Neonatal exposure to bisphenol analogues disrupts genital development in male mice.

The public concern and governmental regulations on bisphenol A (BPA) have stimulated the development and production of alternative analogues to replace BPA in a myriad of applications. Given the endocrine disrupting activities of BPA and potentially other analogues, the present study investigated and compared the effects of neonatal exposure to BPA, BPB, BPE, BPF, and BPS on the genital development in male mice. Pups were injected subcutaneously on the right shoulder in the mornings of postnatal days P0.5, P2, P4, and P6, resulting in a low dose of 0.05 µg/g body weight (bw)/day and a high dose of 10 µg/g bw/day. Mice were sacrificed at predetermined time and evaluated for gene expression levels (3 days after birth or P3), steroid hormone levels (P5), and morphological changes (P21). The results demonstrated that BPA, BPB, BPE, or BPF significantly shortened glans penis length and anogenital distance, while BPS didn't. Testis weight and anogenital distance were also significantly affected by BPA, BPE or BPF. The results also revealed that bisphenol analogues exposure significantly reduced testosterone levels, and altered the expression levels of developmental genes networks in developing penis of mice. Our data demonstrate that selected bisphenol analogues may possess similar endocrine disrupting effects compared to BPA, and exposure to these analogues could affect reproductive development of male mice. This raises the concern on the environmental and health safety of bisphenol analogues applied as industrial BPA replacements.

Prenatal exposure to atrazine induces cryptorchidism and hypospadias in F1 male mouse offspring.

The main objective of the present study was to determine whether prenatal exposure to atrazine could affect testicle descent and penile masculinization. Atrazine has been demonstrated with a variety of endocrine disrupting activities and reproductive toxicities. However, the effects of prenatal atrazine exposure on male offspring's genital malformation, such as hypospadias and cryptorchidism, remain poorly understood. In this study, pregnant ICR mice were gavaged from gestational day 12.5-16.5 with different doses of atrazine. Although no sign of systemic toxicity was observed in F1 male pups, prenatal exposure to 100 mg/kg/day atrazine affected penile morphology, urethral meatus position and descent of testis, and reduced anogenital distance and penile size in postnatal day 21 F1 male pups. The comparative study with an androgen receptor (AR) antagonist vinclozolin suggested that these effects of atrazine on male genital development may not be through antagonism of AR. The results also revealed that atrazine exposure significantly reduced maternal serum testosterone levels, decreased AR nuclear translocation, and altered the expression levels of developmental gene networks in developing penis of mice. Atrazine exposure also affected the expression of insulin-like 3 (Insl3) and steroidogenic gene expression in developing reproductive tract. Therefore, our data indicate that prenatal atrazine exposure can induce hypospadias in F1 mice, likely through disruption of testosterone production, decreasing genomic androgen action, and then altering expression of developmental genes during sexual differentiation. Our data also suggest that prenatal atrazine exposure can induce cryptorchidism in F1 mice, possibly through down regulation of Insl3.

Etiology of Hypospadias: A Comparative Review of Genetic Factors and Developmental Processes Between Human and Animal Models.

Hypospadias is a congenital anomaly of the penis with an occurrence of approximately 1 in 200 boys, but the etiology of the majority of hypospadias has remained unknown. Numerous genes have been reported as having variants in hypospadias patients, and many studies on genetic deletion of key genes in mouse genital development have also been published. Until now, no comparative analysis in the genes related literature has been reported. The basic knowledge of penile development and hypospadias is mainly obtained from animal model studies. Understanding of the differences and similarities between human and animal models is crucial for studies of hypospadias. In this review, mutations and polymorphisms of hypospadias-related genes have been compared between humans and mice, and differential genotype-phenotype relationships of certain genes between humans and mice have been discussed using the data available in PubMed and MGI online databases, and our analysis only revealed mutations in seven out of 43 human hypospadias related genes which have been reported to show similar phenotypes in mutant mice. The differences and similarities in the processes of penile development and hypospadias malformation among human and commonly used animal models suggest that the guinea pig may be a good model to study the mechanism of human penile development and etiology of hypospadias.

Prenatal low-dose methyltestosterone, but not dihydrotestosterone, treatment induces penile formation in female mice and guinea pigs†.

Genital tubercle has bisexual potential before sex differentiation. Females exposed to androgen during sex differentiation show masculinized external genitalia, but the effects of different androgens on tubular urethral and penile formation in females are mostly unknown. In this study, we compared the masculinization effects of commonly used androgens methyltestosterone, dihydrotestosterone, and testosterone on the induction of penile formation in females. Our results suggested that prenatal treatment with low doses of methyltestosterone, but not same doses of dihydrotestosterone or testosterone, could induce penile formation in female mice. The minimum dose of dihydrotestosterone and testosterone for inducing tubular urethral formation in female mice was, respectively, 50 and 20 times higher than that of methyltestosterone. In vivo methyltestosterone treatment induced more nuclear translocation of androgen receptors in genital tubercles of female mice, affected Wnt signaling gene expressions, and then led to similar patterns of cell proliferation and death in developing genital tubercles to those of control males. We further revealed that low-dose methyltestosterone, but not same dose of dihydrotestosterone or testosterone, treatment induced penile formation in female guinea pigs. Exposure of female mouse genital tubercle organ culture to methyltestosterone, dihydrotestosterone, or testosterone could induce nuclear translocation of androgen receptors, suggesting that the differential effect of the three androgens in vivo might be due to the hormonal profile in mother or fetus, rather than the local genital tissue. To understand the differential role of these androgens in masculinization process involved is fundamental to androgen replacement therapy for diseases related to external genital masculinization.

Differential cell proliferation and cell death during the urethral groove formation in guinea pig model.

BACKGROUND: Urethral groove (UG) formation is an important step in penile formation. Because commonly used animal models do not have UG, the mechanisms of UG formation have never been discovered. We aim to discover the cellular mechanism of the UG formation using guinea pig model. METHODS: Histology was used to study the ontogeny of UG. BrdU immunofluorescence was used to label proliferating cells, cell death was determined using LysoTracker Red and TUNEL staining, and stereology was used for quantification. To reveal Shh mRNA expression patterns, in situ hybridization was performed in guinea pig genital tubercles (GTs) and ShhGFPcre-LacZ-reporter mice were used for comparison. RESULTS: Cell proliferation in the outer layers and programmed cell death in the inner layers of urethral epithelium played key roles during urethral canal movement from dorsal to ventral aspect and final opening to form UG. Shh mRNA expression domain shifted out to the ventral surface of GT from proximal throughout to distal in guinea pigs, but was excluded from the ventral surface epithelium in midshaft and distal of mouse GT. CONCLUSION: Differential cell proliferation and cell death in developing urethral epithelium lead to UG formation and Shh expression in ventral surface epithelium of GT may play an important role.

External Genital Development, Urethra Formation, and Hypospadias Induction in Guinea Pig: A Double Zipper Model for Human Urethral Development.

OBJECTIVE: To determine whether the guinea pig phallus would be an appropriate model of human penile development, we characterized the embryology and sexual differentiation of guinea pig external genitalia and attended to induce hypospadias in males and tubular urethra formation in females pharmacologically. MATERIALS AND METHODS: The external genitalia of guinea pig were collected from genital swelling initiation to newborn stages, and scanning electronic microscopy and histology were performed to visualize the morphology and structure. Immunohistochemistry was used to determine the androgen receptor localization. Bicalutamide and methyltestosterone were given to pregnant dams to reveal the role and timing of androgen in guinea pig penile masculinization. RESULTS: Canalization and dorsal-to-ventral movement of the urethral canal develops the urethral groove in both sexes, and then the males perform distal-opening-proximal-closing to form tubular urethra. More nuclear-localized androgen receptor is found in proximal genital tubercles of males than in females at (E) 29. Antiandrogen treatment at E26-E30 can cause hypospadias, and methyltestosterone administration at E27-E31 can induce tubular urethra formation in females. CONCLUSION: Fetal development of the guinea pig phallus is homologous to that of humans. Although guinea pig has structures similar to mouse, the urethral groove and the tubular urethra formation are more similar to humans. Antiandrogen treatment causes hypospadias in males and additional androgen induces tubular urethra formation in females. Thus, guinea pig is an appropriate model for further study of cellular and molecular mechanisms involved in distal-opening-proximal-closing in tubular urethra formation and the evaluation of the pathophysiological processes of hypospadias.

Dose Dependent Dual Effect of Baicalin and Herb Huang Qin Extract on Angiogenesis.

Huang Qin (root of Scutellaria baicalensis) is a widely used herb in different countries for adjuvant therapy of inflammation, diabetes, hypertension, different kinds of cancer and virus related diseases. Baicalin is the main flavonoid in this herb and has been extensively studied for 30 years. The angiogenic effect of herb Huang Qin extract and baicalin was found 13 years ago, however, the results were controversial with pro-angiogenic effect in some studies and anti-angiogenic effect in others. In this paper, the angiogenic effect of baicalin, its aglycone form baicalein and aqueous extract of Huang Qin was studied in chick embryo chorioallantoic membrane (CAM) model. Dose dependent dual effect was found in both aqueous extract and baicalin, but not in baicalein, in which only inhibitory effect was observed. In order to reveal the cellular and molecular mechanism of how baicalin and baicalein affect angiogenesis, cell proliferation and programmed cell death assays were performed in treated CAM. In addition, quantitative PCR array including 84 angiogenesis related genes was used to detect high and low dosage of baicalin and baicalein responsive genes. Low dose baicalin increased cell proliferation in developing blood vessels through upregulation of multiple angiogenic genes expression, but high dose baicalin induced cell death, performing inhibitory effect on angiogenesis. Both high and low dose of baicalein down regulated the expression of multiple angiogenic genes, decreased cell proliferation, and leads to inhibitory effects on angiogenesis.

Molecular Characterization of the Genital Organizer: Gene Expression Profile of the Mouse Urethral Plate Epithelium.

PURPOSE: Lower urinary tract malformations are among the most common congenital anomalies in humans. Molecular genetic studies of mouse external genital development have begun to identify mechanisms that pattern the genital tubercle and orchestrate urethral tubulogenesis. The urethral plate epithelium is an endodermal signaling region that has an essential role in external genital development. However, little is known about the molecular identity of this cell population or the genes that regulate its activity. MATERIALS AND METHODS: We used microarray analysis to characterize differences in gene expression between urethral plate epithelium and surrounding tissue in mouse genital tubercles. In situ hybridizations were performed to map gene expression patterns and ToppCluster (https://toppcluster.cchmc.org/) was used to analyze gene associations. RESULTS: A total of 84 genes were enriched at least 20-fold in urethral plate epithelium relative to surrounding tissue. The majority of these genes were expressed throughout the urethral plate in males and females at embryonic day 12.5 when the urethral plate is known to signal. Functional analysis using ToppCluster revealed genetic pathways with known functions in other organ systems but unknown roles in external genital development. Additionally, a 3-dimensional molecular atlas of genes enriched in urethral plate epithelium was generated and deposited at the GUDMAP (GenitoUrinary Development Molecular Anatomy Project) website (http://gudmap.org/). CONCLUSIONS: We identified dozens of genes previously unknown to be expressed in urethral plate epithelium at a crucial developmental period. It provides a novel panel of genes for analysis in animal models and in humans with external genital anomalies.

Bisphenol Analogues Other Than BPA: Environmental Occurrence, Human Exposure, and Toxicity-A Review.

Numerous studies have investigated the environmental occurrence, human exposure, and toxicity of bisphenol A (BPA). Following stringent regulations on the production and usage of BPA, several bisphenol analogues have been produced as a replacement for BPA in various applications. The present review outlines the current state of knowledge on the occurrence of bisphenol analogues (other than BPA) in the environment, consumer products and foodstuffs, human exposure and biomonitoring, and toxicity. Whereas BPA was still the major bisphenol analogue found in most environmental monitoring studies, BPF and BPS were also frequently detected. Elevated concentrations of BPAF, BPF, and BPS (i.e., similar to or greater than that of BPA) have been reported in the abiotic environment and human urine from some regions. Many analogues exhibit endocrine disrupting effects, cytotoxicity, genotoxicity, reproductive toxicity, dioxin-like effects, and neurotoxicity in laboratory studies. BPAF, BPB, BPF, and BPS have been shown to exhibit estrogenic and/or antiandrogenic activities similar to or even greater than that of BPA. Knowledge gaps and research needs have been identified, which include the elucidation of environmental occurrences, persistence, and fate of bisphenol analogues (other than BPA), sources and pathways for human exposure, effects on reproductive systems and the mammary gland, mechanisms of toxicity from coexposure to multiple analogues, metabolic pathways and products, and the impact of metabolic modification on toxicity.

Timing of androgen receptor disruption and estrogen exposure underlies a spectrum of congenital penile anomalies.

Congenital penile anomalies (CPAs) are among the most common human birth defects. Reports of CPAs, which include hypospadias, chordee, micropenis, and ambiguous genitalia, have risen sharply in recent decades, but the causes of these malformations are rarely identified. Both genetic anomalies and environmental factors, such as antiandrogenic and estrogenic endocrine disrupting chemicals (EDCs), are suspected to cause CPAs; however, little is known about the temporal window(s) of sensitivity to EDCs, or the tissue-specific roles and downstream targets of the androgen receptor (AR) in external genitalia. Here, we show that the full spectrum of CPAs can be produced by disrupting AR at different developmental stages and in specific cell types in the mouse genital tubercle. Inactivation of AR during a narrow window of prenatal development results in hypospadias and chordee, whereas earlier disruptions cause ambiguous genitalia and later disruptions cause micropenis. The neonatal phase of penile development is controlled by the balance of AR to estrogen receptor α (ERα) activity; either inhibition of androgen or augmentation of estrogen signaling can induce micropenis. AR and ERα have opposite effects on cell division, apoptosis, and regulation of Hedgehog, fibroblast growth factor, bone morphogenetic protein, and Wnt signaling in the genital tubercle. We identify Indian hedgehog (Ihh) as a novel downstream target of AR in external genitalia and show that conditional deletion of Ihh inhibits penile masculinization. These studies reveal previously unidentified cellular and molecular mechanisms by which antiandrogenic and estrogenic signals induce penile malformations and demonstrate that the timing of endocrine disruption can determine the type of CPA.

Development and evolution of the unique cetacean dentition.

The evolutionary success of mammals is rooted in their high metabolic rate. A high metabolic rate is sustainable thanks to efficient food processing and that in turn is facilitated by precise occlusion of the teeth and the acquisition of rhythmic mastication. These major evolutionary innovations characterize most members of the Class Mammalia. Cetaceans are one of the few groups of mammals in which precise occlusion has been secondarily lost. Most toothed whales have an increased number of simple crowned teeth that are similar along the tooth row. Evolution toward these specializations began immediately after the time cetaceans transitioned from terrestrial-to-marine environments. The fossil record documents the critical aspects of occlusal evolution of cetaceans, and allows us to pinpoint the evolutionary timing of the macroevolutionary events leading to their unusual dental morphology among mammals. The developmental controls of tooth differentiation and tooth number have been studied in a few mammalian clades, but nothing is known about how these controls differ between cetaceans and mammals that retain functional occlusion. Here we show that pigs, a cetacean relative with regionalized tooth morphology and complex tooth crowns, retain the typical mammalian gene expression patterns that control early tooth differentiation, expressing Bmp4 in the rostral (mesial, anterior) domain of the jaw, and Fgf8 caudally (distal, posterior). By contrast, dolphins have lost these regional differences in dental morphology and the Bmp4 domain is extended into the caudal region of the developing jaw. We hypothesize that the functional constraints underlying mammalian occlusion have been released in cetaceans, facilitating changes in the genetic control of early dental development. Such major developmental changes drive morphological evolution and are correlated with major shifts in diet and food processing during cetacean evolution.

Mediator subunit18 controls flowering time and floral organ identity in Arabidopsis.

Mediator is a conserved multi-protein complex that plays an important role in regulating transcription by mediating interactions between transcriptional activator proteins and RNA polymerase II. Much evidence exists that Mediator plays a constitutive role in the transcription of all genes transcribed by RNA polymerase II. However, evidence is mounting that specific Mediator subunits may control the developmental regulation of specific subsets of RNA polymerase II-dependent genes. Although the Mediator complex has been extensively studied in yeast and mammals, only a few reports on Mediator function in flowering time control of plants, little is known about Mediator function in floral organ identity. Here we show that in Arabidopsis thaliana, MEDIATOR SUBUNIT 18 (MED18) affects flowering time and floral organ formation through FLOWERING LOCUS C (FLC) and AGAMOUS (AG). A MED18 loss-of-function mutant showed a remarkable syndrome of later flowering and altered floral organ number. We show that FLC and AG mRNA levels and AG expression patterns are altered in the mutant. Our results support parallels between the regulation of FLC and AG and demonstrate a developmental role for Mediator in plants.

Developmental basis of sexually dimorphic digit ratios.

Males and females generally have different finger proportions. In males, digit 2 is shorter than digit 4, but in females digit 2 is the same length or longer than digit 4. The second- to fourth-digit (2D:4D) ratio correlates with numerous sexually dimorphic behavioral and physiological conditions. Although correlational studies suggest that digit ratios reflect prenatal exposure to androgen, the developmental mechanism underlying sexually dimorphic digit development remains unknown. Here we report that the 2D:4D ratio in mice is controlled by the balance of androgen to estrogen signaling during a narrow window of digit development. Androgen receptor (AR) and estrogen receptor α (ER-α) activity is higher in digit 4 than in digit 2. Inactivation of AR decreases growth of digit 4, which causes a higher 2D:4D ratio, whereas inactivation of ER-α increases growth of digit 4, which leads to a lower 2D:4D ratio. We also show that addition of androgen has the same effect as inactivation of ER and that addition of estrogen mimics the reduction of AR. Androgen and estrogen differentially regulate the network of genes that controls chondrocyte proliferation, leading to differential growth of digit 4 in males and females. These studies identify previously undescribed molecular dimorphisms between male and female limb buds and provide experimental evidence that the digit ratio is a lifelong signature of prenatal hormonal exposure. Our results also suggest that the 2D:4D ratio can serve as an indicator of disrupted endocrine signaling during early development, which may aid in the identification of fetal origins of adult diseases.

Conservation and divergence of plant LHP1 protein sequences and expression patterns in angiosperms and gymnosperms.

Floral transition is a critical and strictly regulated developmental process in plants. Mutations in Arabidopsis LIKE HETEROCHROMATIN PROTEIN 1 (AtLHP1)/TERMINAL FLOWER 2 (TFL2) result in early and terminal flowers. Little is known about the gene expression, function and evolution of plant LHP1 homologs, except for Arabidopsis LHP1. In this study, the conservation and divergence of plant LHP1 protein sequences was analyzed by sequence alignments and phylogeny. LHP1 expression patterns were compared among taxa that occupy pivotal phylogenetic positions. Several relatively conserved new motifs/regions were identified among LHP1 homologs. Phylogeny of plant LHP1 proteins agreed with established angiosperm relationships. In situ hybridization unveiled conserved expression of plant LHP1 in the axillary bud/tiller, vascular bundles, developing stamens, and carpels. Unlike AtLHP1, cucumber CsLHP1-2, sugarcane SoLHP1 and maize ZmLHP1, rice OsLHP1 is not expressed in the shoot apical meristem (SAM) and the OsLHP1 transcript level is consistently low in shoots. "Unequal crossover" might have contributed to the divergence in the N-terminal and hinge region lengths of LHP1 homologs. We propose an "insertion-deletion" model for soybean (Glycine max L.) GmLHP1s evolution. Plant LHP1 homologs are more conserved than previously expected, and may favor vegetative meristem identity and primordia formation. OsLHP1 may not function in rice SAM during floral induction.

Sonic hedgehog controls growth of external genitalia by regulating cell cycle kinetics.

During embryonic development, cells are instructed which position to occupy, they interpret these cues as differentiation programmes, and expand these patterns by growth. Sonic hedgehog (Shh) specifies positional identity in many organs; however, its role in growth is not well understood. In this study, we show that inactivation of Shh in external genitalia extends the cell cycle from 8.5 to 14.4 h, and genital growth is reduced by ∼75%. Transient Shh signalling establishes pattern in the genital tubercle; however, transcriptional levels of G1 cell cycle regulators are reduced. Consequently, G1 length is extended, leading to fewer progenitor cells entering S-phase. Cell cycle genes responded similarly to Shh inactivation in genitalia and limbs, suggesting that Shh may regulate growth by similar mechanisms in different organ systems. The finding that Shh regulates cell number by controlling the length of specific cell cycle phases identifies a novel mechanism by which Shh elaborates pattern during appendage development.

Differential gene expression in Arabidopsis wild-type and mutant anthers: insights into anther cell differentiation and regulatory networks.

In flowering plants, the anther contains highly specialized reproductive and somatic cells that are required for male fertility. Genetic studies have uncovered several genes that are important for anther development. However, little information is available regarding most genes active during anther development, including possible relationships between these genes and genetically defined regulators. In Arabidopsis, two previously isolated male-sterile mutants display dramatically altered anther cell differentiation patterns. The sporocyteless (spl)/nozzle (nzz) mutant is defective in the differentiation of primary sporogenous cells into microsporocytes, and does not properly form the anther wall. The excess microsporocytes1 (ems1)/extrasporogenous cells (exs) mutants produce excess microsporocytes at the expense of the tapetum. To gain additional insights into microsporocyte and tapetum differentiation and to uncover potential genetic interactions, expression profiles were compared between wild-type anthers (stage 4-6) and those of the spl or ems1 mutants. A total of 1954 genes were found to be differentially expressed in the ems1 and/or spl anthers, and these were grouped into 14 co-expression clusters. The presence of genes with known and predicted functions in specific clusters suggests potential functions for other genes in the same cluster. To obtain clues about possible co-regulation within co-expression clusters, we searched for shared cis-regulatory motifs in putative promoter regions. Our analyses were combined with data from previous studies to develop a model of the anther gene regulatory network. This model includes hypotheses that can be tested experimentally to gain further understanding of the mechanisms controlling anther development.

SIAMESE, a plant-specific cell cycle regulator, controls endoreplication onset in Arabidopsis thaliana.

Recessive mutations in the SIAMESE (SIM) gene of Arabidopsis thaliana result in multicellular trichomes harboring individual nuclei with a low ploidy level, a phenotype strikingly different from that of wild-type trichomes, which are single cells with a nuclear DNA content of approximately 16C to 32C. These observations suggested that SIM is required to suppress mitosis as part of the switch to endoreplication in trichomes. Here, we demonstrate that SIM encodes a nuclear-localized 14-kD protein containing a cyclin binding motif and a motif found in ICK/KRP (for Interactors of Cdc2 kinase/Kip-related protein) cell cycle inhibitor proteins. Accordingly, SIM was found to associate with D-type cyclins and CDKA;1. Homologs of SIM were detected in other dicots and in monocots but not in mammals or fungi. SIM proteins are expressed throughout the shoot apical meristem, in leaf primordia, and in the elongation zone of the root and are localized to the nucleus. Plants overexpressing SIM are slow-growing and have narrow leaves and enlarged epidermal cells with an increased DNA content resulting from additional endocycles. We hypothesize that SIM encodes a plant-specific CDK inhibitor with a key function in the mitosis-to-endoreplication transition.

Functional diversification of MYB23 and GL1 genes in trichome morphogenesis and initiation.

The functional diversification of duplicated genes is one of the driving forces in evolution. To understand the molecular mechanisms of gene diversification, we studied the functional relationship of the two Arabidopsis paralogous MYB-related genes GL1 and MYB23. We show that MYB23 controls trichome branching and trichome initiation at leaf edges. The latter is controlled redundantly together with GL1. We show that the two proteins are functionally equivalent during trichome initiation but not during trichome branching. RT-PCR and reporter construct analysis revealed spatial, temporal and genetic differences in transcriptional regulation of the GL1 and MYB23 genes. Presented data indicate that the diversification of GL1 and MYB23 gene functions occurred at the level of cis-regulatory sequences with respect to trichome initiation, and that, in parallel, the diversification with respect to regulation of trichome branching also involved changes in respective proteins.

The PRETTY FEW SEEDS2 gene encodes an Arabidopsis homeodomain protein that regulates ovule development.

The PRETTY FEW SEEDS2 gene encodes a homeodomain protein that regulates ovule development. In peptide alignments spanning the homeodomain and the WOX domain, PFS2 shared 95% amino acid identity with the PRESSED FLOWER and WUSCHEL proteins. In the pfs2-1 allele, the integuments display morphological abnormalities and 95% of the embryo sacs fail to develop properly, which results in reduced fecundity. PFS2 transcripts were most abundant in developing ovules, which accounts for the ovule phenotype in pfs2 mutants. In addition, PFS2 transcripts were present in developing primordia and differentiating organs, but, interestingly, they were absent during cell maturation. Ectopic PFS2 expression interfered with differentiation of primordia from meristems. For most plants, this resulted in fasciated stems, altered phyllotaxy, a cessation of primordia differentiation, or a combination of these. In the plants that made ovules, ectopic PFS2 expression blocked megaspore mother cell differentiation and often impeded polarized growth of the outer integument. PFS2 activity altered AGAMOUS expression, which accounts for some of the gain- and loss-of-function phenotypes. Based on analyses presented here, PFS2 affects either ovule patterning or differentiation.