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Wnt family member 9b (Wnt9b) has been demonstrated as a valuable marker for breast cancer diagnosis in surgical pathology. In this study, we examined the utility of Wnt9b in diagnosing metastatic breast carcinoma in cytology samples. Cell blocks from fine needle aspirations (FNA) and fluid specimens of 96 metastatic breast carcinomas and 123 primary and metastatic non-breast neoplasms from various organ systems were evaluated by Wnt9b and GATA3 immunohistochemistry (IHC). Wnt9b and GATA3 were positive in 81.3% and 92.7% of metastatic breast carcinomas, respectively. Conversely, 93.5% and 90.0% of non-breast, non-urothelial carcinomas were negative for Wnt9b and GATA3, respectively. Wnt9b expression was positive in rare gastrointestinal, gynecological, lung, pancreas, and salivary gland tumors. All twenty-eight urothelial carcinomas were negative for Wnt9b, while twenty-six (92.9%) were positive for GATA3. Wnt9b was slightly less sensitive but more specific than GATA3 in diagnosing metastatic breast cancer in cytology samples. Particularly, Wnt9b shows higher specificity in differentiating breast and urothelial primaries. The combined use of Wnt9b and GATA3 may increase diagnostic accuracy. [ABSTRACT FROM AUTHOR]

Orofacial clefts have been associated with specific cleft candidate genes which encode regulatory proteins required for orofacial region development. Cleft candidate genes encode proteins involved with the cleft morphopathogenesis process, but their exact interactions and roles are relatively unclear in human cleft tissue. This study evaluates the presence and correlations of Sonic Hedgehog (SHH), SRY-Box Transcription Factor 3 (SOX3), Wingless-type Family Member 3A (WNT3A) and 9B (WNT9B) protein containing cells in different cleft tissue. Non-syndromic cleft-affected tissue was subdivided into three groups—unilateral cleft lip (UCL) (n = 36), bilateral cleft lip (BCL) (n = 13), cleft palate (CP) (n = 26). Control tissue was obtained from five individuals. Immunohistochemistry was implemented. The semi-quantitative method was used. Non-parametric statistical methods were applied. A significant decrease in SHH was found in BCL and CP tissue. SOX3, WNT3A and WNT9B had a significant decrease in all clefts. Statistically significant correlations were found. The significant decrease in SHH could be associated with BCL and CP pathogenesis. SOX3, WNT3A and WNT9B could have morphopathogenetic involvement in UCL, BCL, and CP. Similar correlations imply the presence of similar pathogenetic mechanisms in different cleft variations. [ABSTRACT FROM AUTHOR]

BackgroundCleft lip and palate is one of the most common human birth defects, but the underlying etiology is poorly understood. The A/WySn mouse is a spontaneously occurring model of multigenic clefting in which 20% to 30% of individuals develop an orofacial cleft. Recent work has shown altered methylation at a specific retrotransposon insertion downstream of the Wnt9b locus in clefting animals, which results in decreased Wnt9b expression.ResultsUsing a newly developed protocol that allows us to measure morphology, gene expression, and DNA methylation in the same embryo, we relate gene expression in an individual embryo directly to its three-dimensional morphology for the first time. We find that methylation at the retrotransposon relates to Wnt9b expression and morphology. IAP methylation relates to shape of the nasal process in a manner consistent with clefting. Embryos with low IAP methylation exhibit increased among-individual variance in facial shape.ConclusionsMethylation and gene expression relate nonlinearly to nasal process morphology. Individuals at one end of a continuum of phenotypic states display a clinical phenotype and increased phenotypic variation. Variable penetrance and expressivity in this model is likely determined both by among-individual variation in methylation and changes in phenotypic robustness along the underlying liability distribution for orofacial clefting.

WNT9B plays a key role in the development of the mammalian urogenital system. It is essential for the induction of mesonephric and metanephric tubules, the regulation of renal tubule morphogenesis, and the regulation of renal progenitor cell expansion and differentiation. To our knowledge, WNT9B has not been associated with renal defects in humans; however, WNT9B−/− mice have renal agenesis/hypoplasia and reproductive tract abnormalities. We report four individuals from two unrelated consanguineous families with bilateral renal agenesis/hypoplasia/dysplasia and homozygous variants in WNT9B. The proband from Family 1 has bilateral renal cystic dysplasia and chronic kidney disease. He has two deceased siblings who presented with bilateral renal hypoplasia/agenesis. The three affected family members were homozygous for a missense variant in WNT9B (NM_003396.2: c.949G>A/p.(Gly317Arg)). The proband from Family 2 has renal hypoplasia/dysplasia, chronic kidney disease, and is homozygous for a nonsense variant in WNT9B (NM_003396.2: c.11dupC/p.(Pro5Alafs*52)). Two of her siblings died in the neonatal period, one confirmed to be in the context of oligohydramnios. The proband's unaffected brother is also homozygous for the nonsense variant in WNT9B, suggesting nonpenetrance. We propose a novel association of WNT9B and renal anomalies in humans. Further study is needed to delineate the contribution of WNT9B to genitourinary anomalies in humans. [ABSTRACT FROM AUTHOR]

Aim: This study sought to evaluate the relationship between height of an individual and the presence of impaction of maxillary and mandibular third molars, and to determine the role of genetics in third molar impaction. Materials and Methods: This was a case–control study, with cases consisted of 200 subjects with third molar impactions; and 200 controls without third molar impactions. Height of subjects was measured, and saliva samples were collected from all the subjects. DNA was extracted from saliva samples. To investigate the role of selected genes in the etiology of third molar impactions, Taqman Genotyping using SNPs identified for jaw growth, height and tooth agenesis was employed. Five candidate genes were investigated using 11 markers (SNPs). Results: The mean height of cases was significantly lower than that of the control subjects (p = 0.04). No difference was found in allele frequency between cases and controls for 10 of the 11 SNPs. However, for rs6504591 the p value was near significance (p = 0.07) with odd ratio of 2.131. Subjects with lower third molar impactions were significantly shorter than those who have fully erupted third molars. Conclusions: Subjects with lower third molar impactions were significantly shorter than those who have fully erupted third molars. We observed that individuals with third molar impaction tend to have T allele at the locus, suggesting that the T allele at the locus may increase the risk for having an impacted third molar. The rs6504591 G/T variation on human chromosome 17 (WNT9B gene) appears to increase risk by twofolds for impaction albeit with inability to detect significance due to small sample size. [ABSTRACT FROM AUTHOR]

The R-spondin (RSPO) family of proteins potentiate canonical WNT/β-catenin signaling and may provide a mechanism to fine-tune the strength of canonical WNT signaling. Although several in vitro studies have clearly demonstrated the potentiation of canonical WNT signaling by RSPOs, whether this potentiation actually occurs in normal development and tissue function in vivo still remains poorly understood. Here, we provide clear evidence of the potentiation of canonical WNT signaling by RSPO during mouse facial development by analyzing compound Wnt9b and Rspo2 gene knockout mice and utilizing ex vivo facial explants. Wnt9b;Rspo2 double mutant mice display facial defects and dysregulated gene expression pattern that are significantly more severe than and different from those of Wnt9b or Rspo2 null mutant mice. Furthermore, we found suggestive evidence that the LGR4/5/6 family of the RSPO receptors may play less critical roles in WNT9b:RSPO2 cooperation. Our results suggest that RSPO-induced cooperation is a key mechanism for fine-tuning canonical WNT/β-catenin signaling in mouse facial development.

Objectives: The CL/Fr mouse displays cleft lip and palate (CLP) at a rate of 35%. The clf1 mutation is associated with CLP in related "A" strain mice and affects the gene Wnt9b. The purpose of this study was to determine tissue specific expression of Wnt9b during facial prominence morphogenesis in CL/Fr mice and provide new details concerning gene variants associated with CLP. Methods: Facial prominences from CLP(-) and CLP(+) CL/Fr and 3H1 wild-type (WT) mice at embryonic day 11.5 (E11.5) were collected for expression assays (DNA microarray analysis, qRT-PCR, immunostaining, and in situ hybridization). A modified Chi square test was used to analyze microarray data while a student t-test was used to statistically compare qRT-PCR values (p<0.05). Results: There was a partial and variable loss of Wnt9b in facial prominences of E11.5 CLP susceptible CL/Fr mice, with a greater loss associated with CLP(+). Two genes in the clf2 locus, Adcy2 and Ube2q11 also showed decreased expression. Two regulators of palatogenesis, Runx2 and Osr2 were significantly downregulated, while an inhibitor of cell proliferation, somatostatin (Sst), was elevated in CLP(+) relative to CLP(-) mice. Conclusion: Results indicate a role for Wnt9b in the pathogenesis of CLP and supports previous reports concerning its involvement with CLP in "A" strain mice. Misexpression of Sst suggests that it may be a downstream target of Wnt9b causing reduced overall growth possibly hindering fusion of facial prominences and contributing to the development of CLP. [ABSTRACT FROM AUTHOR]

Mayer-Rokitansky-Küster-Hauser syndrome ( MRKHS) is a well-known malformation pattern of the Müllerian ducts ( MDs) characterized by congenital absence of the uterus and vagina. To date, most cases remain unexplained at molecular level. As female Wnt9b-/- mice show a MRKHS-like phenotype, WNT9B has emerged as a promising candidate gene for this disease. We performed retrospective sequence analyses of WNT9B in 226 female patients with disorders of the MDs, including 109 patients with MRKHS, as well as in 135 controls. One nonsense mutation and five likely pathogenic missense mutations were detected in WNT9B. Five of these mutations were found in cases with MRKHS accounting for 4.6% of the patients with this phenotype. No pathogenic mutations were detected in the control group (p = 0.017). Interestingly, all of the MRKHS patients with a WNT9B mutation were classified as MRKHS type 1, representing 8.5% of the cases from this subgroup. In previous studies, two of the patients with a WNT9B mutation were found to carry either an additional deletion of LHX1 or a missense mutation in TBX6. We conclude that mutations in WNT9B were frequently associated with MRKHS in our cohort and some cases may be explained by a digenic disease model. [ABSTRACT FROM AUTHOR]

Background: The heritable multifactorial etiology of human nonsyndromic cleft lip with or without cleft palate (CL ± P) is not understood. CL ± P occurs in 15% of neonates in the homozygous A/WySn mouse strain, with a multifactorial genetic etiology, the clf1 and clf2 variant genes. Clf1 acts as a mutant allele of Wnt9b but its coding sequence is normal. An IAP (intracisternal A particle) retrotransposon inserted near the Wnt9b gene is associated with clf1. Methods: Transcription of noncoding sequence between the IAP and the Wnt9b gene was examined in A/WySn embryos. The levels of Wnt9b transcript and of an 'IAP antisense' transcript initiated in the IAP and extending into the noncoding interval were assayed in A/WySn and C57BL/6J whole embryos or heads across embryonic days 8 to 12. Methylation of the 5′ LTR of the IAP was examined in E12 A/WySn embryo heads. Results: Mean Wnt9b transcript levels were lower in A/WySn than in C57BL/6J at all ages examined and lower in CL ± P embryos than in their normal littermates. The 'IAP antisense' transcript was found in all A/WySn embryos and was highest in CL ± P embryos. The IAP at Wnt9b was generally unmethylated in CL ± P embryos and approximately 50% methylated in normal littermates. Conclusion: The clf1 mutation in A/WySn is a 'metastable epiallele', in which stochastic deficiency in some individuals of DNA methylation of a retrotransposon uniquely inserted near the Wnt9b gene allows transcriptional activity of the retrotransposon and interference with transcription from Wnt9b. Methylation of metastable epialleles should be investigated in human nonsyndromic CL ± P. Birth Defects Research (Part A) 100:772-788, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The Müllerian duct gives rise to female reproductive organs, such as the oviduct and uterus. During gestation, the Wolffian duct, which generates male reproductive organs and the kidney, is formed, and the Müllerian duct then elongates caudally along the preformed Wolffian duct. Anatomical separation of these two ducts in chick embryos demonstrated that the Wolffian duct is required for Müllerian duct formation. Likewise, a few reports supported this notion in mice, including studies on Wnt9b mutant mice and Wolffian duct-specific Lhxl deletion. However, anatomical ablation of the Wolffian duct has not been established in mice. In this study, we addressed the importance of the interaction between these two reproductive ducts, by generating mice that specifically expressed a diphtheria toxin subunit in the Wolffian duct. While this genetic ablation of the Wolffian duct resulted in kidney hypoplasia/agenesis in both male and female mutant mice, the female mutant mice lacked the uterus, which is derived from the Müllerian duct. At mid-gestation, the Müllerian duct was truncated at the level where the mutant Wolffian duct was prematurely terminated, meaning that Müllerian duct elongation was dependent on the preformed Wolffian duct. However, Wnt9b expression in the Wolffian duct and the resultant canonical Wnt activity, as well as Lhx1 expression, were not affected in the mutant mice. These results suggest that the Wolffian duct regulates Müllerian duct elongation by currently unidentified mechanisms that are independent of canonical Wnt signaling or Lhx1 expression. [ABSTRACT FROM AUTHOR]

The WNT9B gene is a common organizing signal regulating different segments of the mammalian urogenital system and plays a primary rote in the development of the female reproductive tract. The aim of the present work was to examine the presence of WNT mutations in a population of women with Müllerian duct abnormalities (MDA) in order to elucidate whether mutations in WNT9B are causative for MDA in Chinese women. Initially, 191 Chinese MDA patients and 192 healthy individuals (controls) were recruited. All coding regions were amplified by PCR and sequenced to search for variants. To verify the initial results, the numbers of patients and ethnic-matched controls were expanded to 542 and 563, respectively. One known single-nucleotide polymorphism and four novel variants were identified in the first stage: two were synonymous; the other two were rare nonsynonymous novel variants (c.566G>A (p.Arg189Gln) and c.773G>A (p.Arg258His)). None of the four novel variants was found in controls. In the second stage, both novel nonsynonymous variants were detected in MDA cases and controls. The results indicate that mutations in the coding sequence of WNT9B are not responsible for MDA in the Chinese population. [ABSTRACT FROM AUTHOR]

Mayer--Rokitansky-Küster-Hauser (MRKH) syndrome is a rare congenital female genital anomaly, which is caused by aplasia of the caudal portion of the Miillerian duct. The WNT9B gene encodes a secretory glycoprotein essential for the caudal extension of the MuUerian duct during embryonic development in mice. Coding regions and exon/intron boundaries of the WNT9B gene were amplified and sequenced in 42 Chinese women with MRKH syndrome and 42 controls. Two novel heterozygous mutations were identified, which were absent in controls. One was a missense mutation in exon 1, and the other was located in the 3'-untranslated region. Both variants were detected in one out of 42 patients. The two novel mutations may be pathogenic variants in MRKH patients and warrant further functional study. [ABSTRACT FROM AUTHOR]

Abstract: Wnt activity is critical in craniofacial morphogenesis. Dysregulation of Wnt/β-catenin signaling results in significant alterations in the facial form, and has been implicated in cleft palate phenotypes in mouse and man. In zebrafish, we show that wnt9a is expressed in the pharyngeal arch, oropharyngeal epithelium that circumscribes the ethmoid plate, and ectodermal cells superficial to the lower jaw structures. Alcian blue staining of morpholino-mediated knockdown of wnt9a results in loss of the ethmoid plate, absence of lateral and posterior parachordals, and significant abrogation of the lower jaw structures. Analysis of cranial neural crest cells in the sox10:eGFP transgenic demonstrates that the wnt9a is required early during pharyngeal development, and confirms that the absence of Alcian blue staining is due to absence of neural crest derived chondrocytes. Molecular analysis of genes regulating cranial neural crest migration and chondrogenic differentiation suggest that wnt9a is dispensable for early cranial neural crest migration, but is required for chondrogenic development of major craniofacial structures. Taken together, these data corroborate the central role for Wnt signaling in vertebrate craniofacial development, and reveal that wnt9a provides the signal from the pharyngeal epithelium to support craniofacial chondrogenic morphogenesis in zebrafish. [Copyright &y& Elsevier]

The members of the Wnt family of secreted factors have oncogenic potential and important roles as developmental regulators. We report an analysis of mouse Wnt9b (also called Wnt15 and Wnt14b), including its cDNA sequence, chromosomal mapping, epithelial cell transforming activity, adult and embryonic tissue expression patterns, and evolution. We also deduced the full-length amino acid sequence of its close relative, Wnt9a (also called Wnt14), from unannotated genomic DNA sequences in GenBank. Full-length comparisons among Wnt amino acid sequences provide evidence that Wnt9b and Wnt9a are close paralogs of each other and are orthologs of Wnt9 genes from shark and hagfish. Mapping Wnt9b to The Jackson Laboratory BSS interspecific backcross panel places it at 63.0 cM on chromosome 11. Sequence comparisons of two pairs of linked Wnt genes (the Wnt9a–Wnt3a pair and the Wnt9b–Wnt3 pair) suggest that they arose from the relatively recent duplication of a single ancestral Wnt gene pair, confirming the close paralogous relationship of Wnt9a and Wnt9b. Wnt9b expression is primarily restricted to the kidney in the adult mouse, with lower levels detected in the preputial gland, liver, and mammary gland. Testing of staged whole mouse embryos from 9.5 to 17.5 days of gestation showed expression at all stages with a peak at day 10.5. In situ hybridization analysis showed expression in most but not all tissues of the 16.5-day embryo. No significant elevation of Wnt9b expression was detected in 29 mouse mammary tumor virus-induced tumors. Overexpression of Wnt9b in C57MG mammary epithelial cells caused small transformed foci in cell monolayers and a moderate morphological transformation in pooled colonies compared with Wnt1. [Copyright &y& Elsevier]

Zebrafish are an established research organism that has made many contributions to our understanding of vertebrate tissue and organ development, yet there are still significant gaps in our understanding of the genes that regulate gonad development, sex, and reproduction. Unlike the development of many organs, such as the brain and heart that form during the first few days of development, zebrafish gonads do not begin to form until the larval stage (≥5 days post-fertilization). Thus, forward genetic screens have identified very few genes required for gonad development. In addition, bulk RNA-sequencing studies that identify genes expressed in the gonads do not have the resolution necessary to define minor cell populations that may play significant roles in the development and function of these organs. To overcome these limitations, we have used single-cell RNA sequencing to determine the transcriptomes of cells isolated from juvenile zebrafish ovaries. This resulted in the profiles of 10,658 germ cells and 14,431 somatic cells. Our germ cell data represents all developmental stages from germline stem cells to early meiotic oocytes. Our somatic cell data represents all known somatic cell types, including follicle cells, theca cells, and ovarian stromal cells. Further analysis revealed an unexpected number of cell subpopulations within these broadly defined cell types. To further define their functional significance, we determined the location of these cell subpopulations within the ovary. Finally, we used gene knockout experiments to determine the roles of foxl2l and wnt9b for oocyte development and sex determination and/or differentiation, respectively. Our results reveal novel insights into zebrafish ovarian development and function, and the transcriptome profiles will provide a valuable resource for future studies., Competing Interests: YL, MK, MM, LC, SS, SW, CK, SH, NA, ID, CJ, BD No competing interests declared, (© 2022, Liu et al.)

Summary Hemodynamic forces play an essential epigenetic role in heart valve development, but how they do so is not known. Here, we show that the shear-responsive transcription factor KLF2 is required in endocardial cells to regulate the mesenchymal cell responses that remodel cardiac cushions to mature valves. Endocardial Klf2 deficiency results in defective valve formation associated with loss of Wnt9b expression and reduced canonical WNT signaling in neighboring mesenchymal cells, a phenotype reproduced by endocardial-specific loss of Wnt9b . Studies in zebrafish embryos reveal that wnt9b expression is similarly restricted to the endocardial cells overlying the developing heart valves and is dependent upon both hemodynamic shear forces and klf2a expression. These studies identify KLF2-WNT9B signaling as a conserved molecular mechanism by which fluid forces sensed by endothelial cells direct the complex cellular process of heart valve development and suggest that congenital valve defects may arise due to subtle defects in this mechanotransduction pathway. [ABSTRACT FROM AUTHOR]

The Müllerian ducts are part of the embryonic urogenital system. They give rise to mature structures that serve a critical function in the transport and development of the oocyte and/or embryo. In most vertebrates, both sexes initially develop Müllerian ducts during embryogenesis, but they regress in males under the influence of testis-derived Anti-Müllerian Hormone (AMH). A number of regulatory factors have been shown to be essential for proper duct development, including Bmp and Wnt signaling molecules, together with homeodomain transcription factors such as PAX2 and LIM1. Later in development, the fate of the ducts diverges between males and females and is regulated by AMH and Wnt signaling molecules (duct regression in males) and Hox genes (duct patterning in females). Most of the genes and molecular pathways known to be involved in Müllerian duct development have been elucidated through animal models, namely, the mouse and chicken. In addition, genetic analysis of humans with reproductive tract disorders has further defined molecular mechanisms of duct formation and differentiation. However, despite our current understanding of Müllerian duct development, some questions remain to be answered at the molecular genetic level. This article is categorized under: Early Embryonic Development > Development to the Basic Body Plan., (© 2018 Wiley Periodicals, Inc.)

The article offers information on a developmental biology research which shows that Sall1-dependent signals affect Wnt signaling and ureter tip fate to initiate kidney development. Development of the metanephric kidney depends on precise control of branching of the ureteric bud. It was found that Sall1-dependent signals from the metanephric mesenchyme are required to modulate ureteric bud tip Wnt patterning in order to initiate branching.