list of known meiotic genes from Arabidopsis.ConclusionsTaken together, we present a high-resolution analysis of the transcriptome landscape in early meiosis of an important crop plant, providing support for choosing genes for detailed characterization of recombination initiation and regulation of early meiosis. Our data also reveal an important connection between meiotic processes and altered/increased energy production.

Homoeologous Chromosome Sorting and Progression of Meiotic Recombination in Brassica napus: Ploidy Does Matter!

Meiotic recombination is the fundamental process that produces balanced gametes and generates diversity within species. For successful meiosis, crossovers must form between homologous chromosomes. This condition is more difficult to fulfill in allopolyploid species, which have more than two sets of related chromosomes (homoeologs). Here, we investigated the formation, progression, and completion of several key hallmarks of meiosis in Brassica napus (AACC), a young polyphyletic allotetraploid crop species with closely related homoeologous chromosomes. Altogether, our results demonstrate a precocious and efficient sorting of homologous versus homoeologous chromosomes during early prophase I in two representative B. napus accessions that otherwise show a genotypic difference in the progression of homologous recombination. More strikingly, our detailed comparison of meiosis in near isogenic allohaploid and euploid plants showed that the mechanism(s) promoting efficient chromosome sorting in euploids is adjusted to promote crossover formation between homoeologs in allohaploids. This suggests that, in contrast to other polyploid species, chromosome sorting is context dependent in B. napus.

Trimethylation of Histone H3 Lysine 36 by Human Methyltransferase PRDM9 Protein

PRDM9 (PR domain-containing protein 9) is a meiosis-specific protein that trimethylates H3K4 and controls the activation of recombination hot spots. It is an essential enzyme in the progression of early meiotic prophase. Disruption of the PRDM9 gene results in sterility in mice. In human, several PRDM9 SNPs have been implicated in sterility as well. Here we report on kinetic studies of H3K4 methylation by PRDM9 in vitro indicating that PRDM9 is a highly active histone methyltransferase catalyzing mono-, di-, and trimethylation of the H3K4 mark. Screening for other potential histone marks, we identified H3K36 as a second histone residue that could also be mono-, di-, and trimethylated by PRDM9 as efficiently as H3K4. Overexpression of PRDM9 in HEK293 cells also resulted in a significant increase in trimethylated H3K36 and H3K4 further confirming our in vitro observations. Our findings indicate that PRDM9 may play critical roles through H3K36 trimethylation in cells.

STAG3 is a strong candidate gene for male infertility

Oligo- and azoospermia are severe forms of male infertility. However, known genetic factors account only for a small fraction of the cases. Recently, whole-exome sequencing in a large consanguineous family with inherited premature ovarian failure (POF) identified a homozygous frameshift mutation in the STAG3 gene leading to a premature stop codon. STAG3 encodes a meiosis-specific subunit of the cohesin complex, a large proteinaceous ring with DNA-entrapping ability that ensures sister chromatid cohesion and enables correct synapsis and segregation of homologous chromosomes during meiosis. The pathogenicity of the STAG3 mutations was functionally validated with a loss-of-function mouse model for STAG3 in oogenesis. However, and since none of the male members of this family was homozygous for the mutant allele, we only could hypothesized its putative involvement in male infertility. In this report, we show that male mice devoid of Stag3 display a severe meiotic phenotype that includes a meiotic arrest at zygonema-like shortening of their chromosome axial elements/lateral elements, partial loss of centromeric cohesion at early prophase and maintenance of the ability to initiate but not complete RAD51- and DMC1-mediated double-strand break repair, demonstrating that STAG3 is a crucial cohesin subunit in mammalian gametogenesis and supporting our proposal that STAG3 is a strong candidate gene for human male infertility.

Mutant Cohesin in Premature Ovarian Failure

Premature ovarian failure is a major cause of female infertility. The genetic causes of this disorder remain unknown in most patients. Using whole-exome sequence analysis of a large consanguineous family with inherited premature ovarian failure, we identified a homozygous 1-bp deletion inducing a frameshift mutation in STAG3 on chromosome 7. STAG3 encodes a meiosis-specific subunit of the cohesin ring, which ensures correct sister chromatid cohesion. Female mice devoid of Stag3 are sterile, and their fetal oocytes are arrested at early prophase I, leading to oocyte depletion at 1 week of age.

The APC/C activator FZR1 is essential for meiotic prophase I in mice

Fizzy-related 1 (FZR1) is an activator of the Anaphase promoting complex/cyclosome (APC/C) and an important regulator of the mitotic cell division cycle. Using a germ-cell-specific conditional knockout model we examined its role in entry into meiosis and early meiotic events in both sexes. Loss of APC/C(FZR1) activity in the male germline led to both a mitotic and a meiotic testicular defect resulting in infertility due to the absence of mature spermatozoa. Spermatogonia in the prepubertal testes of such mice had abnormal proliferation and delayed entry into meiosis. Although early recombination events were initiated, male germ cells failed to progress beyond zygotene and underwent apoptosis. Loss of APC/C(FZR1) activity was associated with raised cyclin B1 levels, suggesting that CDK1 may trigger apoptosis. By contrast, female FZR1Δ mice were subfertile, with premature onset of ovarian failure by 5 months of age. Germ cell loss occurred embryonically in the ovary, around the time of the zygotene-pachytene transition, similar to that observed in males. In addition, the transition of primordial follicles into the growing follicle pool in the neonatal ovary was abnormal, such that the primordial follicles were prematurely depleted. We conclude that APC/C(FZR1) is an essential regulator of spermatogonial proliferation and early meiotic prophase I in both male and female germ cells and is therefore important in establishing the reproductive health of adult male and female mammals.

Molecular characterization of collaborator of ARF (CARF) as a DNA damage response and cell cycle checkpoint regulatory protein

CARF is an ARF-binding protein that has been shown to regulate the p53–p21–HDM2 pathway. CARF overexpression was shown to cause growth arrest of human cancer cells and premature senescence of normal cells through activation of the p53 pathway. Because replicative senescence involves permanent withdrawal from the cell cycle in response to DNA damage response-mediated signaling, in the present study we investigated the relationship between CARF and the cell cycle and whether it is involved in the DNA damage response. We demonstrate that the half-life of CARF protein is less than 60min, and that in cycling cells CARF levels are highest in G2 and early prophase. Serially passaged normal human skin and stromal fibroblasts showed upregulation of CARF during replicative senescence. Induction of G1 growth arrest and senescence by a variety of drugs was associated with increase in CARF expression at the transcriptional and translational level and was seen to correlate with increase in DNA damage response and checkpoint proteins, ATM, ATR, CHK1, CHK2, γH2AX, p53 and p21. Induction of growth arrest by oncogenic RAS and shRNA-mediated knockdown of TRF2 in cancer cells also caused upregulation of CARF. We conclude that CARF is associated with DNA damage response and checkpoint signaling pathways.

PCC-like induction of mitosis in Chara vulgaris antheridia initiating differentiation of spermatozoids in the darkness

The objects of this research were the antheridial filament cells of <em>Chara vulgaris</em> which after 5-day mitodepressing treatment of the darkness reactivated mitosis (without light stimulation) in the result of the physiological state evocing the initiation of spermiogenesis in the antheridium. The course of these divisions was observed in relation to control cells cultivated in the natural photoperiod L:D = 14:10. It was shown that the passage of dark-arrested cells from early G<sub>2</sub> (VII stage) to the early prophase in the cells linked to the base of filaments by plasmodesmata with spermatids initiating spermiogenesis, resembles the prematuraly condensed chromosomes (PCC). In this way a by-pass of the cells in the stage VII to early prophase occurs without reconstruction of granular components of nucleoli and with condensed stage of mitochondria characterising the dark-arrested cells. In opposition to this phenomenon in the control cell cycle and in light-induced reactivation of mitosis in the dark-arrested cells, initiation of the prophase follows the series of changes of the nuclear ultrastructure (VIII stage - decondensation of chromatin, IX and X stages nuclei with reticulate chromatin). At the early prophase, irrespective of the physiological conditions, the electron-transparent space appears between nuclear envelope and the chromatin as the effect of solublisation of lamins. The fine fibrils pass this space and link chromatin to the inner nuclear membrane.

Aggregations of organelles in meiotic cells of higher plants

During early prophase I in microsporocytes and sporocytes of various plants all mitochondria and plastids aggregate in a group, where some plastids seem to undergo division. This group desintegrates by middle prophase I. Further aggregations of plastids and mitochondria occur in microsporogenesis and sporogenesis is of a simultaneous type. Organelles aggregate the second time at the end of prophase 1 and during or after telophase I they form a dense equatorial plate which lasts until telophase IL Since the phragmoplast is dismantled after telophase I and there is no cytokinesis, organelles aggregated in the plate apparently prevent merging of the nuclei and spindles of meiosis II, thus taking over a role of a phragmoplast and cell wall. In some plants after telophase II organelle aggregation changes shape and occupies the planes where cell walls will be built in simultaneous cytokinesis. Positioning of plastids and mitochondria along these planes may facilitate their equal apportionment among the postmeiotic cells.

Ultrastructural changes during megasporogenesis in Epipactis (Orchidaceae)

Plastids were temporarily localized within the micropylar portion of the early first prophase <em>Epipactis</em> meiocyte. Some of these plastids were observed in close proximity to the nuclear envelope. With the exception of this short period plastids were distributed randomly in the meiocyte. During late prophase, starch-containing plastids become cup-shaped and depleted of starch. Plastids were found within both dyad cells and all cells of the tetrad. Elongated segments of ER cisternae in various configurations were present. The chalazal wall of the prophase meiocyte differed from other walls in the presence of the ingrowths and plasmodesmata. The micropylar portion of the nuclear envelope at some stages of the I prophase seemed to be devoid of pores whereas the chalazal part contained numerous pores. These structural characters reflect a polar differentiation of the meiocyte along a micropylar-chalazal axis.

Meiotic studies in some species of tribe Cichorieae (Asteraceae) from Western Himalayas.

The present paper deals with meiotic studies in 15 species belonging to 6 genera of the tribe Cichorieae from various localities of Western Himalayas. The chromosome number has been reported for the first time in Hieracium crocatum (2n = 10) and Lactuca lessertiana (2n = 2x = 16). Further, intraspecific variability has been reported for the first time in H. umbellatum (2n = 2x = 10 and 2n = 6x = 54), Tragopogon dubius (2n = 2x = 14 and 2n = 4x = 28), and T. gracilis (2n = 2x = 14). The chromosome report of 2n = 2x = 10 in Youngia tenuifolia is made for the first time in India. Maximum numbers of the populations show laggards, chromosome stickiness, and cytomixis from early prophase to telophase-II, leading to the formation of aneuploid cells or meiocytes with double chromosome number. Such meiotic abnormalities produce unreduced pollen grains and the reduced pollen viability.

Meiotic Cohesin SMC1β Provides Prophase I Centromeric Cohesion and Is Required for Multiple Synapsis-Associated Functions

Cohesin subunit SMC1β is specific and essential for meiosis. Previous studies showed functions of SMC1β in determining the axis-loop structure of synaptonemal complexes (SCs), in providing sister chromatid cohesion (SCC) in metaphase I and thereafter, in protecting telomere structure, and in synapsis. However, several central questions remained unanswered and concern roles of SMC1β in SCC and synapsis and processes related to these two processes. Here we show that SMC1β substantially supports prophase I SCC at centromeres but not along chromosome arms. Arm cohesion and some of centromeric cohesion in prophase I are provided by non-phosphorylated SMC1α. Besides supporting synapsis of autosomes, SMC1β is also required for synapsis and silencing of sex chromosomes. In absence of SMC1β, the silencing factor γH2AX remains associated with asynapsed autosomes and fails to localize to sex chromosomes. Microarray expression studies revealed up-regulated sex chromosome genes and many down-regulated autosomal genes. SMC1β is further required for non-homologous chromosome associations observed in absence of SPO11 and thus of programmed double-strand breaks. These breaks are properly generated in Smc1β−/− spermatocytes, but their repair is delayed on asynapsed chromosomes. SMC1α alone cannot support non-homologous associations. Together with previous knowledge, three main functions of SMC1β have emerged, which have multiple consequences for spermatocyte biology: generation of the loop-axis architecture of SCs, homologous and non-homologous synapsis, and SCC starting in early prophase I.

Loss Of NIPA Leads To Defects In The Repair Of DNA Double Strand Breaks In Mice

Regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry.Using a conditional knockout strategy we inactivated the gene encoding Nipa. Nipa-deficient animals are viable, but show a lower birth rate and a reduced body weight. Furthermore, Nipa-deficient males were sterile due to a block of spermatogenesis during meiotic prophase. Virtually no spermatocytes progress beyond a late-zygotene to early-pachytene stage and reach an aberrant stage, with synaptonemal complex disassembly and incomplete synapsis. Nipa-/- females are sub-fertile with an early and severe meiotic defect during embryogenesis with extensive apoptosis in early prophase (E13.5-E14.5).Here we report, that Nipa-/- meiocytes exhibit persistent cytological markers for DNA double strand break repair proteins (like DMC1, RAD51) in meiotic prophase with more than twice as many DMC1 foci as control animals. Kinetic analysis of the first wave of spermatogenesis showed increased DMC1/RAD51 foci in Nipa-/- cells as soon as early-pachynema cells appear (13-14 days post partum). Moreover, we show that Nipa deficiency does not lead to a defect in meiotic sex chromosome inactivation despite epithelial stage IV apoptosis. Nipa-deficient spermatocytes exhibit numerous abnormalities in staining of chromosome axis associated proteins (like SYCP3 and STAG3) indicating that chromosome axis defects were associated with compromised chromosome axis integrity leading to overt chromosome fragmentation.Further in vitro analyses with bleomycin treated MEFs displayed high pH2AX levels in cells lacking NIPA. Repair of DNA DSB seemed to be abolished in these cells as the pH2AX-level were sustained and still visible after 90 min of timecourse, where wildtype cells already repaired sides of DNA Damage. Consistent with these findings NIPA-deficient spleen cells showed compromised DNA Damage repair measured in a comet assay with a significantly longer olive tail moment in NIPA knockout cells under repair conditions.Taken together, the phenotype of Nipa-knockout mice is a definitive proof of the meiotic significance of NIPA and our results show a new, unsuspected role of NIPA in chromosome stability and the repair of DNA double strand breaks. Disclosures:No relevant conflicts of interest to declare.

Centromere Pairing in Early Meiotic Prophase Requires Active Centromeres and Precedes Installation of the Synaptonemal Complex in Maize

Pairing of homologous chromosomes in meiosis is critical for their segregation to daughter cells. In most eukaryotes, clustering of telomeres precedes and facilitates chromosome pairing. In several species, centromeres also form pairwise associations, known as coupling, before the onset of pairing. We found that, in maize (Zea mays), centromere association begins at the leptotene stage and occurs earlier than the formation of the telomere bouquet. We established that centromere pairing requires centromere activity and the sole presence of centromeric repeats is not sufficient for pairing. In several species, homologs of the ZIP1 protein, which forms the central element of the synaptonemal complex in budding yeast (Saccharomyces cerevisiae), play essential roles in centromere coupling. However, we found that the maize ZIP1 homolog ZYP1 installs in the centromeric regions of chromosomes after centromeres form associations. Instead, we found that maize structural maintenance of chromosomes6 homolog forms a central element of the synaptonemal complex, which is required for centromere associations. These data shed light on the poorly understood mechanism of centromere interactions and suggest that this mechanism may vary somewhat in different species.

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 (Wisp3 GFP-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 Wisp3 GFP-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. Wisp3 GFP-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 Wisp3 GFP-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.