Therapeutic Approaches Targeting PAX3-FOXO1 and Its Regulatory and Transcriptional Pathways in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a family of soft tissue cancers that are related to the skeletal muscle lineage and predominantly occur in children and young adults. A specific chromosomal translocation t(2;13)(q35;q14) that gives rise to the chimeric oncogenic transcription factor PAX3-FOXO1 has been identified as a hallmark of the aggressive alveolar subtype of RMS. PAX3-FOXO1 cooperates with additional molecular changes to promote oncogenic transformation and tumorigenesis in various human and murine models. Its expression is generally restricted to RMS tumor cells, thus providing a very specific target for therapeutic approaches for these RMS tumors. In this article, we review the recent understanding of PAX3-FOXO1 as a transcription factor in the pathogenesis of this cancer and discuss recent developments to target this oncoprotein for treatment of RMS.

The miR-146b-3p/PAX8/NIS Regulatory Circuit Modulates the Differentiation Phenotype and Function of Thyroid Cells during Carcinogenesis.

The presence of differentiated thyroid cells in thyroid cancer is critical for the antitumor response to radioactive iodide treatment, and loss of the differentiated phenotype is a key hallmark of iodide-refractory metastatic disease. The role of microRNAs (miRNA) in fine-tuning gene expression has become a major regulatory mechanism by which developmental and pathologic processes occur. In this study, we performed next-generation sequencing and expression analysis of eight papillary thyroid carcinomas (PTC) to comprehensively characterize miRNAs involved in loss of differentiation. We found that only a small set of abundant miRNAs is differentially expressed between PTC tissue and normal tissue from the same patient. In addition, we integrated computational prediction of potential targets and mRNA sequencing and identified a master miRNA regulatory network involved in essential biologic processes such as thyroid differentiation. Both mature products of mir-146b (miR-146b-5p and -3p) were among the most abundantly expressed miRNAs in tumors. Specifically, we found that miR-146b-3p binds to the 3'-untranslated region of PAX8 and sodium/iodide symporter (NIS), leading to impaired protein translation and a subsequent reduction in iodide uptake. Furthermore, our findings show that miR-146b and PAX8 regulate each other and share common target genes, thus highlighting a novel regulatory circuit that governs the differentiated phenotype of PTC. In conclusion, our study has uncovered the existence of a miR-146b-3p/PAX8/NIS regulatory circuit that may be exploited therapeutically to modulate thyroid cell differentiation and iodide uptake for improved treatment of advanced thyroid cancer.

Identification of the receptor tyrosine kinases (RTKs)-oriented functional targets of miR-206 by an antibody-based protein array.

This study demonstrated the feasibility and benefit of an antibody-based experimental approach to identify microRNA functional targets from hundreds of predicted genes using miR-206 as an example. Using a receptor tyrosine kinase (RTK) antibody array, we identified 7 phosphorylated RTKs that were significantly differentially regulated after miR-206-mimic transfection. We then focused on MET, the most varied RTK, and bioinformatically constructed a MET-centred network using computationally predicted miR-206 targets. Within this network, we analyzed two validated targets, PAX3 and SNX2, and one candidate target, EIF4E, may account for the inhibitory effect of miR-206 on MET phosphorylation. Luciferase and Western-blot assays indicated that EIF4E was a direct target of miR-206. This concept may also be applicable for other microRNAs and other antibody array platforms.

The homeodomain of Eyeless regulates cell growth and antagonizes the paired domain-dependent retinal differentiation function.

Pax6 and its Drosophila homolog Eyeless (Ey) play essential roles during eye development. Ey/Pax6 contains two distinct DNA binding domains, a Paired domain (PD) and a Homeodomain (HD). While Ey/Pax6 PD is required for the expression of key regulators of retinal development, relatively little is known about the HD-dependent Ey function. In this study, we used the UAS/GAL4 system to determine the functions of different Ey domains on cell growth and on retinal development. We showed that Ey can promote cell growth, which requires the HD but not the PD. In contrast, the ability of Ey to activate Ato expression and induce ectopic eye formation requires the PD but not the HD. Interestingly, deletion of the HD enhanced Ey-dependent ectopic eye induction while overexpression of the HD only Ey forms antagonizes ectopic eye induction. These studies revealed a novel function of Ey HD on cell growth and a novel antagonistic effect of Ey HD on Ey PD-dependent eye induction. We further show the third helix of the Ey HD can directly interact with the RED subdomain in Ey PD and that deletion of the HD increased the binding of Ey PD to its target. These results suggest that the direct interaction between the HD and the PD potentially mediates their antagonistic effects. Since different Ey splicing forms are expressed in overlapping regions during normal development, we speculate that the expression ratios of the different Ey splice forms potentially contribute to the regulation of growth and differentiation of these tissues.

Neoblast specialization in regeneration of the planarian Schmidtea mediterranea.

Planarians can regenerate any missing body part in a process requiring dividing cells called neoblasts. Historically, neoblasts have largely been considered a homogeneous stem cell population. Most studies, however, analyzed neoblasts at the population rather than the single-cell level, leaving the degree of heterogeneity in this population unresolved. We combined RNA sequencing of neoblasts from wounded planarians with expression screening and identified 33 transcription factors transcribed in specific differentiated cells and in small fractions of neoblasts during regeneration. Many neoblast subsets expressing distinct tissue-associated transcription factors were present, suggesting candidate specification into many lineages. Consistent with this possibility, klf, pax3/7, and FoxA were required for the differentiation of cintillo-expressing sensory neurons, dopamine-ß-hydroxylase-expressing neurons, and the pharynx, respectively. Together, these results suggest that specification of cell fate for most-to-all regenerative lineages occurs within neoblasts, with regenerative cells of blastemas being generated from a highly heterogeneous collection of lineage-specified neoblasts.

Suppression of PAX6 promotes cell proliferation and inhibits apoptosis in human retinoblastoma cells.

The aim of this study was to investigate the role of the transcription factor, PAX6, in the development of retinoblastoma. The expression of endogenous PAX6 was knocked down using PAX6-specific lentivirus in two human retinoblastoma cell lines, SO-Rb50 and Y79. Cell proliferation functional assays and apoptotic assays were performed on the cells in which PAX6 was knocked down. The results revealed that PAX6 knockdown efficiency was significant (P<0.01, n=3) in the SO-Rb50 and Y79 cells. The inhibition of PAX6 reduced tumor cell apoptosis (P<0.05, n=3), but induced cell cycle S phase arrest (SO-Rb50; P<0.05, n=3) and G2/M phase arrest (Y79; P<0.05, n=3). Western blot analysis indicated that the inhibition of PAX6 increased the levels of the anti-apoptotic proteins, Bcl-2, proliferating cell nuclear antigen (PCNA) and CDK1, but reduced the levels of the pro-apoptotic proteins, BAX and p21. In conclusion, our data demonstrate that the suppression of PAX6 increases proliferation and decreases apoptosis in human retinoblastoma cells by regulating several cell cycle and apoptosis biomarkers.

Stage-dependent requirement of neuroretinal Pax6 for lens and retina development.

The physical contact of optic vesicle with head surface ectoderm is an initial event triggering eye morphogenesis. This interaction leads to lens specification followed by coordinated invagination of the lens placode and optic vesicle, resulting in formation of the lens, retina and retinal pigmented epithelium. Although the role of Pax6 in early lens development has been well documented, its role in optic vesicle neuroepithelium and early retinal progenitors is poorly understood. Here we show that conditional inactivation of Pax6 at distinct time points of mouse neuroretina development has a different impact on early eye morphogenesis. When Pax6 is eliminated in the retina at E10.5 using an mRx-Cre transgene, after a sufficient contact between the optic vesicle and surface ectoderm has occurred, the lens develops normally but the pool of retinal progenitor cells gradually fails to expand. Furthermore, a normal differentiation program is not initiated, leading to almost complete disappearance of the retina after birth. By contrast, when Pax6 was inactivated at the onset of contact between the optic vesicle and surface ectoderm in Pax6(Sey/flox) embryos, expression of lens-specific genes was not initiated and neither the lens nor the retina formed. Our data show that Pax6 in the optic vesicle is important not only for proper retina development, but also for lens formation in a non-cell-autonomous manner.

All-trans-retinoid acid (ATRA) may have inhibited chondrogenesis of primary hind limb bud mesenchymal cells by downregulating Pitx1 expression.

Despite frequently well-established role of all-trans-retinoid acid (ATRA) in congenital limb deformities, its mechanism of action, thus far, is still ambiguous. Pitx1, which is expressed in the hindlimb bud mesenchyme, or its pathways may be etiologically responsible for the increased incidence of clubfoot. Here, we sought to investigate the mechanisms whereby Pitx1 regulated chondrogenesis of hindlimb bud mesenchymal cells in vitro. E12.5 embryonic rat hind limb bud mesenchymal cells were treated with ATRA at appropriate concentrations. Cell Counting Kit-8 (CCK-8) assay was performed to evaluate cell proliferation. Hematoxylin-safranin-O-fast-green staining assays were used to observe cartilage nodules, and Pitx1 expression was examined by immunofluorescent microscopy. Real-time quantitative PCR and immunoblotting assays were applied to determine the mRNA expressions of Pitx1, Sox9 and type II collagen (Col2al), respectively. The results showed that ATRA inhibited the proliferation of hind limb bud cells dose-dependently. ATRA also induced a dose-dependent reduction in the number of cartilage nodules and the area of cartilage nodules compared with controls. Our real-time quantitative RT-PCR assays revealed that the mRNA expression of Pitx1, Sox9 and Col2al were significantly downregulated by ATRA. Furthermore, our immunofluorescent microscopy and Western blotting assays indicated that Pitx1 was mainly expressed in the cartilage nodules and the levels of Pitx1, Sox9 and Col2al were also downregulated by ATRA dose-dependently. The results indicated that ATRA may decrease chondrogenesis of hind limb bud mesenchymal cells by inhibiting cartilage-specific molecules, such as Sox9 and Col2al, via downregulating Pitx1 expression.

PITX1, a specificity determinant in the HIF-1α-mediated transcriptional response to hypoxia.

Hypoxia is an important developmental cue for multicellular organisms but it is also a contributing factor for several human pathologies, such as stroke, cardiovascular diseases and cancer. In cells, hypoxia activates a major transcriptional program coordinated by the Hypoxia Inducible Factor (HIF) family. HIF can activate more than one hundred targets but not all of them are activated at the same time, and there is considerable cell type variability. In this report we identified the paired-like homeodomain pituitary transcription factor (PITX1), as a transcription factor that helps promote specificity in HIF-1α dependent target gene activation. Mechanistically, PITX1 associates with HIF-1ß and it is important for the induction of certain HIF-1 dependent genes but not all. In particular, PITX1 controls the HIF-1α-dependent expression of the histone demethylases; JMJD2B, JMJD2A, JMJD2C and JMJD1B. Functionally, PITX1 is required for the survival and proliferation responses in hypoxia, as PITX1 depleted cells have higher levels of apoptotic markers and reduced proliferation. Overall, our study identified PITX1 as a key specificity factor in HIF-1α dependent responses, suggesting PITX1 as a protein to target in hypoxic cancers.

Small molecule inhibition of PAX3-FOXO1 through AKT activation suppresses malignant phenotypes of alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma comprises a rare highly malignant tumor presumed to be associated with skeletal muscle lineage in children. The hallmark of the majority of alveolar rhabdomyosarcoma is a chromosomal translocation that generates the PAX3-FOXO1 fusion protein, which is an oncogenic transcription factor responsible for the development of the malignant phenotype of this tumor. Alveolar rhabdomyosarcoma cells are dependent on the oncogenic activity of PAX3-FOXO1, and its expression status in alveolar rhabdomyosarcoma tumors correlates with worst patient outcome, suggesting that blocking this activity of PAX3-FOXO1 may be an attractive therapeutic strategy against this fusion-positive disease. In this study, we screened small molecule chemical libraries for inhibitors of PAX3-FOXO1 transcriptional activity using a cell-based readout system. We identified the Sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCA) inhibitor thapsigargin as an effective inhibitor of PAX3-FOXO1. Subsequent experiments in alveolar rhabdomyosarcoma cells showed that activation of AKT by thapsigargin inhibited PAX3-FOXO1 activity via phosphorylation. Moreover, this AKT activation appears to be associated with the effects of thapsigargin on intracellular calcium levels. Furthermore, thapsigargin inhibited the binding of PAX3-FOXO1 to target genes and subsequently promoted its proteasomal degradation. In addition, thapsigargin treatment decreases the growth and invasive capacity of alveolar rhabdomyosarcoma cells while inducing apoptosis in vitro. Finally, thapsigargin can suppress the growth of an alveolar rhabdomyosarcoma xenograft tumor in vivo. These data reveal that thapsigargin-induced activation of AKT is an effective mechanism to inhibit PAX3-FOXO1 and a potential agent for targeted therapy against alveolar rhabdomyosarcoma.

MicroRNA-10b pleiotropically regulates invasion, angiogenicity and apoptosis of tumor cells resembling mesenchymal subtype of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a heterogeneous disease despite its seemingly uniform pathology. Deconvolution of The Cancer Genome Atlas's GBM gene expression data has unveiled the existence of distinct gene expression signature underlying discrete GBM subtypes. Recent conflicting findings proposed that microRNA (miRNA)-10b exclusively regulates glioma growth or invasion but not both. We showed that silencing of miRNA-10b by baculoviral decoy vectors in a glioma cell line resembling the mesenchymal subtype of GBM reduces its growth, invasion and angiogenesis while promoting apoptosis in vitro. In an orthotopic human glioma mouse model, inhibition of miRNA-10b diminishes the invasiveness, angiogenicity and growth of the mesenchymal subtype-like glioma cells in the brain and significantly prolonged survival of glioma-bearing mice. We demonstrated that the pleiotropic nature of miRNA-10b was due to its suppression of multiple tumor suppressors, including TP53, FOXO3, CYLD, PAX6, PTCH1, HOXD10 and NOTCH1. In particular, siRNA-mediated knockdown experiments identified TP53, PAX6, NOTCH1 and HOXD10 as invasion regulatory genes in our mesenchymal subtype-like glioma cells. By interrogating the REMBRANDT, we noted that dysregulation of many direct targets of miRNA-10b was associated with significantly poorer patient survival. Thus, our study uncovers a novel role for miRNA-10b in regulating angiogenesis and suggests that miRNA-10b may be a pleiotropic regulator of gliomagenesis.

A phosphatidylinositol 3-kinase-Pax3 axis regulates Brn-2 expression in melanoma.

Deregulation of transcription arising from mutations in key signaling pathways is a hallmark of cancer. In melanoma, the most aggressive and lethal form of skin cancer, the Brn-2 transcription factor (POU3F2) regulates proliferation and invasiveness and lies downstream from mitogen-activated protein kinase (MAPK) and Wnt/ß-catenin, two melanoma-associated signaling pathways. In vivo Brn-2 represses expression of the microphthalmia-associated transcription factor, MITF, to drive cells to a more stem cell-like and invasive phenotype. Given the key role of Brn-2 in regulating melanoma biology, understanding the signaling pathways that drive Brn-2 expression is an important issue. Here, we show that inhibition of phosphatidylinositol 3-kinase (PI3K) signaling reduces invasiveness of melanoma cells in culture and strongly inhibits Brn-2 expression. Pax3, a transcription factor regulating melanocyte lineage-specific genes, directly binds and regulates the Brn-2 promoter, and Pax3 expression is also decreased upon PI3K inhibition. Collectively, our results highlight a crucial role for PI3K in regulating Brn-2 and Pax3 expression, reveal a mechanism by which PI3K can regulate invasiveness, and imply that PI3K signaling is a key determinant of melanoma subpopulation diversity. Together with our previous work, the results presented here now place Brn-2 downstream of three melanoma-associated signaling pathways.

Pancreas-enriched miRNA refines endocrine cell differentiation.

Genome-encoded microRNAs (miRNAs) provide a post-transcriptional regulatory layer that is important for pancreas development. However, how specific miRNAs are intertwined into the transcriptional network, which controls endocrine differentiation, is not well understood. Here, we show that microRNA-7 (miR-7) is specifically expressed in endocrine precursors and in mature endocrine cells. We further demonstrate that Pax6 is an important target of miR-7. miR-7 overexpression in developing pancreas explants or in transgenic mice led to Pax6 downregulation and inhibition of α- and ß-cell differentiation, resembling the molecular changes caused by haploinsufficient expression of Pax6. Accordingly, miR-7 knockdown resulted in Pax6 upregulation and promoted α- and ß-cell differentiation. Furthermore, Pax6 downregulation reversed the effect of miR-7 knockdown on insulin promoter activity. These data suggest a novel miR-7-based circuit that ensures precise control of endocrine cell differentiation.

Genome-wide analysis of Pax8 binding provides new insights into thyroid functions.

BACKGROUND: The transcription factor Pax8 is essential for the differentiation of thyroid cells. However, there are few data on genes transcriptionally regulated by Pax8 other than thyroid-related genes. To better understand the role of Pax8 in the biology of thyroid cells, we obtained transcriptional profiles of Pax8-silenced PCCl3 thyroid cells using whole genome expression arrays and integrated these signals with global cis-regulatory sequencing studies performed by ChIP-Seq analysis RESULTS: Exhaustive analysis of Pax8 immunoprecipitated peaks demonstrated preferential binding to intragenic regions and CpG-enriched islands, which suggests a role of Pax8 in transcriptional regulation of orphan CpG regions. In addition, ChIP-Seq allowed us to identify Pax8 partners, including proteins involved in tertiary DNA structure (CTCF) and chromatin remodeling (Sp1), and these direct transcriptional interactions were confirmed in vivo. Moreover, both factors modulate Pax8-dependent transcriptional activation of the sodium iodide symporter (Nis) gene promoter. We ultimately combined putative and novel Pax8 binding sites with actual target gene expression regulation to define Pax8-dependent genes. Functional classification suggests that Pax8-regulated genes may be directly involved in important processes of thyroid cell function such as cell proliferation and differentiation, apoptosis, cell polarity, motion and adhesion, and a plethora of DNA/protein-related processes. CONCLUSION: Our study provides novel insights into the role of Pax8 in thyroid biology, exerted through transcriptional regulation of important genes involved in critical thyrocyte processes. In addition, we found new transcriptional partners of Pax8, which functionally cooperate with Pax8 in the regulation of thyroid gene transcription. Besides, our data demonstrate preferential location of Pax8 in non-promoter CpG regions. These data point to an orphan CpG island-mediated mechanism that represents a novel role of Pax8 in the transcriptional output of the thyrocyte.

Possible role of Pax-6 in promoting breast cancer cell proliferation and tumorigenesis.

Pax 6, a member of the paired box (Pax) family, has been implicated in oncogenesis. However, its therapeutic potential has been never examined in breast cancer. To explore the role of Pax6 in breast cancer development, a lentivirus based short hairpin RNA (shRNA) delivery system was used to knockdown Pax6 expression in estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells. Effect of Pax6 silencing on breast cancer cell proliferation and tumorigenesis was analyzed. Pax6-RNAi-lentivirus infection remarkably downregulated the expression levels of Pax6 mRNA and protein in MCF-7 and MDA-MB-231 cells. Accordingly, the cell viability, DNA synthesis, and colony formation were strongly suppressed, and the tumorigenesis in xenograft nude mice was significantly inhibited. Moreover, tumor cells were arrested at G0/G1 phase after Pax6 was knocked down. Pax6 facilitates important regulatory roles in breast cancer cell proliferation and tumor progression, and could serve as a diagnostic marker for clinical investigation.

L1-CAM expression in ccRCC correlates with shorter patients survival times and confers chemoresistance in renal cell carcinoma cells.

Conflicting data exist about the expression of L1 cell adhesion molecule (L1-CAM) in clear cell renal cell carcinoma (ccRCC). To determine the clinical usefulness of L1-CAM as a therapeutic or prognostic marker molecule in renal cancer patients, we analyzed its expression on a cohort of 282 renal cell carcinoma (RCC) patients. L1-CAM expression was found in 49.5% of 282 renal cancer tissues. Importantly, L1-CAM expression in patients with ccRCC was associated with significantly shorter patient survival time. We further present evidence that L1-CAM was involved in the resistance against therapeutic reagents like rapamycin, sunitinib and cisplatin. The downregulation of L1-CAM expression decreased renal cancer cell proliferation and reduced the expression of cyclin D1. In addition, we found out that Von Hippel-Lindau (VHL) deficiency was accompanied by a downregulation of the transcription factor PAX8 and L1-CAM. In normal renal tissue, PAX8 and L1-CAM were co-expressed in collecting duct cells. Importantly, the downregulation of PAX8 by small interfering RNA increased the expression of L1-CAM and concomitantly induced the migration of renal cancer cells. Furthermore, we observed in 65.3% of 282 RCC patients a downregulation of PAX8 expression. With chromatin immunoprecipitation analysis, we additionally demonstrate that PAX8 can bind to the promoter of L1-CAM and we further observed that the downregulation of PAX8 was accompanied by increased L1-CAM expression in a high fraction of ccRCC patients. In summary, we show that VHL and PAX8 are involved in the regulation of L1-CAM in renal cancer and L1-CAM represents an important therapeutic and prognostic marker protein for the treatment of ccRCC.

Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer.

BACKGROUND & AIMS: Aberrant activation of the RAS pathway is ubiquitous in human hepatocarcinogenesis, but the molecular mechanisms leading to RAS induction in the absence of RAS mutations remain under-investigated. We defined the role of Ras GTPase activating proteins (GAPs) in the constitutive activity of Ras signaling during human hepatocarcinogenesis. METHODS: The mutation status of RAS genes and RAS effectors was assessed in a collection of human hepatocellular carcinomas (HCC). Levels of RAS GAPs (RASA1-4, RASAL1, nGAP, SYNGAP1, DAB2IP, and NF1) and the RASAL1 upstream inducer PITX1 were determined by real-time RT-PCR and immunoblotting. The promoter and genomic status of RASAL1, DAB2IP, NF1, and PITX1 were assessed by methylation assays and microsatellite analysis. Effects of RASAL1, DAB2IP, and PITX1 on HCC growth were evaluated by transfection and siRNA analyses of HCC cell lines. RESULTS: In the absence of Ras mutations, downregulation of at least one RAS GAP (RASAL1, DAB2IP, or NF1) was found in all HCC samples. Low levels of DAB2IP and PITX1 were detected mostly in a HCC subclass from patients with poor survival, indicating that these proteins control tumor aggressiveness. In HCC cells, reactivation of RASAL1, DAB2IP, and PITX1 inhibited proliferation and induced apoptosis, whereas their silencing increased proliferation and resistance to apoptosis. CONCLUSIONS: Selective suppression of RASAL1, DAB2IP, or NF1 RAS GAPs results in unrestrained activation of Ras signaling in the presence of wild-type RAS in HCC.

Inhibition of PAX3 by TGF-beta modulates melanocyte viability.

The protein encoded by paired-box homeotic gene 3 (PAX3) is a key regulator of the microphthalmia-associated transcription factor (Mitf) in the melanocyte lineage. Here, we show that PAX3 expression in skin is directly inhibited by TGF-beta/Smads. UV irradiation represses TGF-beta in keratinocytes, and the repression of TGF-beta/Smads upregulates PAX3 in melanocytes, which is associated with a UV-induced melanogenic response and consequent pigmentation. Furthermore, the TGF-beta-PAX3 signaling pathway interacts with the p53-POMC/MSH-MC1R signaling pathway, and both are crucial in melanogenesis. The activation of p53-POMC/MSH-MC1R signaling is required for the UV-induced melanogenic response because PAX3 functions in synergy with SOX10 in a cAMP-response element (CRE)-dependent manner to regulate the transcription of Mitf. This study will provide a rich foundation for further research on skin cancer prevention by enabling us to identify targeted small molecules in the signaling pathways of the UV-induced melanogenic response that are highly likely to induce naturally protective pigmentation.

Function of Pax1 and Pax9 in the sclerotome of medaka fish.

We examined the expression and functions of Pax1 and Pax9 in a teleost fish, the medaka Oryzias latipes. While Pax1 and Pax9 show distinct expression in the sclerotome in amniotes, we could not detect the differential expression of Pax1 and Pax9 in the developing sclerotome of the medaka. Furthermore, unlike the mouse, in which Pax1 is essential for development of the vertebral body, and where the neural arch is formed independent of either Pax1 or Pax9, our morpholino knockdown experiments revealed that both Pax1 and Pax9 are indispensable for the development of the vertebral body and neural arch. Therefore, we conclude that after gene duplication, Pax1 and Pax9 subfunctionalize their roles in the sclerotome independently in teleosts and amniotes. In Stage-30 embryo, Pax9 was strongly expressed in the posterior mesoderm, as was also observed for mouse Pax9. Since this expression was not detected for Pax1 in the mouse or fish, this new expression in the posterior mesoderm likely evolved in Pax9 of ancestral vertebrates after gene duplication. Two-month-old fish injected with Pax9 morpholino oligonucleotide showed abnormal morphology in the tail hypural skeletal element, which may have been related to this expression.