Circular RNA circ-PITX1 promotes the progression of glioblastoma by acting as a competing endogenous RNA to regulate miR-379-5p/MAP3K2 axis.

As the most fatal disease in human central nerve system, glioblastoma has attracted increasing attention. Unfortunately, the prognosis for patients with glioblastoma still quite unfavorable. Recent years, circular RNAs (circRNAs) have been identified to be associated with carcinogenesis due to their abnormal expression. However, the detailed molecular mechanism of circRNAs in regulating cancer progression is still unclear. This study focused on the potential mechanism of circ-PITX1 in glioblastoma. Herein, circ-PITX1 was found to be upregulated in glioblastoma and could mediate glioblastoma tissues and cell lines. Functionally, downregulation of circ-PITX1 hampered cell proliferation and accelerated cell apoptosis. Through mechanism investigation, we identified the cytoplasmic localization of circ-PITX1 and its molecular sponge role. The interactions between circ-PITX1 and miR-379-5p as well as between miR-379-5p and MAP3K2 were demonstrated. Thus, we confirmed that circ-PITX1 exerted as a competing endogenous RNA (ceRNA) in glioblastoma by sponging miR-379-5p to elevate MAP3K2 expression. Rescue assays demonstrated that MAP3K2 rescued the proliferation and apoptosis mediated by the silencing of circ-PITX1. Collectively, our study elucidated a novel molecular pathway and its functions in glioblastoma.

Generation and Phenotype Identification of PAX4 Gene Knockout Rabbit by CRISPR/Cas9 System.

Paired-homeodomain transcription factor 4 (PAX4) gene encodes a transcription factor which plays an important role in the generation, differentiation, development, and survival of insulin-producing ß-cells during mammalian pancreas development. PAX4 is a key diabetes mellitus (DM) susceptibility gene, which is associated with many different types of DM, including T1DM, T2DM, maturity onset diabetes of the young 9 (MODY9) and ketosis prone diabetes. In this study, a novel PAX4 gene knockout (KO) model was generated through co-injection of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) mRNA/sgRNA into rabbit zygotes. Typical phenotypes of growth retardation, persistent hyperglycemia, decreased number of insulin-producing ß cells and increased number of glucagon-producing α cells were observed in the homozygous PAX4 KO rabbits. Furthermore, DM associated phenotypes including diabetic nephropathy, hepatopathy, myopathy and cardiomyopathy were also observed in the homozygous PAX4 KO rabbits but not in the wild type (WT) controls and the heterozygous PAX4 KO rabbits. In summary, this is the first PAX4 gene KO rabbit model generated by CRISPR/Cas9 system. This novel rabbit model may provide a new platform for function study of PAX4 gene in rabbit and gene therapy of human DM in clinical trails.

PAX9 regulates squamous cell differentiation and carcinogenesis in the oro-oesophageal epithelium.

PAX9 is a transcription factor of the PAX family characterized by a DNA-binding paired domain. Previous studies have suggested a potential role of PAX9 in squamous cell differentiation and carcinogenesis of the oro-oesophageal epithelium. However, its functional roles in differentiation and carcinogenesis remain unclear. In this study, Pax9 deficiency in mouse oesophagus promoted cell proliferation, delayed cell differentiation, and altered the global gene expression profile. Ethanol exposure downregulated PAX9 expression in human oesophageal epithelial cells in vitro and mouse forestomach and tongue in vivo. We further showed that PAX9 was downregulated in human oro-oesophageal squamous cell carcinoma (OESCC), and its downregulation was associated with alcohol drinking and promoter hypermethylation. Moreover, ad libitum feeding with a liquid diet containing ethanol for 40 weeks or Pax9 deficiency promoted N-nitrosomethylbenzylamine-induced squamous cell carcinogenesis in mouse tongue, oesophagus, and forestomach. In conclusion, PAX9 regulates squamous cell differentiation in the oro-oesophageal epithelium. Alcohol drinking and promoter hypermethylation are associated with PAX9 silencing in human OESCC. PAX9 downregulation may contribute to alcohol-associated oro-oesophageal squamous cell carcinogenesis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pineal hypoplasia, reduced melatonin and sleep disturbance in patients with PAX6 haploinsufficiency.

In rodent studies, paired box 6 (PAX6) appears to play an important role in the development of the pineal, the primary source of the circadian regulating hormone, melatonin. Pineal hypoplasia has been previously reported in patients with PAX6 haploinsufficiency (+/−); however, pineal measurement, melatonin concentrations and sleep quality have not been reported. This cross-sectional descriptive study examined pineal volume, melatonin secretion and sleep disturbance in 37 patients with PAX6+/− (age 15.3 ± 9.9 years) and 17 healthy controls (16.0 ± 7.2 years), within an inpatient setting at the Clinical Research Center of the National Institutes of Health, Bethesda, Maryland, USA. Pineal volume was evaluated by magnetic resonance imaging. Diurnal serum cortisol, serum melatonin and urine 6-sulphatoxymelatonin concentrations were measured by enzyme-linked immunosorbent assay. The Child Sleep Habits Questionnaire was administered for patients <13 years old. Pineal volume was fivefold lower in PAX6+/− versus controls (mean ± SD: 25 ± 15 versus 129 ± 50 µL, P < 0.001). Midnight serum cortisol was similar in PAX6+/− versus controls (P = 0.14). Midnight serum melatonin was > twofold lower in PAX6+/− versus controls [median (25th-75 th): 28 (22-42) versus 71 (46-88) pg mL-(1), P < 0.001]. First morning void urinary 6-sulphatoxymelatonin was fourfold lower in PAX6+/− versus controls [11 (6-26) versus 45 (34-61) ng mg(-1) Cr, P = 0.001]. Child Sleep Habits Questionnaire score was higher in PAX6+/− versus controls (48 ± 6 versus 41 ± 5, P = 0.03). The current findings suggest that PAX6+/− is associated with smaller pineal size, lower melatonin secretion and greater parental report of sleep disturbances in children. Further studies are needed to explore the potential use of melatonin replacement for improving sleep quality in patients with PAX6+/−.

The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos.

Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.

Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients.

Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans.

The paired box transcription factor Pax8 is essential for function and survival of adult thyroid cells.

The transcription factor Pax8 is already known to be essential at very early stages of mouse thyroid gland development, before the onset of thyroid hormone production. In this paper we show, using a conditional inactivation strategy, that the removal of the Pax8 protein late in gland development results in severe hypothyroidism, consequent to a reduced gland size and a deranged differentiation. These results demonstrate that Pax8 is also an essential player in controlling survival and differentiation of adult thyroid follicular cells.

De novo 2q36.1q36.3 interstitial deletion involving the PAX3 and EPHA4 genes in a fetus with spina bifida and cleft palate.

BACKGROUND: Interstitial 2q36 deletion is a rare event. Only two previously published cases of 2q36 deletions were characterized using array-CGH. This is the first case diagnosed prenatally. METHODS: We report on the prenatal diagnosis of a 2q36.1q36.3 interstitial deletion in a fetus with facial dysmorphism, spina bifida, and cleft palate. RESULTS: Array-CGH analysis revealed a 5.6 Mb interstitial deletion of the long arm of chromosome 2q36.1q36.3, including the PAX3 and EPHA4 genes. CONCLUSION: The present study reinforces the hypothesis that PAX3 haploinsufficiency may be associated with neural tube defects in humans and suggests that the EPHA4 gene might be implicated during palate development. This report also illustrates the added value of array-CGH to detect cryptic chromosomal imbalances in malformed fetuses and to improve genetic counseling prenatally.

Targeting of Slc25a21 is associated with orofacial defects and otitis media due to disrupted expression of a neighbouring gene.

Homozygosity for Slc25a21(tm1a(KOMP)Wtsi) results in mice exhibiting orofacial abnormalities, alterations in carpal and rugae structures, hearing impairment and inflammation in the middle ear. In humans it has been hypothesised that the 2-oxoadipate mitochondrial carrier coded by SLC25A21 may be involved in the disease 2-oxoadipate acidaemia. Unexpectedly, no 2-oxoadipate acidaemia-like symptoms were observed in animals homozygous for Slc25a21(tm1a(KOMP)Wtsi) despite confirmation that this allele reduces Slc25a21 expression by 71.3%. To study the complete knockout, an allelic series was generated using the loxP and FRT sites typical of a Knockout Mouse Project allele. After removal of the critical exon and neomycin selection cassette, Slc25a21 knockout mice homozygous for the Slc25a21(tm1b(KOMP)Wtsi) and Slc25a21(tm1d(KOMP)Wtsi) alleles were phenotypically indistinguishable from wild-type. This led us to explore the genomic environment of Slc25a21 and to discover that expression of Pax9, located 3' of the target gene, was reduced in homozygous Slc25a21(tm1a(KOMP)Wtsi) mice. We hypothesize that the presence of the selection cassette is the cause of the down regulation of Pax9 observed. The phenotypes we observed in homozygous Slc25a21(tm1a(KOMP)Wtsi) mice were broadly consistent with a hypomorphic Pax9 allele with the exception of otitis media and hearing impairment which may be a novel consequence of Pax9 down regulation. We explore the ramifications associated with this particular targeted mutation and emphasise the need to interpret phenotypes taking into consideration all potential underlying genetic mechanisms.

Postnatal manipulation of Pax6 dosage reverses congenital tissue malformation defects.

Aniridia is a congenital and progressive panocular condition with poor visual prognosis that is associated with brain, olfactory, and pancreatic abnormalities. Development of aniridia is linked with nonsense mutations that result in paired box 6 (PAX6) haploinsufficiency. Here, we used a mouse model of aniridia to test the hypothesis that manipulation of Pax6 dosage through a mutation-independent nonsense mutation suppression strategy would limit progressive, postnatal damage in the eye. We focused on the nonsense suppression drugs 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid (ataluren) and gentamicin. Remarkably, we demonstrated that nonsense suppression not only inhibited disease progression but also stably reversed corneal, lens, and retinal malformation defects and restored electrical and behavioral responses of the retina. The most successful results were achieved through topical application of the drug formulation START (0.9% sodium chloride, 1% Tween 80, 1% powdered ataluren, 1% carboxymethylcellulose), which was designed to enhance particle dispersion and to increase suspension viscosity. These observations suggest that the eye retains marked developmental plasticity into the postnatal period and remains sensitive to molecular remodeling. Furthermore, these data indicate that other neurological developmental anomalies associated with dosage-sensitive genetic mutations may be reversible through nonsense suppression therapeutics.

Pax6 downregulation mediates abnormal lineage commitment of the ocular surface epithelium in aqueous-deficient dry eye disease.

Keratinizing squamous metaplasia (SQM) of the ocular surface is a blinding consequence of systemic autoimmune disease and there is no cure. Ocular SQM is traditionally viewed as an adaptive tissue response during chronic keratoconjunctivitis sicca (KCS) that provokes pathological keratinization of the corneal epithelium and fibrosis of the corneal stroma. Recently, we established the autoimmune regulator-knockout (Aire KO) mouse as a model of autoimmune KCS and identified an essential role for autoreactive CD4+ T cells in SQM pathogenesis. In subsequent studies, we noted the down-regulation of paired box gene 6 (Pax6) in both human patients with chronic KCS associated with Sjögren's syndrome and Aire KO mice. Pax6 encodes a pleiotropic transcription factor guiding eye morphogenesis during development. While the postnatal function of Pax6 is largely unknown, we hypothesized that its role in maintaining ocular surface homeostasis was disrupted in the inflamed eye and that loss of Pax6 played a functional role in the initiation and progression of SQM. Adoptive transfer of autoreactive T cells from Aire KO mice to immunodeficient recipients confirmed CD4+ T cells as the principal downstream effectors promoting Pax6 downregulation in Aire KO mice. CD4+ T cells required local signaling via Interleukin-1 receptor (IL-1R1) to provoke Pax6 loss, which prompted a switch from corneal-specific cytokeratin, CK12, to epidermal-specific CK10. The functional role of Pax6 loss in SQM pathogenesis was indicated by the reversal of SQM and restoration of ocular surface homeostasis following forced expression of Pax6 in corneal epithelial cells using adenovirus. Thus, tissue-restricted restoration of Pax6 prevented aberrant epidermal-lineage commitment suggesting adjuvant Pax6 gene therapy may represent a novel therapeutic approach to prevent SQM in patients with chronic inflammatory diseases of the ocular surface.

BAF chromatin remodeling complex: cortical size regulation and beyond.

The multi-subunit chromatin remodeling BAF complex controls different developmental processes. Using cortex-specific conditional knockout and overexpression mouse models, we have recently reported that BAF170, a subunit of the vertebrate BAF chromatin remodeling complex, interacts with transcription factor (TF) Pax6 to control cortical size and volume. The mechanistic basis includes suppression of the expression of Pax6 target genes, which are required for genesis of cortical intermediate progenitors (IPs) and specification of late neuronal subtype identity. In addition, we showed that a dynamic competition between BAF170 and BAF155 subunits within the BAF complex during progression of neurogenesis is a primary event in modulating the size of the mammalian cortex. Here, we present additional insights into the interaction between the BAF complex and TF Pax6 in the genesis of IPs of the developing cortex. Furthermore, we show that such competition between BAF170 and BAF155 is involved as well in the determination of the size of the embryonic body. Our results add new insights into a cell-intrinsic mechanism, mediated by the chromatin remodeling BAF complex that controls vertebrate body shape and size.

Sensory cortex limits cortical maps and drives top-down plasticity in thalamocortical circuits.

The primary somatosensory cortex (S1) contains a complete body map that mirrors the subcortical maps developed by peripheral sensory input projecting to the sensory hindbrain, the thalamus and then S1. Peripheral changes during development alter these maps through 'bottom-up' plasticity. Unknown is how S1 size influences map organization and whether an altered S1 map feeds back to affect subcortical maps. We show that the size of S1 in mice is significantly reduced by cortex-specific deletion of Pax6, resulting in a reduced body map and loss of body representations by an exclusion of later-differentiating sensory thalamocortical input. An initially normal sensory thalamus was repatterned to match the aberrant S1 map by apoptotic deletion of thalamic neurons representing body parts with axons excluded from S1. Deleted representations were rescued by altering competition between thalamocortical axons using sensory deprivation or increasing the size of S1. Thus, S1 size determined the resolution and completeness of body maps and engaged 'top-down' plasticity that repatterned the sensory thalamus to match S1.

Pax6 directly down-regulates Pcsk1n expression thereby regulating PC1/3 dependent proinsulin processing.

BACKGROUND: Heterozygous paired box6 (Pax6) mutations lead to abnormal glucose metabolism in mice older than 6 months as well as in human beings. Our previous study found that Pax6 deficiency caused down-expression of prohormone convertase 1/3 (Pcsk1), resulting in defective proinsulin processing. As a protein cleaving enzyme, in addition to its expression, the activity of PC1/3 is closely related to its function. We therefore hypothesize that Pax6 mutation alters the activity of PC1/3, which affects proinsulin processing. METHODOLOGY/PRINCIPAL FINDINGS: Using quantitative RT-PCR, western blot and enzyme assay, we found that PC1/3 C-terminal cleavage and its activity were compromised in Pax6 R266Stop mutant mice, and the expression of Pcsk1n, a potent inhibitor of PC1/3, was elevated by Pax6 deficiency in the mutant mice and MIN6 cells. We confirmed the effect of proSAAS, the protein encoded by Pcsk1n, on PC1/3 C-terminal cleavage and its activity by Pcsk1n RNAi in MIN6 cells. Furthermore, by luciferase-reporter analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assay, we revealed that Pax6 bound to Pcsk1n promoter and directly down-regulated its expression. Finally, by co-transfecting Pax6 siRNA with Pcsk1n siRNA, we showed that Pax6 knock-down inhibited proinsulin processing and that this effect could be rescued by proSAAS down-regulation. These findings confirm that Pax6 regulates proinsulin processing partially through proSAAS-mediated PC1/3 processing and activity. CONCLUSIONS/SIGNIFICANCE: Collectively, the above experiments demonstrate that Pax6 can directly down-regulate Pcsk1n expression, which negatively affects PC1/3 C-terminal cleavage and activity and subsequently participates in proinsulin processing. We identified proSAAS as a novel down-regulated target of Pax6 in the regulation of glucose metabolism. This study also provides a complete molecular mechanism for the Pax6 deficiency-caused diabetes.

Reduced proliferation and excess astrogenesis of Pax6 heterozygous neural stem/progenitor cells.

Neural stem/progenitor cells (NSPCs) are generated in early embryonic brains and maintained to produce neurons and glial cells in the central nervous system throughout the lifespan. A transcription factor Pax6 is a pivotal player in various neurodevelopmental processes. Previously, we have shown that Pax6 heterozygous rodents have defects in hippocampal neurogenesis and production of olfactory bulb interneurons. However, characters of NSPCs derived from Pax6 heterozygous rodents have not been studied in vitro. Here we examined the maintenance/proliferation and differentiation of Pax6 heterozygous mutant (rSey(2)/+) rat NSPCs in the neurosphere culture system. We found that the proliferative activity of NSPCs derived from rSey(2)/+ rats was reduced after serial passages. We also observed an excess astrogenesis in serially passaged NSPCs from rSey(2)/+ rats. These results show that Pax6 is essential for maintaining NSPCs and determining their differentiation fates.

The homeodomain-containing transcription factors Arx and Pax4 control enteroendocrine subtype specification in mice.

Intestinal hormones are key regulators of digestion and energy homeostasis secreted by rare enteroendocrine cells. These cells produce over ten different hormones including GLP-1 and GIP peptides known to promote insulin secretion. To date, the molecular mechanisms controlling the specification of the various enteroendocrine subtypes from multipotent Neurog3(+) endocrine progenitor cells, as well as their number, remain largely unknown. In contrast, in the embryonic pancreas, the opposite activities of Arx and Pax4 homeodomain transcription factors promote islet progenitor cells towards the different endocrine cell fates. In this study, we thus investigated the role of Arx and Pax4 in enteroendocrine subtype specification. The small intestine and colon of Arx- and Pax4-deficient mice were analyzed using histological, molecular, and lineage tracing approaches. We show that Arx is expressed in endocrine progenitors (Neurog3(+)) and in early differentiating (ChromograninA(-)) GLP-1-, GIP-, CCK-, Sct- Gastrin- and Ghrelin-producing cells. We noted a dramatic reduction or a complete loss of all these enteroendocrine cell types in Arx mutants. Serotonin- and Somatostatin-secreting cells do not express Arx and, accordingly, the differentiation of Serotonin cells was not affected in Arx mutants. However, the number of Somatostatin-expressing D-cells is increased as Arx-deficient progenitor cells are redirected to the D-cell lineage. In Pax4-deficient mice, the differentiation of Serotonin and Somatostatin cells is impaired, as well as of GIP and Gastrin cells. In contrast, the number of GLP-1 producing L-cells is increased concomitantly with an upregulation of Arx. Thus, while Arx and Pax4 are necessary for the development of L- and D-cells respectively, they conversely restrict D- and L-cells fates suggesting antagonistic functions in D/L cell allocation. In conclusion, these finding demonstrate that, downstream of Neurog3, the specification of a subset of enteroendocrine subtypes relies on both Arx and Pax4, while others depend only on Arx or Pax4.

Fetal neural tube stem cells from Pax3 mutant mice proliferate, differentiate, and form synaptic connections when stimulated with folic acid.

Although maternal intake of folic acid (FA) prevents neural tube defects in 70% of the population, the exact mechanism of prevention has not been elucidated. We hypothesized that FA affects neural stem cell (NSC) proliferation and differentiation. This hypothesis was examined in a folate-responsive spina bifida mouse model, Splotch (Sp(-/-)), which has a homozygous loss-of-function mutation in the Pax3 gene. Neurospheres were generated with NSCs from the lower lumbar neural tube of E10.5 wild-type (WT) and Sp(-/-) embryos, in the presence and absence of FA. In the absence of FA, the number of neurospheres generated from Sp(-/-) embryos compared with WT was minimal (P<0.05). Addition of FA to Sp(-/-) cultures increased the expression of a Pax3 downstream target, fgfr4, and rescued NSC proliferative potential, as demonstrated by a significant increase in neurosphere formation (P<0.01). To ascertain if FA affected cell differentiation, FA-stimulated Sp(-/-) neurospheres were allowed to differentiate in the continued presence or absence of FA. Neurospheres from both conditions expressed multi-potent stem cell characteristics and the same differentiation potential as WT. Further, multiple neurospheres from both WT and FA-stimulated Sp(-/-) cell cultures formed extensive synaptic connections. On the whole, FA-mediated rescue of neural tube defects in Sp(-/-) embryos promotes NSC proliferation at an early embryonic stage. FA-stimulated Sp(-/-) neurospheres differentiate and form synaptic connections, comparable to WT.

The defects in development and apoptosis of cardiomyocytes in mice lacking the transcriptional factor Pax-8.

BACKGROUND: Cardiac-specific deletion of ALK3 is lethal in mid-gestation with ventricular septum malformations (VSM). This study was designed to define the Pax-8's role in heart development and cardiomyocyte apoptosis. METHODS: Pathologic changes in the hearts of Pax-8 or ALK3 knockout and wild type control mice were determined by light and electron microscopy. Analysis of cardiomyocyte apoptosis was performed by TUNEL. The effect of Pax-8 gene deficiency on caspase-3 activity was examined after transfecting Pax-8 siRNA into cultured myoblast cell line. RESULTS: Mice with ALK3 or Pax-8 gene knockout but not wild type control animals showed the development of VSM. Increased cardiomyocyte apoptosis was found in homozygotes. Echocardiography showed that Pax-8 homozygote mice developed malfunction of the heart. Furthermore, the caspase-3 activity was significantly higher in the cells treated with Pax-8 siRNA as compared to those treated with negative control siRNA in H9C2 (2-1) cell line. CONCLUSIONS: The Pax-8 gene may play a crucial role in heart development and regulating cardiocyte apoptosis. Knockout of Pax-8 may exert a similar effect on myocardial morphology and apoptosis as those seen in ALK3 knockouts. Furthermore, the ventricular septum malformations could be partially attributed to accelerated cardiomyocyte apoptosis.

Arx and Nkx2.2 compound deficiency redirects pancreatic alpha- and beta-cell differentiation to a somatostatin/ghrelin co-expressing cell lineage.

BACKGROUND: Nkx2.2 and Arx represent key transcription factors implicated in the specification of islet cell subtypes during pancreas development. Mice deficient for Arx do not develop any alpha-cells whereas beta- and delta-cells are found in considerably higher numbers. In Nkx2.2 mutant animals, alpha- and beta-cell development is severely impaired whereas a ghrelin-expressing cell population is found augmented.Notably, Arx transcription is clearly enhanced in Nkx2.2-deficient pancreata. Hence in order to precise the functional link between both factors we performed a comparative analysis of Nkx2.2/Arx single- and double-mutants but also of Pax6-deficient animals. RESULTS: We show that most of the ghrelin+ cells emerging in pancreata of Nkx2.2- and Pax6-deficient mice, express the alpha-cell specifier Arx, but also additional beta-cell related genes. In Nkx2.2-deficient mice, Arx directly co-localizes with iAPP, PC1/3 and Pdx1 suggesting an Nkx2.2-dependent control of Arx in committed beta-cells. The combined loss of Nkx2.2 and Arx likewise results in the formation of a hyperplastic ghrelin+ cell population at the expense of mature alpha- and beta-cells. Surprisingly, such Nkx2.2-/-Arx- ghrelin+ cells also express the somatostatin hormone. CONCLUSIONS: Our data indicate that Nkx2.2 acts by reinforcing the transcriptional networks initiated by Pax4 and Arx in early committed beta- and alpha-cell, respectively. Our analysis also suggests that one of the coupled functions of Nkx2.2 and Pax4 is to counteract Arx gene activity in early committed beta-cells.

Thyroid hormone controls cone opsin expression in the retina of adult rodents.

Mammalian retinas display an astonishing diversity in the spatial arrangement of their spectral cone photoreceptors, probably in adaptation to different visual environments. Opsin expression patterns like the dorsoventral gradients of short-wave-sensitive (S) and middle- to long-wave-sensitive (M) cone opsin found in many species are established early in development and thought to be stable thereafter throughout life. In mouse early development, thyroid hormone (TH), through its receptor TRß2, is an important regulator of cone spectral identity. However, the role of TH in the maintenance of the mature cone photoreceptor pattern is unclear. We here show that TH also controls adult cone opsin expression. Methimazole-induced suppression of serum TH in adult mice and rats yielded no changes in cone numbers but reversibly altered cone patterns by activating the expression of S-cone opsin and repressing the expression of M-cone opsin. Furthermore, treatment of athyroid Pax8(-/-) mice with TH restored a wild-type pattern of cone opsin expression that reverted back to the mutant S-opsin-dominated pattern after termination of treatment. No evidence for cone death or the generation of new cones from retinal progenitors was found in retinas that shifted opsin expression patterns. Together, this suggests that opsin expression in terminally differentiated mammalian cones remains subject to control by TH, a finding that is in contradiction to previous work and challenges the current view that opsin identity in mature mammalian cones is fixed by permanent gene silencing.