Aristaless-like Homeobox Research Articles

Correction: A 20 bp Duplication in Exon 2 of the Aristaless-Like Homeobox 4 Gene (ALX4) Is the Candidate Causative Mutation for Tibial Hemimelia Syndrome in Galloway Cattle

Correction: A 20 bp Duplication in Exon 2 of the Aristaless-Like Homeobox 4 Gene (ALX4) Is the Candidate Causative Mutation for Tibial Hemimelia Syndrome in Galloway Cattle

Bilateral Foramina Parietalia Permagna - A Calvarial Defect Caused by Haploinsufficiency of the Msh Homeobox 2 Gene: A Case Report and Current Literature Review.

Foramina parietalia permagna (FPP) is a rare anatomical defect that affects the parietal bones of the human skull. FPP is characterized by symmetric perforations on either side of the skull, which are caused by insufficient ossification during embryogenesis. These openings are typically abnormally large and can range from a few millimeters to several centimeters in diameter. Enlarged foramina are often discovered incidentally during anatomical or radiological examinations and in most cases left untreated unless symptoms develop. Although this calvarial defect is usually asymptomatic, it may be accompanied by neurological or vascular conditions that can have clinical significance in certain cases. FPP is an inherited disorder and arises due to mutations in either Msh homeobox 2 (MSX2) or aristaless-like homeobox 4 (ALX4) genes. In almost all cases, one parent is affected. Clinical findings and diagnostic imaging typically contribute to determine the diagnosis.

Aristaless-like homeobox-3 is wound induced and promotes a low-Wnt environment required for planarian head regeneration.

The planarian Schmidtea mediterranea is a well-established model of adult regeneration, which is dependent on a large population of adult stem cells called neoblasts. Upon amputation, planarians undergo transcriptional wounding programs and coordinated stem cell proliferation to give rise to missing tissues. Interestingly, the Wnt signaling pathway is key to guiding what tissues are regenerated, yet less known are the transcriptional regulators that ensure proper activation and timing of signaling pathway components. Here, we have identified an aristaless-like homeobox transcription factor, alx-3, that is enriched in a population of putative neural-fated progenitor cells at homeostasis, and is also upregulated in stem cells and muscle cells at anterior-facing wounds upon amputation. Knockdown of alx-3 results in failure of head regeneration and patterning defects in amputated tail fragments. alx-3 is required for the expression of several early wound-induced genes, including the Wnt inhibitor notum, which is required to establish anterior polarity during regeneration. Together, these findings reveal a role for alx-3 as an early wound-response transcriptional regulator in both muscle cells and stem cells that is required for anterior regeneration by promoting a low-Wnt environment.

Effect of polycyclic aromatic hydrocarbons on homeobox gene expression during embryonic development of cuttlefish, Sepia officinalis

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants (POPs) commonly found in marine environments. Their bioaccumulation can cause harm to aquatic organisms, including invertebrates, particularly during the early stages of embryonic development. In this study, we evaluated, for the first time, the patterns of PAH accumulation in both capsule and embryo of common cuttlefish (Sepia officinalis). In addition, we explored the effects of PAHs by analysing the expression profiles of seven homeobox genes [i.e., gastrulation brain homeobox (GBX), paralogy group labial/Hox1 (HOX1), paralogy group Hox3 (HOX3), dorsal root ganglia homeobox (DRGX), visual system homeobox (VSX), aristaless-like homeobox (ARX) and LIM-homeodomain transcription factor (LHX3/4)]. We found that PAH levels in egg capsules were higher than those observed in chorion membranes (35.1 ± 13.3 ng/g vs 16.4 ± 5.9 ng/g). Furthermore, PAHs were also found in perivitellin fluid (11.5 ± 5.0 ng/ml). Naphthalene and acenaphthene were the congeners present at highest concentrations in each analysed egg component suggesting higher bioaccumulation rates. Embryos with high concentrations of PAHs also showed a significant increase in mRNA expression for each of the analysed homeobox genes. In particular, we observed a 15-fold increase in the ARX expression levels. Additionally, the statistically significant variation in homeobox gene expression patterns was accompanied by a concomitant increase in mRNA levels of both aryl hydrocarbon receptor (AhR) and estrogen receptor (ER). These findings suggest that bioaccumulation of PAHs may modulate developmental processes of cuttlefish embryos by targeting homeobox gene-mediated transcriptional outcomes. Mechanisms underlying the upregulation of homeobox genes could be related to the ability of PAHs to directly activate AhR- or ER-related signaling pathways.

Circular RNA Circ_0000098 Elevates ALX4 Expression via Adsorbing miR-1204 to Inhibit the Progression of Hepatocellular Carcinoma.

BackgroundCircular RNAs (CircRNAs) feature prominently in the progression of various cancers. However, the biological functions of many circRNAs in hepatocellular carcinoma (HCC) are far from fully clarified. This work is performed to decipher the function of circ_0000098 (circSLC30A7) in modulating the progression of HCC and its molecular mechanism.MethodsMicroarray data (GSE97332) were available from the Gene Expression Omnibus (GEO) database, and circRNA differentially expressed in HCC tissues was screened out by GEO2R tool. Circ_0000098, microRNA-1204 (miR-1204), and aristaless-like homeobox-4 (ALX4) mRNA expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8), scratch wound healing, and Transwell assays were adopted to determine proliferation, migration, and invasion of HCC cells. ALX4 protein, E-cadherin, N-cadherin, and Vimentin expressions were evaluated by Western blot. In addition, the targeting relationship between miR-1204 and circ_0000098 or ALX4 was studied with dual-luciferase reporter assay and RIP assay.ResultsCirc_0000098 expression level was markedly declined in HCC tissues and cells, and its underexpression was associated with larger tumor size of HCC patients. Knocking down circ_0000098 observably promoted the multiplication, migration, invasion, and epithelial-mesenchymal transition (EMT) of Huh7 and SMMC-7721 cells. Additionally, circ_0000098 was mainly distributed in the cytoplasm of HCC cells, and up-regulated ALX4 expression through competitively decoying miR-1204.ConclusionCirc_0000098, as a competitive endogenous RNA (ceRNA) of miR-1204, upregulates ALX4 expression and suppresses the growth, migration, invasion, and EMT of HCC cells.

ALX-Related Frontonasal Dysplasias: Clinical Characteristics and Surgical Management.

The term frontonasal dysplasia (FND) represents a spectrum of anomalies and its genetics have not been well defined. Recently, the critical role of the aristaless-like homeobox (ALX) gene family on the craniofacial development has been discovered. In the present study, we aimed to propose a systematic surgical treatment plan for the ALX-related FNDs according to the genotypic classification as well as demonstrating their clinical characteristics to help surgeons diagnose the underlying pathology accurately. Single-institution retrospective. Tertiary health care. Eighty-nine FND cases were evaluated. Eight of them had ALX1-related FND3, 3 had ALX3-related FND1, and 2 had ALX4-related FND2. Phenotype characteristics of ALX-related FNDs were evaluated, and relevant surgical interventions were assessed. The ALX1-related FND3 phenotype is striking due to the involvement of the eyes in addition to the presence of hypertelorism, facial clefts, and nasal deformities. A widened philtrum and prominent philtral columns are remarkable features of the ALX3-related FND1, whereas the ALX4-related FND2 has more severe deformities: severe hypertelorism, brachycephaly, large parietal bone defects, broad nasal dorsum, and alopecia. Facial bipartition, box osteotomies, eyelid coloboma repair, cleft lip and palate repair, nasal reconstruction, and fronto-orbital advancement can be performed in ALX-related FNDs based on the characteristics of each subtype. This genetic classification system will help surgeon diagnose patients with FND with unique features and draw a roadmap for their treatment with a better surgical perspective.

Variants in ALX4 and their association with genitourinary defects.

Genitourinary anomalies occur in approximately 1% of humans, but in most cases, the cause is unknown. Aristaless-like homeobox 4 (ALX4) is an important homeodomain transcription factor. ALX4 mutations in humans and mouse have been associated with craniofacial defects and genitourinary anomalies such as cryptorchidism and epispadias. To investigate the presence and the functional impact of ALX4 variants in patients with genitourinary defects. Two separate patient cohorts were analyzed. One includes clinical exome-sequencing (ES) data from 7500 individuals. The other includes 52 ALX4 Sanger-sequenced individuals with bladder exstrophy-epispadias complex (BEEC). Dual luciferase assays were conducted to investigate the functional transcriptional impact of ALX4 variants in HeLa cells and HEK293 cells. A total of 41 distinct ALX4 heterozygous missense variants were identified in the ES cohort with 15 variants present as recurrent in multiple patients. p.G369E and p.L373F were the only two present in individuals with genitourinary defects. A p.L373F heterozygous variant was also identified in one of the 52 individuals in the BEEC cohort. p.L373F and p.G369E were tested in vitro as both are considered damaging by MutationTaster, although only p.G369E was considered damaging by PolyPhen-2. p.L373F did not alter transcriptional activity in HeLa and HEK293 cells. p.G369E caused a significant 3.4- and 1.8-fold decrease in transcriptional activities relative to wild-type ALX4 in HEK293 and HeLa cells, respectively. Our study supports the idea that transcription factors like ALX4 could influence the normal development of the GU tract in humans as demonstrated in mouse models as ALX4 variant p.G369E (predicted pathogenic by multiple databases) affects ALX4 function in vitro. Variant p.L373F (predicted pathogenic by only MutationTaster) did not affect ALX4 function in vitro. Exon-sequence information and mouse genetics provide important insights into the complex mechanisms driving genitourinary defects allowing the association of transcriptional defects with congenital disorders.

MiR-1470 regulates cell proliferation and apoptosis by targeting ALX4 in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. MicroRNAs (miRNAs) have been proven to play essential roles in different cancers, including HCC. The current study was mainly focused on the role of miR-1470 in HCC progression. MethodsQuantitative real-time PCR (qRT-PCR) was performed to detect the expression levels of miR-1470 and Aristaless-like homeobox-4 (ALX4). The CCK-8 and EdU assays were used to examine cell proliferation. Flow cytometric analysis was used to elucidate the cell cycle and cell apoptosis. A xenograft tumor assay was carried out to verify the effect of miR-1470 on tumor formation in vivo. ResultsAccording to the qRT-PCR assay, miR-1470 was proven to be overexpressed in HCC. As shown by the CCK-8 assay, EdU assay and flow cytometric analysis, miR-1470 overexpression promoted cell proliferation and inhibited cell apoptosis. ALX4 was proven via a dual luciferase reporter assay to be a downstream target gene of miR-1470. ALX4 was downregulated in HCC. The results of a rescue assay revealed that miR-1470 had an oncogenic role in HCC by regulating ALX4. ConclusionmiR-1470 exhibits an oncogenic role in HCC by targeting ALX4. The data from our study may provide novel insight for the identification of new biomarkers and treatment strategies for HCC.

The evolution of a new cell type was associated with competition for a signaling ligand.

There is presently a very limited understanding of the mechanisms that underlie the evolution of new cell types. The skeleton-forming primary mesenchyme cells (PMCs) of euechinoid sea urchins, derived from the micromeres of the 16-cell embryo, are an example of a recently evolved cell type. All adult echinoderms have a calcite-based endoskeleton, a synapomorphy of the Ambulacraria. Only euechinoids have a micromere-PMC lineage, however, which evolved through the co-option of the adult skeletogenic program into the embryo. During normal development, PMCs alone secrete the embryonic skeleton. Other mesoderm cells, known as blastocoelar cells (BCs), have the potential to produce a skeleton, but a PMC-derived signal ordinarily prevents these cells from expressing a skeletogenic fate and directs them into an alternative developmental pathway. Recently, it was shown that vascular endothelial growth factor (VEGF) signaling plays an important role in PMC differentiation and is part of a conserved program of skeletogenesis among echinoderms. Here, we report that VEGF signaling, acting through ectoderm-derived VEGF3 and its cognate receptor, VEGF receptor (VEGFR)-10-Ig, is also essential for the deployment of the skeletogenic program in BCs. This VEGF-dependent program includes the activation of aristaless-like homeobox 1 (alx1), a conserved transcriptional regulator of skeletogenic specification across echinoderms and an example of a "terminal selector" gene that controls cell identity. We show that PMCs control BC fate by sequestering VEGF3, thereby preventing activation of alx1 and the downstream skeletogenic network in BCs. Our findings provide an example of the regulation of early embryonic cell fates by direct competition for a secreted signaling ligand, a developmental mechanism that has not been widely recognized. Moreover, they reveal that a novel cell type evolved by outcompeting other embryonic cell lineages for an essential signaling ligand that regulates the expression of a gene controlling cell identity.

Epigenetic silencing of ALX4 regulates microcystin-LR induced hepatocellular carcinoma through the P53 pathway

Recent studies have shown that microcystin-LR (MC-LR) is one of the principal factors that cause liver cancer. Previously we have found that Aristaless-like Homeobox 4 (ALX4) was differentially expressed in MC-LR-induced malignant transformed L02 cells. However, the expression regulation, role and molecular mechanism of ALX4 during the process of liver cancer induced by MC-LR are still unclear. The expression of ALX4 was detected by quantitative reverse-transcription PCR and Western blot in MC-LR induced malignantly transformed cell and rat models. Methylation status of ALX4 promoter region was evaluated by methylation-specific PCR and bisulfite genomic sequencing. The anti-tumor effects of ALX4 on MC-LR induced liver cancer were identified in vitro and in vivo. ALX4 expression was progressively down-regulated in MC-LR-induced malignantly transformed L02 cells and the MC-LR exposed rat models. ALX4 promoter regions were highly methylated in malignantly transformed cells, while treatment with demethylation agent 5-aza-dC significantly increased ALX4 expression. Functional studies showed that overexpression of ALX4 inhibits cell proliferation, migration, invasion and metastasis in vitro and in vivo, blocks the G1/S phase and promotes the apoptosis. Conversely, knockdown of ALX4 promotes cell proliferation, migration and invasion. Mechanism study found that ALX4 exerts its antitumor function through the P53 pathway, C-MYC and MMP9. More importantly, ALX4 expression level showed a negative relation with serum MC-LR levels in patients with hepatocellular carcinoma. Our results suggested that ALX4 was inactivated by DNA methylation and played a tumor suppressor function through the P53 pathway in MC-LR induced liver cancer.

Downregulated long noncoding RNA LINC00313 inhibits the epithelial-mesenchymal transition, invasion, and migration of thyroid cancer cells through inhibiting the methylation of ALX4.

Thyroid cancer represents one of the prevalent endocrine cancer with relatively high incidence rate around the world, accompanied by unchanged fatality rate. We probe into the specific role of LINC00313 in mediation of cellular processes of thyroid cancer including proliferation, migration, and invasion through the methylation of aristaless-like homeobox 4 (ALX4). Thyroid cancer-related long noncoding RNAs (lncRNAs) and genes were analyzed by microarray-based analysis. The antitumor effect of LINC00313 was examined with the gain- and loss-of-function experiments. In addition, the binding of LINC00313 and the promoter region of ALX4, and the interaction of LINC00313 with methylation-related proteins were detected. Later, xenograft tumors in nude mice were induced expecting to dig out the modulatory function of LINC00313 in tumor growth of thyroid carcinoma. The microarray-based analysis manifested that LINC00313 was overexpressed, whereas ALX4 was downregulated in thyroid cancer, the results of which were also verified in thyroid cancer tissues. Besides, our results demonstrated that LINC00313 bound to the ALX4 promoter region, and LINC00313 recruited DNMT1 and DNMT3B proteins to promote the methylation of ALX4 promoter region, thus suppressing the ALX4 expression. Finally, the downregulation of LINC00313 and upregulation of ALX4 repressed the AKT/mTOR signaling axis, thus inhibiting proliferative, migratory, invasive abilities as well as epithelial-to-mesenchymal transition (EMT) of thyroid cancer cells. Collectively, downregulated LINC00313 suppresses cell proliferation, migration, as well as invasion of thyroid cancer by inhibiting the methylation of ALX4 and increasing its expression by inactivation of the AKT/mTOR signaling pathway.

Comprehensive analysis of gene expression profiles associated with proliferative diabetic retinopathy.

Proliferative diabetic retinopathy (PDR) is characterized by neovascularization on the surface of the retina or the optic disc, which is associated with environmental and genetic factors. However, its regulatory mechanism remains to be fully elucidated, particularly at a multiomics level. In the present study, a comprehensive analysis was performed of the gene expression profile of fibrovascular membranes (FVMs) associated with PDR, including an analysis of differentially expressed genes, functional enrichment, and regulation of transcription factors (TFs). As a result, novel marker genes of PDR were identified, including flavin containing monooxygenase 2. Furthermore, several common or specific genes, pathways and TFs have been recovered for active and inactive FVMs. In the present study, lymphoid enhancer binding factor 1 (LEF1) was identified as an upregulator in active and inactive FVMs, which is capable of activating or repressing target genes, including claudin 2, secreted phosphoprotein 1 (SPP1), and aristaless-like homeobox 4. It was demonstrated that the Wnt/β-catenin effector LEF1 regulating SPP1 is potentially important in PDR. The results of the present study may provide novel insights into the molecular mechanisms underlying the pathophysiology of PDR.

Overexpression of ALK4 inhibits cell proliferation and migration through the inactivation of JAK/STAT3 signaling pathway in glioma

Aristaless-like homeobox 4 (ALK4) is a member of ALK proteins family and plays an important role in tumorigenesis. However, the expression and function of ALK4 in glioma remain largely unknown. The aim of our study was to elucidate its expression pattern in human glioma tissues and cell lines, as well as its functions in glioma cells. Our results demonstrated that ALK4 was lowly expressed in human glioma tissues and cell lines. Additionally, overexpression of ALK4 significantly suppressed the proliferation, migration and invasion of glioma cells, as well as inhibited the epithelial-mesenchymal transition (EMT) phenotype in glioma cells. Furthermore, overexpression of ALK4 significantly downregulated the phosphorylation levels of JAK2 and STAT3 in U87 cells. STAT3 inhibitor (Niclosamide) obviously enhanced ALK4-inhibted glioma cell proliferation and invasion. In conclusion, we demonstrated that overexpression of ALK4 suppressed glioma cell proliferation, migration and invasion through the inactivation of JAK/STAT3 signaling pathway. Thus, ALK4 may be a potential therapeutic target for the treatment of glioma.

Abstract IA16: Current and emerging molecular diagnostic assays for colorectal cancer

Abstract IA16: Current and emerging molecular diagnostic assays for colorectal cancer

Overexpression of Aristaless-Like Homeobox-4 Inhibits Proliferation, Invasion, and EMT in Hepatocellular Carcinoma Cells.

Aristaless-like homeobox-4 (ALX4), a member of the Aristaless-like homeobox family, has been found to be involved in tumor cell proliferation, migration, and invasion. However, the role of ALX4 in hepatocellular carcinoma (HCC) remains largely unclear. Therefore, in this study we investigated the effects of ALX4 on HCC. The study results indicated that the expression of ALX4 was downregulated in HCC tissues and cell lines. Furthermore, we demonstrated that overexpression of ALX4 inhibited the proliferation, invasion, and epithelial–mesenchymal transition (EMT) in HCC cells. We also found that ALX4 had an inhibitory effect on the sonic hedgehog (Shh) signaling pathway. Taken together, the results suggest that ALX4 may be a promising target for HCC treatment.

Glucose-dependent downregulation of glucagon gene expression mediated by selective interactions between ALX3 and PAX6 in mouse alpha cells.

The stimulation of glucagon secretion in response to decreased glucose levels has been studied extensively. In contrast, little is known about the regulation of glucagon gene expression in response to fluctuations in glucose concentration. Paired box 6 (PAX6) is a key transcription factor that regulates the glucagon promoter by binding to the G1 and G3 elements. Here, we investigated the role of the transcription factor aristaless-like homeobox 3 (ALX3) as a glucose-dependent modulator of PAX6 activity in alpha cells. Experiments were performed in wild-type or Alx3-deficient islets and alphaTC1 cells. We used chromatin immunoprecipitations and electrophoretic mobility shift assays for DNA binding, immunoprecipitations and pull-down assays for protein interactions, transfected cells for promoter activity, and small interfering RNA and quantitative RT-PCR for gene expression. Elevated glucose concentration resulted in stimulated expression of Alx3 and decreased glucagon gene expression in wild-type islets. In ALX3-deficient islets, basal glucagon levels were non-responsive to changes in glucose concentration. In basal conditions ALX3 bound to the glucagon promoter at G3, but not at G1. ALX3 could form heterodimers with PAX6 that were permissive for binding to G3 but not to G1. Thus, increasing the levels of ALX3 in response to glucose resulted in the sequestration of PAX6 by ALX3 for binding to G1, thus reducing glucagon promoter activation and glucagon gene expression. Glucose-stimulated expression of ALX3 in alpha cells provides a regulatory mechanism for the downregulation of glucagon gene expression by interfering with PAX6-mediated transactivation on the glucagon G1 promoter element.

Differential expression of aristaless-like homeobox 4: a potential marker for gastric adenocarcinoma.

The objective of this experiment was to evaluate the ALX-4 mRNA expression level in different stages of human gastric adenocarcinoma compared to the gastric cancer stem cells (GCSC) and gastric cancer cell line, MKN-45. Gastric cancer is the second most common cancer in the world today, leading approximately to 3-10% of all cancer-related deaths. Identification of specific biomarkers could be a crucial approach to improve diagnosis and treatment of this cancer type. Recent findings emphasized on the up-regulation of Aristaless-Like Homeobox 4 (ALX-4) gene expression in several tumors. MKN-45 cell culture was prepared, and gastric cancer stem cell (GCSC) isolation was performed by flowcytometry. Then, 37 fresh gastric tissue samples from cancer patient were subjected for expression analysis by quantitative RT-PCR, prior to any therapeutic intervention in the comparative study for evaluation of ALX-4 gene expression. GCSCs with cuboidal shape as well as a positive expression of CD105, CD44, CD90 and negative for CD45, CD34 markers were identified. Overexpression of ALX-4 was detected in 46% (3.351±2.94, P<0.05) of gastric cancer tissue specimens and GCSCs (4.31±0.04, P<0.005). The mRNA expression level of ALX-4 in MKN-45 gastric cancer cell line was 2.81±0.07 (P<0.005). We determined that ALX-4 mRNA level significantly correlated with the tumor grade (P=0.004), stage (p=0.000153), but not gender (P= 0.06). These results documented the important role of ALX-4 in GCSCs, as an oncogene in progressive cancer, and valuable target in the treatment of drug resistant tumors.

Aristaless-Like Homeobox protein 1 (ALX1) variant associated with craniofacial structure and frontonasal dysplasia in Burmese cats

Frontonasal dysplasia (FND) can have severe presentations that are medically and socially debilitating. Several genes are implicated in FND conditions, including Aristaless-Like Homeobox 1 (ALX1), which is associated with FND3. Breeds of cats are selected and bred for extremes in craniofacial morphologies. In particular, a lineage of Burmese cats with severe brachycephyla is extremely popular and is termed Contemporary Burmese. Genetic studies demonstrated that the brachycephyla of the Contemporary Burmese is a simple co-dominant trait, however, the homozygous cats have a severe craniofacial defect that is incompatible with life. The craniofacial defect of the Burmese was genetically analyzed over a 20 year period, using various genetic analysis techniques. Family-based linkage analysis localized the trait to cat chromosome B4. Genome-wide association studies and other genetic analyses of SNP data refined a critical region. Sequence analysis identified a 12bp in frame deletion in ALX1, c.496delCTCTCAGGACTG, which is 100% concordant with the craniofacial defect and not found in cats not related to the Contemporary Burmese.

A 20 bp Duplication in Exon 2 of the Aristaless-Like Homeobox 4 Gene (ALX4) Is the Candidate Causative Mutation for Tibial Hemimelia Syndrome in Galloway Cattle.

Aristaless-like homeobox 4 (ALX4) gene is an important transcription regulator in skull and limb development. In humans and mice ALX4 mutations or loss of function result in a number of skeletal and organ malformations, including polydactyly, tibial hemimelia, omphalocele, biparietal foramina, impaired mammary epithelial morphogenesis, alopecia, coronal craniosynostosis, hypertelorism, depressed nasal bridge and ridge, bifid nasal tip, hypogonadism, and body agenesis. Here we show that a complex skeletal malformation of the hind limb in Galloway cattle together with other developmental anomalies is a recessive autosomal disorder most likely caused by a duplication of 20 bp in exon 2 of the bovine ALX4 gene. A second duplication of 34 bp in exon 4 of the same gene has no known effect, although both duplications result in a frameshift and premature stop codon leading to a truncated protein. Genotyping of 1,688 Black/Red/Belted/Riggit Galloway (GA) and 289 White Galloway (WGA) cattle showed that the duplication in exon 2 has allele frequencies of 1% in GA and 6% in WGA and the duplication in exon 4 has frequencies of 23% in GA and 38% in WGA. Both duplications were not detected in 876 randomly selected German Holstein Friesian and 86 cattle of 21 other breeds. Hence, we have identified a candidate causative mutation for tibial hemimelia syndrome in Galloway cattle and selection against this mutation can be used to eliminate the mutant allele from the breed.

Pdx1 and USF transcription factors co-ordinately regulate Alx3 gene expression in pancreatic β-cells.

Alterations in transcription factors expressed in insulin-producing islet β-cells generate pancreatic dysfunction leading to diabetes. The homeodomain transcription factor Alx3 (aristaless-like homeobox 3) expressed in pancreatic islets participates in the regulated expression of several islet genes, and its deficiency in mice leads to islet cell apoptosis and glucose intolerance. In the present study, we investigated the mechanisms that regulate expression of Alx3in pancreatic islets at the transcriptional level. We found that the Alx3 promoter contains at least eight putative regulatory elements with an E-box consensus sequence, three of which were determined to be functional and required for Alx3 promoter activity by mutational analysis in transfected MIN6 β-cells. We determined that these E-box elements are recognized by the basic helix-loop-helix transcription factors USF1 (upstream stimulatory factor 1) and USF2. We also identified a highly conserved A-box in the Alx3 promoter that is recognized by the islet-specific transcription factor Pdx1 (pancreatic and duodenal homeobox 1). Pdx1-mediated transactivation of the Alx3 promoter requires the integrity of the three functional E-boxes and the co-operation with USF transcription factors bound to them. The results from the present study indicate that Pdx1 contributes to the transcriptional transactivation of Alx3in pancreatic β-cells by acting in co-ordination with USF1 and USF2.