Hyperglycemia effects on epigenetic regulation of pax3 during the early embryonic development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Novel variants in the KIT and PAX3 genes in horses with white-spotted coat colour phenotypes.

Variants in the EDNRB, KIT, MITF, PAX3 and TRPM1 genes are known to cause white spotting phenotypes in horses, which can range from the common white markings up to completely white horses. In this study, we investigated these candidate genes in 169 horses with white spotting phenotypes not explained by the previously described variants. We identified a novel missense variant, PAX3:p.Pro32Arg, in Appaloosa horses with a splashed white phenotype in addition to their leopard complex spotting patterns. We also found three novel variants in the KIT gene. The splice site variant c.1346+1G>A occurred in a Swiss Warmblood horse with a pronounced depigmentation phenotype. The missense variant p.Tyr441Cys was present in several part-bred Arabians with sabino-like depigmentation phenotypes. Finally, we provide evidence suggesting that the common and widely distributed KIT:p.Arg682His variant has a very subtle white-increasing effect, which is much less pronounced than the effect of the other described KIT variants. We termed the new KIT variants W18-W20 to provide a simple and unambiguous nomenclature for future genetic testing applications.

cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer of striated muscle characterized in 60% of cases by a t(2;13)(q35;q14). This results in the fusion of PAX3, a developmental transcription factor required for limb myogenesis, with FKHR, a member of the forkhead family of transcription factors. The resultant PAX3-FKHR gene possesses transforming properties; however, the effects of this chimeric oncogene on gene expression are largely unknown. To investigate the actions of these transcription factors, both Pax3 and PAX3-FKHR were introduced into NIH 3T3 cells, and the resultant gene expression changes were analyzed with a murine cDNA microarray containing 2,225 elements. We found that PAX3-FKHR but not PAX3 activated a myogenic transcription program including the induction of transcription factors MyoD, Myogenin, Six1, and Slug as well as a battery of genes involved in several aspects of muscle function. Notable among this group were the growth factor gene Igf2 and its binding protein Igfbp5. Relevance of this model was suggested by verification that three of these genes (IGFBP5, HSIX1, and Slug) were also expressed in alveolar rhabdomyosarcoma cell lines. This study utilizes cDNA microarrays to elucidate the pattern of gene expression induced by an oncogenic transcription factor and demonstrates the profound myogenic properties of PAX3-FKHR in NIH 3T3 cells.

Embryonic expression of the tumor-associated PAX3-FKHR fusion protein interferes with the developmental functions of Pax3

A unique chromosomal translocation involving the genes PAX3 and FKHR is characteristic of most human alveolar rhabdomyosarcomas. The resultant chimeric protein fuses the PAX3 DNA-binding domains to the transactivation domain of FKHR, suggesting that PAX3-FKHR exerts its role in alveolar rhabdomyosarcomas through dysregulation of PAX3-specific target genes. Here, we have produced transgenic mice in which PAX3-FKHR expression was driven by mouse Pax3 promoter/enhancer sequences. Five independent lines expressed PAX3-FKHR in the dorsal neural tube and lateral dermomyotome. Each line exhibited phenotypes that correlated with PAX3-FKHR expression levels and predominantly involved pigmentary disturbances of the abdomen, hindpaws, and tail, with additional neurological related alterations. Phenotypic severity could be increased by reducing Pax3 levels through matings with Pax3-defective Splotch mice, and interference between PAX3 and PAX3-FKHR was apparent in transcription reporter assays. These data suggest that the tumor-associated PAX3-FKHR fusion protein interferes with normal Pax3 developmental functions as a prelude to transformation.

The hybrid PAX3-FKHR fusion protein of alveolar rhabdomyosarcoma transforms fibroblasts in culture.

Pediatric alveolar rhabdomyosarcoma is characterized by a chromosomal translocation that fuses parts of the PAX3 and FKHR genes. PAX3 codes for a transcriptional regulator that controls developmental programs, and FKHR codes for a forkhead-winged helix protein, also a likely transcription factor. The PAX3-FKHR fusion product retains the DNA binding domains of the PAX3 protein and the putative activator domain of the FKHR protein. The PAX3-FKHR protein has been shown to function as a transcriptional activator. Using the RCAS retroviral vector, we have introduced the PAX3-FKHR gene into chicken embryo fibroblasts. Expression of the PAX3-FKHR protein in these cells leads to transformation: the cells become enlarged, grow tightly packed and in multiple layers, and acquire the ability for anchorage-independent growth. This cellular transformation in vitro will facilitate studies on the mechanism of PAX3-FKHR-induced oncogenesis.

Pax3 modulates expression of the c-Met receptor during limb muscle development.

Pax3 is a transcription factor whose expression has been used as a marker of myogenic precursor cells arising in the lateral somite destined to migrate to and populate the limb musculature. Accruing evidence indicates that the embryologic origins of axial and appendicular muscles are distinct, and limb muscle abnormalities in both mice and humans harboring Pax3 mutations support this distinction. The mechanisms by which Pax3 affects limb muscle development are unknown. The tyrosine kinase receptor for hepatocyte growth factor/scatter factor encoded by the c-met protooncogene is also expressed in limb muscle progenitors and, like Pax-3, is required in the mouse for limb muscle development. Here, we show that c-met expression is markedly reduced in the lateral dermomyotome of Splotch embryos lacking Pax3. We show that Pax3 can stimulate c-met expression in cultured cells, and we identify a potential Pax3 binding site in the human c-MET promoter that may contribute to direct transcriptional regulation. In addition, we have found that several cell lines derived from patients with rhabdomyosarcomas caused by a t(2;13) chromosomal translocation activating PAX3 express c-MET, whereas those rhabdomyosarcoma cell lines examined without the translocation do not. These results are consistent with a model in which Pax3 modulates c-met expression in the lateral dermomyotome, a function that is required for the appropriate migration of these myogenic precursors to the limb where the ligand for c-met (hepatocyte growth factor/scatter factor) is expressed at high levels.

Interactions between endothelin receptor B and transcription factors Sox10 and Pax3 in the melanocyte lineage

Genetic interactions that underlie developmental processes such as cell differentiation and pattern formation are complex and difficult to elucidate. Neural Crest (NC) cells and their derivatives offer an optimal system in which to probe for these complex interactions as they acquire different cell fates and constitute a variety of structures. The transcription factors Sox10 and Pax3 as well as the transmembrane receptor Endothelin receptor b (Ednrb) are temporally and spatially co-expressed early in NC cells and mutations in these genes lead to similar hypopigmentation phenotypes due to a reduced number of NC-derived melanocyte precursors, the melanoblasts. The goal of this study was to establish whether Sox10 and Ednrb or Pax3 and Ednrb interact to promote normal murine melanocyte development. Crosses of Sox10 or Pax3 with Ednrb heterozygous mutants showed that the double heterozygous hypopigmentation phenotype was significantly more pronounced than phenotypes of single heterozygotes, implying that a synergistic interaction exists between Sox10 and Ednrb and Pax3 and Ednrb. This interaction was further explored by the attempt to rescue the Sox10 and Pax3 hypopigmentation phenotypes by the transgenic addition of Ednrb to melanoblasts. Pigmentation was completely restored in the Sox10 and partially restored in the Pax3 mutant mice. The comparison of the number of melanoblasts in transgenic and non-transgenic Sox10 mutant embryos showed that the transgenic rescue occurred as early as E11.5, a critical time for melanoblast population expansion. Cell survival assays indicated that the rescue was not due to an effect of the transgene on melanoblast survival. A novel phenotype arose when studying the interaction between Ednrb and Pax3. Newborns appeared normal but by 3.5 weeks of age, the affected pups were smaller than normal littermates and developed a dome-shaped head; some also developed thoracic kyphosis. Affected pups were dead by 4 weeks of age: 80% were Pax3Sp/+ and 75% were female. When compared to normal littermates, affected mice had brains with enlarged 4th ventricles and more glia while skeletal staining showed kyphosis, wider rib cages and pelvic differences. An epistatic interaction resulting from the mixing of genetic backgrounds that is exacerbated in the presence of Pax3 heterozygosity is suspected.

Mus Musculus Neural Crest Development: Is Pax3 Regulating the Expression of Endothelin Receptor B?

The vertebrate Neural Crest (NC) is formed during early embryonic development at the neurulation stage. This group of multi potent cells gives rise to a variety of derivatives such as the skin's pigmented cells (Melanocytes), the peripheral nervous system with its associated components, and the endocrine cells of the adrenal medulla amongst others. There are several molecular mechanisms that underlie the development and migration of NC derived cells. For example, during melanocyte differentiation and migration the Endothelin Receptor B and its ligand Endothelin 3 (EdnrB/Edn3), the kit/ Steel factor and the FGF receptor I FGF pathways amongst others play important roles. Additionally, several transcription factors such as Pax3, SoxlO and Mitfalso intervene during the NC cells differentiation processes. In this work, the possible regulatory interaction of Pax3 and EdnrB was assessed by in situ hybridization methods with EdnrB, SoxlO and Dct riboprobes in Pax3 homozygous embryos. To further characterize this interaction, genetic crosses between Pax3 heterozygous mutants and EdnrB heterozygous animals were established. Coat pigmentation was used as an indicator of genetic interaction on the progeny. Experimental results indicated that Pax3 does not directly regulate the expression of EdnrB during neural crest development but interact to produce normal coat color. I propose two possible models to explain the epistatic relationship of Pax3 and EdnrB during normal melanocyte development.

Interactions between Endothelin Receptor B and Transcription Factors Sox10 and Pax3 in the Melanocyte Lineage

Genetic interactions that underlie developmental processes such as cell differentiation and pattern formation are complex and difficult to elucidate. Neural Crest (NC) cells and their derivatives offer an optimal system in which to probe for these complex interactions as they acquire different cell fates and constitute a variety of structures. The transcription factors Sox10 and Pax3 as well as the transmembrane receptor Endothelin receptor b (Ednrb) are temporally and spatially co-expressed early in NC cells and mutations in these genes lead to similar hypopigmentation phenotypes due to a reduced number of NC-derived melanocyte precursors, the melanoblasts. The goal of this study was to establish whether Sox10 and Ednrb or Pax3 and Ednrb interact to promote normal murine melanocyte development. Crosses of Sox10 or Pax3 with Ednrb heterozygous mutants showed that the double heterozygous hypopigmentation phenotype was significantly more pronounced than phenotypes of single heterozygotes, implying that a synergistic interaction exists between Sox10 and Ednrb and Pax3 and Ednrb. This interaction was further explored by the attempt to rescue the Sox10 and Pax3 hypopigmentation phenotypes by the transgenic addition of Ednrb to melanoblasts. Pigmentation was completely restored in the Sox10 and partially restored in the Pax3 mutant mice. The comparison of the number of melanoblasts in transgenic and non-transgenic Sox10 mutant embryos showed that the transgenic rescue occurred as early as E11.5, a critical time for melanoblast population expansion. Cell survival assays indicated that the rescue was not due to an effect of the transgene on melanoblast survival. A novel phenotype arose when studying the interaction between Ednrb and Pax3. Newborns appeared normal but by 3.5 weeks of age, the affected pups were smaller than normal littermates and developed a dome-shaped head; some also developed thoracic kyphosis. Affected pups were dead by 4 weeks of age: 80% were Pax3Sp/+ and 75% were female. When compared to normal littermates, affected mice had brains with enlarged 4th ventricles and more glia while skeletal staining showed kyphosis, wider rib cages and pelvic differences. An epistatic interaction resulting from the mixing of genetic backgrounds that is exacerbated in the presence of Pax3 heterozygosity is suspected.

Pax3 expression enhances PDGF-B-induced brainstem gliomagenesis and characterizes a subset of brainstem glioma.

High-grade Brainstem Glioma (BSG), also known as Diffuse Intrinsic Pontine Glioma (DIPG), is an incurable pediatric brain cancer. Increasing evidence supports the existence of regional differences in gliomagenesis such that BSG is considered a distinct disease from glioma of the cerebral cortex (CG). In an effort to elucidate unique characteristics of BSG, we conducted expression analysis of mouse PDGF-B-driven BSG and CG initiated in Nestin progenitor cells and identified a short list of expression changes specific to the brainstem gliomagenesis process, including abnormal upregulation of paired box 3 (Pax3). In the neonatal mouse brain, Pax3 expression marks a subset of brainstem progenitor cells, while it is absent from the cerebral cortex, mirroring its regional expression in glioma. Ectopic expression of Pax3 in normal brainstem progenitors in vitro shows that Pax3 inhibits apoptosis. Pax3-induced inhibition of apoptosis is p53-dependent, however, and in the absence of p53, Pax3 promotes proliferation of brainstem progenitors. In vivo, Pax3 enhances PDGF-B-driven gliomagenesis by shortening tumor latency and increasing tumor penetrance and grade, in a region-specific manner, while loss of Pax3 function extends survival of PDGF-B-driven;p53-deficient BSG-bearing mice by 33%. Importantly, Pax3 is regionally expressed in human glioma as well, with high PAX3 mRNA characterizing 40% of human BSG, revealing a subset of tumors that significantly associates with PDGFRA alterations, amplifications of cell cycle regulatory genes, and is exclusive of ACVR1 mutations. Collectively, these data suggest that regional Pax3 expression not only marks a novel subset of BSG but also contributes to PDGF-B-induced brainstem gliomagenesis.

A Novel Mouse Model of Diffuse Intrinsic Pontine Glioma Initiated in Pax3-Expressing Cells.

Diffuse intrinsic pontine glioma (DIPG) is a rare and incurable brain tumor that arises predominately in children and involves the pons, a structure that along with the midbrain and medulla makes up the brainstem. We have previously developed genetically engineered mouse models of brainstem glioma using the RCAS/Tv-a system by targeting PDGF-B overexpression, p53 loss, and H3.3K27M mutation to Nestin-expressing brainstem progenitor cells of the neonatal mouse. Here we describe a novel mouse model targeting these same genetic alterations to Pax3-expressing cells, which in the neonatal mouse pons consist of a Pax3+/Nestin+/Sox2+ population lining the fourth ventricle and a Pax3+/NeuN+ parenchymal population. Injection of RCAS-PDGF-B into the brainstem of Pax3-Tv-a mice at postnatal day 3 results in 40% of mice developing asymptomatic low-grade glioma. A mixture of low- and high-grade glioma results from injection of Pax3-Tv-a;p53(fl/fl) mice with RCAS-PDGF-B and RCAS-Cre, with or without RCAS-H3.3K27M. These tumors are Ki67+, Nestin+, Olig2+, and largely GFAP- and can arise anywhere within the brainstem, including the classic DIPG location of the ventral pons. Expression of the H3.3K27M mutation reduces overall H3K27me3 as compared with tumors without the mutation, similar to what has been previously shown in human and mouse tumors. Thus, we have generated a novel genetically engineered mouse model of DIPG, which faithfully recapitulates the human disease and represents a novel platform with which to study the biology and treatment of this deadly disease.

A genome-wide association study identifies five loci influencing facial morphology in Europeans

Inter-individual variation in facial shape is one of the most noticeable phenotypes in humans, and it is clearly under genetic regulation; however, almost nothing is known about the genetic basis of normal human facial morphology. We therefore conducted a genome-wide association study for facial shape phenotypes in multiple discovery and replication cohorts, considering almost ten thousand individuals of European descent from several countries. Phenotyping of facial shape features was based on landmark data obtained from three-dimensional head magnetic resonance images (MRIs) and two-dimensional portrait images. We identified five independent genetic loci associated with different facial phenotypes, suggesting the involvement of five candidate genes-PRDM16, PAX3, TP63, C5orf50, and COL17A1-in the determination of the human face. Three of them have been implicated previously in vertebrate craniofacial development and disease, and the remaining two genes potentially represent novel players in the molecular networks governing facial development. Our finding at PAX3 influencing the position of the nasion replicates a recent GWAS of facial features. In addition to the reported GWA findings, we established links between common DNA variants previously associated with NSCL/P at 2p21, 8q24, 13q31, and 17q22 and normal facial-shape variations based on a candidate gene approach. Overall our study implies that DNA variants in genes essential for craniofacial development contribute with relatively small effect size to the spectrum of normal variation in human facial morphology. This observation has important consequences for future studies aiming to identify more genes involved in the human facial morphology, as well as for potential applications of DNA prediction of facial shape such as in future forensic applications.

Genetic analysis of an Indian family with members affected with Waardenburg syndrome and Duchenne muscular dystrophy

Purpose: Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. Methods: Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. Results: Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. Conclusions: This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.

非洲爪蟾Nkx6.3在原肠运动和神经嵴诱导调控中的功能及爪蟾神经管腹侧图式形成的研究

神经嵴（neural crest）是一类脊椎动物特有的多潜能迁移细胞。这一类细胞历经“表皮—间充质”转换（EMT），与神经管背侧的其它细胞分离，经由不同路线迁移，定位于胚胎外周各处，后分化为不同的细胞类型包括外周神经系统、颅面骨骼系统及色素细胞等。神经嵴的发育是一个多途径多步骤的过程，受多种信号通路及转录因子调控。这些调控因子相互调节形成精密网络，可被划分为三个主要层次类群：分泌性信号分子（BMP、Wnt、FGF、Delta）、神经板边界特异基因（Msx、Pax3/7、 Zic1、Dlx3/5）、神经嵴特异基因（Snail/Slug、AP-2、FoxD3、Twist、Id、cMyc、Sox9/10）。本文第一章主要概述不同组织来源的各种分泌信号在神经嵴诱导中的作用以及他们之间的整合调控。 Nkx6家族蛋白是一类进化上保守的转录因子，在脊椎动物中枢神经系统（CNS)的图式形成和胰腺的发育中有重要作用。在第二章，我们描述了非洲爪蟾中Nkx6家族基因的克隆及其表达图式。与小鼠和鸡中的同源基因类似，爪蟾的Nkx6家族基因在胚胎发育过程中主要表达于中枢神经系统和前部内胚层组织。其中Nkx6.1和Nkx6.2在神经胚期神经板表达重合，晚期都表达于后脑和脊髓的腹侧。Nkx6.3从卵裂期到神经胚早期都表达于非神经外胚层，而尾芽期表达于后脑后部和腮弓。在内胚层中，Nkx6.2在尾芽期表达于底索。在蝌蚪期，Nkx6家族的三个基因分别表达于前部内胚层的衍生物，包括胰腺、胃、食道和肺。 Nkx6.3是最近发现的Nkx6家族新成员，它在爪蟾中的表达与Nkx6.1和Nkx6.2有了较大分歧。在第三章，我们通过功能获得及功能缺失实验来探讨Nkx6.3在爪蟾早期发育中的功能。我们发现原肠期前过量或抑制Nkx6.3表达都会影响胚胎原肠运动的正常进行。我们通过动物帽延伸实验证明Nkx6.3参与了细胞运动。半定量RT-PCR结果显示，Nkx6.3可以调控一些粘附分子的表达。以上结果说明Nkx6.3通过调控粘附分子的转录而参与细胞运动的调控。我们还发神经嵴（neural crest）是一类脊椎动物特有的多潜能迁移细胞。这一类细胞历经“表皮—间充质”转换（EMT），与神经管背侧的其它细胞分离，经由不同路线迁移，定位于胚胎外周各处，后分化为不同的细胞类型包括外周神经系统、颅面骨骼系统及色素细胞等。神经嵴的发育是一个多途径多步骤的过程，受多种信号通路及转录因子调控。这些调控因子相互调节形成精密网络，可被划分为三个主要层次类群：分泌性信号分子（BMP、Wnt、FGF、Delta）、神经板边界特异基因（Msx、Pax3/7、 Zic1、Dlx3/5）、神经嵴特异基因（Snail/Slug、AP-2、FoxD3、Twist、Id、cMyc、Sox9/10）。本文第一章主要概述不同组织来源的各种分泌信号在神经嵴诱导中的作用以及他们之间的整合调控。 Nkx6家族蛋白是一类进化上保守的转录因子，在脊椎动物中枢神经系统（CNS)的图式形成和胰腺的发育中有重要作用。在第二章，我们描述了非洲爪蟾中Nkx6家族基因的克隆及其表达图式。与小鼠和鸡中的同源基因类似，爪蟾的Nkx6家族基因在胚胎发育过程中主要表达于中枢神经系统和前部内胚层组织。其中Nkx6.1和Nkx6.2在神经胚期神经板表达重合，晚期都表达于后脑和脊髓的腹侧。Nkx6.3从卵裂期到神经胚早期都表达于非神经外胚层，而尾芽期表达于后脑后部和腮弓。在内胚层中，Nkx6.2在尾芽期表达于底索。在蝌蚪期，Nkx6家族的三个基因分别表达于前部内胚层的衍生物，包括胰腺、胃、食道和肺。 Nkx6.3是最近发现的Nkx6家族新成员，它在爪蟾中的表达与Nkx6.1和Nkx6.2有了较大分歧。在第三章，我们通过功能获得及功能缺失实验来探讨Nkx6.3在爪蟾早期发育中的功能。我们发现原肠期前过量或抑制Nkx6.3表达都会影响胚胎原肠运动的正常进行。我们通过动物帽延伸实验证明Nkx6.3参与了细胞运动。半定量RT-PCR结果显示，Nkx6.3可以调控一些粘附分子的表达。以上结果说明Nkx6.3通过调控粘附分子的转录而参与细胞运动的调控。我们还发现，在爪蟾胚胎中Nkx6.3的过表达或抑制表达都导致神经嵴标记基因表达降低。进一步研究发现，32细胞期在不同部位注射Nkx6.3 mRNA可以异位诱导或抑制Slug的表达。动物帽实验显示，Nkx6.3单独过表达可以诱导神经嵴发生，而迄今为止转录因子中只有Snail1具有这一单独诱导能力。在爪蟾胚胎及动物帽中，过表达Nkx6.3都可以诱导Fgf8、Wnt8而抑制BMP4的转录，而且Nkx6.3对这些分泌因子的调控方式是不同的。4细胞期过表达Nkx6.3的胚胎，在促进Fgf8和Wnt8而抑制BMP4的同时，却抑制神经板边界特异基因Msx1、Pax3和神经嵴特异基因Slug的表达，说明Nkx6.3对神经嵴的诱导调控在神经板边界基因层次还存在抑制作用。32细胞过表达Nkx6.3会细胞自主性抑制以及细胞非自主诱导Msx1、Pax3、Slug的表达。Nkx6.3异位诱导Dlx5却抑制Dlx3的表达，说明Dlx5可能是Nkx6.3负调控的直接靶基因。由此，我们提出Nkx6.3的神经嵴诱导调控分为两个层次：分泌信号分子水平的正调控和神经板边界决定水平的负调控。在脊椎动物的神经发生过程中，神经管背腹不同层次形成不同的神经元。这些神经元细胞的命运由背腹起源的多种形态发生素决定。形态发生素通过浓度梯度确定了一组转录因子在神经管背腹不同层次的特异表达，这些基因的组合调控决定了神经前体细胞的命运。然而，这些转录因子是如何解读形态发生素梯度信号的还不是很清楚。第四章，我们通过对神经管腹侧特异表达的转录因子的调控区进行预测，确定了可能调控这些基因表达的保守区段。此外，我们改进了爪蟾转基因操作，并用这一技术确证了Nkx6.2的调控区域。Dbx1、Nkx2.2及Pax6的转录调控区已在小鼠或爪蟾中报道过。由此我们得到了两对在神经管背腹图式中相互作用的转录因子的调控区域：Nkx6.2和Dbx1、Nkx2.2和Pax6。通过对Nkx6.2和Dbx1的调控保守区的转录因子结合位点的预测，我们发现这四个基因以及Wnt信号之间存在大量的相互调控。然而在这两个基因的调控区，我们没有发现Gli的调控位点，暗示这两个基因可能不受Shh的直接调控。我们还克隆了Dbx家族的两个基因，并检测了它们的时空特异性表达，发现Dbx2是母源性表达的，而Dbx1是合子型基因。这两个基因的表达图式相似，都在神经板中线两侧成线状表达，尾芽期在神经管中部表达。过表达Dbx2抑制神经元的初级分化，说明它可能与Dbx1一样具有维持神经板细胞未分化状态的功能。Dbx2的过表达还抑制Nkx6.2及Dbx1的表达，说明它们可能一起参与了神经管腹侧图式的调控。