Novel forms of Paired-like homeodomain transcription factor 2 (PITX2): Generation by alternative translation initiation and mRNA splicing

<p>Abstract</p> <p>Background</p> <p>Members of the <it>Paired</it>-like homeodomain transcription factor (<it>PITX</it>) gene family, particularly <it>PITX1 </it>and <it>PITX2</it>, play important roles in normal development and in differentiated cell functions. Three major isoforms of PITX2 were previously reported to be produced through both alternative mRNA splicing (<it>PITX2A </it>and <it>PITX2B</it>) and alternative promoter usage (<it>PITX2C</it>). The proteins derived from these mRNAs contain identical homeodomain and carboxyl termini. Differences in the amino-termini of the proteins may confer functional differences in some contexts.</p> <p>Results</p> <p>Here, we report the identification of two novel PITX2 isoforms. First, we demonstrate that the <it>Pitx2c </it>mRNA generates two protein products, PITX2Cα and PITX2Cβ, via alternative translation initiation. Second, we identified a novel mRNA splice variant, <it>Pitx2b2</it>, which uses the same 5' splice donor in intron 2 as <it>Pitx2b </it>(hereafter referred to as <it>Pitx2b1</it>), but employs an alternative 3' splice acceptor, leading to an in-frame deletion of 39 base pairs relative to <it>Pitx2b1</it>. <it>Pitx2b2 </it>mRNA is expressed in both murine and human pituitary. The data show that in a murine gonadotrope cell line and adult murine pituitary what was previously thought to be PITX2B1 is actually PITX2Cβ, or perhaps PITX2B2. PITX2B1 is expressed at lower levels than previously thought. PITX2Cβ and PITX2B2 activate gonadotrope-specific gene promoter-reporters similarly to known PITX2 isoforms.</p> <p>Conclusion</p> <p>We have identified and characterized two novel isoforms of PITX2, generated by alternative translation initiation (PITX2Cβ) and alternative mRNA splicing (PITX2B2). These proteins show similar DNA binding and <it>trans</it>-activation functions as other PITX2 isoforms <it>in vitro</it>, though their conservation across species suggests that they may play distinct, as yet unidentified, roles <it>in vivo</it>.</p

PITX2 gain-of-function induced defects in mouse forelimb development

BACKGROUND: Limb development and patterning originate from a complex interplay between the skeletal elements, tendons, and muscles of the limb. One of the genes involved in patterning of limb muscles is the homeobox transcription factor Pitx2 but its role in forelimb development is uncharacterized. Pitx2 is expressed in the majority of premature presumptive forelimb musculature at embryonic day 12.5 and then maintained throughout embryogenesis to adult skeletal muscle. RESULTS: To further study the role of Pitx2 in forelimb development we have generated transgenic mice that exhibit a pulse of PITX2 over-expression at embryonic day 13.5 and 14.5 in the developing forelimb mesenchyme. These mice exhibit a distal misplacement of the biceps brachii insertion during embryogenesis, which twists the forelimb musculature resulting in severe skeletal malformations. The skeletal malformations have some similarities to the forearm deformities present in Leri-Weill dyschondrosteosis. CONCLUSION: Taken together, the tendon, muscle, and bone anomalies further support a role of Pitx2 in forelimb development and may also shed light on the interaction between the skeletal elements and muscles of the limb during embryogenesis

Effects on differentiation of embryonic ventral midbrain progenitors by Lmx1a, MSX1, Ngn2, and Pitx3.

Neurons derived from neural stem cells could potentially be used for cell therapy in neurodegenerative disorders, such as Parkinson's disease. To achieve controlled differentiation of neural stem cells, we expressed transcription factors involved in the development of midbrain dopaminergic neurons in rat and human neural progenitors. Using retroviral-mediated transgene delivery, we overexpressed Lmx1a (LIM homeobox transcription factor 1, alpha), Msx1 (msh homeobox homolog 1), Ngn2 (neurogenin 2), or Pitx3 (paired-like homeodomain transcription factor 3) in neurospheres derived from embryonic day 14.5 rat ventral mesencephalic progenitors. We also expressed either Lmx1a or Msx1 in the human embryonic midbrain-derived progenitor cell line NGC-407. Rat cells transduced with Ngn2 exited the cell cycle and expressed the neuronal marker microtubule-associated protein 2 and catecholamine-neuron protein vesicular monoamine transporter 2. Interestingly, Pitx3 downregulated the expression of SOX2 (SRY-box containing gene 2) and Nestin, altered cell morphology, but never induced neuronal or glial differentiation. Ngn2 exhibited a strong neuron-inducing effect. In contrast, few Lmx1a-transduced cells matured into neurons, and Msx1 overexpression promoted oligodendrogenesis rather than neuronal differentiation. Importantly, none of these four genes, alone or in combination, enhanced differentiation of rat neural stem cells into dopaminergic neurons. Notably, the overexpression of Lmx1a, but not Msx1, in human neural progenitors increased the yield of tyrosine hydroxylase-immunoreactive cells by threefold. Together, we demonstrate that induced overexpression of transcription factor genes has profound and specific effects on the differentiation of rat and human midbrain progenitors, although few dopamine neurons are generated

The channels for important new knowledge to medical doctors. The case of Helicobacter pylori and stomach and duodenal ulcers

Neðst á síðunni er hægt að nálgast greinina í heild sinni með því að smella á hlekkinn View/OpenA retrospective survey of the flow of information to medical doctors as regards the relationship between Helicobacter pylori and stomach and duodenal ulcer and other gastrointestinal diseases. This is the Icelandic part of a joint study in five Nordic countries. Objective: The objective of the research was to assess the effectiveness of different sources of information, to measure the length of time it takes for the information to spread and influence medical practice. Material and methods: The information was collected with the help of questionnaires that were sent to 159 general practitioners (GP) and 110 physicians in three medical specialities. Among the questions asked were when and how the information had reached the respondents and when and how it had influenced their medical practices. Results: The knowledge about Helicobacter pylori had generally reached medical doctors six to eight years after it first appeared in the medical journals and had three years later led to changes in the routine examinations and treatment. The specialists got the news one to three years earlier than the GPs and also started to prescribe antibiotics one to three years earlier. The most frequently cited source of information was international medical journals, then scientific conferences, colleagues and The Icelandic Medical Journal. The most important source was considered to be international medical journals, then scientific conferences and colleagues. A certain difference was found between GPs and the specialist doctors. More GPs said they had got information from the pharmaceutical industry or through The Icelandic Medical Journal and relied on clinical diagnosis. More specialist doctors considered the most important source of new knowledge to be the colleagues, they also said they used endoscopy and took tissue samples more often and more often considered it correct to do so. Conclusions: Only a decade after the first reports on Helicobacter pylori appeared in medical journals most Icelandic doctors had got the knowledge and were prescribing appropriate treatment, the specialist doctors in the lead. International medical journals spread the news most effectively but The Icelandic Medical Journal played only a minor role. The question is if the process could have been accelerated any further by some more hitting Icelandic news and by more definite initiative in framing guidelines.Lýst er afturskyggnri rannsókn á upplýsingaflæði til lækna af tengslum Helicobacter pylori við sár í maga og skeifugörn og fleiri meltingarfærasjúkdóma. Þetta er íslenski hlutinn af sameiginlegri rannsókn í fimm Norðurlöndum. Tilgangur: Tilgangur rannsóknarinnar var að fá fram mynd af virkni dreifingarleiða faglegrar þekkingar og sjá hve lengi merk tíðindi eru að berast og valda breytingum á hefbundnum vinnubrögðum. Efniviður og aðferðir: Sendir voru spurningalistar til 159 íslenskra heimilislækna og 110 lækna í þremur sérgreinum. Meðal annars var spurt hvenær og hvaðan umrædd frétt barst, hvenær og hvernig hún breytti vinnubrögðum við greiningu og meðferð. Niðurstöður: Tíðindin um H. pylori höfðu almennt borist læknum sex til átta árum eftir fyrstu skrif um þau í fagritum og þremur árum síðar höfðu þau leitt til viðeigandi breytinga á rannsóknum og meðferð. Sérgreinalæknarnir fengu fréttirnar einu til þremur árum á undan heimilislæknunum og fóru einnig einu til þremur árum á undan þeim að nota sýklalyf í meðferð sýrusára. Fróðleiksuppspretturnar, sem flestir nefndu, voru erlend fagrit, þá vísindaráðstefnur, starfssystkini og íslenskt fagrit. Mikilvægustu heimild töldu flestir erlend fagrit, þá vísindaráðstefnur og starfssystkini. Viss munur kom fram á heimilislæknahópnum og sérgreinalæknahópnum. Fleiri heimilislæknar sögðust fá fréttir frá lyfjaiðnaðinum, fleiri lesa þær í Læknablaðinu og treysta fleiri á klíníska sjúkdómsgreiningu. Sérgreinalæknarnir töldu fleiri mikilvægustu heimildina vera starfssystkini, einnig sögðust þeir fleiri nota og telja rétt að nota speglanir og vefjasýnatökur en heimilislæknarnir. Ályktanir: Aðeins áratugi eftir birtingu fyrstu greina í fagritum um Helicobacter pylori höfðu íslenskir læknar aflað sér þekkingar og tekið upp viðeigandi meðferð, og fóru sérgreinalæknar þar eðlilega fyrir. Fréttin barst flestum með erlendu fagriti, en hlutverk innlends fagrits var lítið. Spurning er hvort hægt hefði verið að hraða þessu ferli enn frekar með markvissari innlendri fréttamiðlun og frumkvæði að gerð vinnureglna

Neurogenin2 Directs Granule Neuroblast Production and Amplification while NeuroD1 Specifies Neuronal Fate during Hippocampal Neurogenesis

The specification and differentiation of dentate gyrus granule neurons in the hippocampus require temporally and spatially coordinated actions of both intrinsic and extrinsic molecules. The basic helix-loop-helix transcription factor Neurogenin2 (Ngn2) and NeuroD1 are key regulators in these processes. Based on existing classification, we analyzed the molecular events occurring during hippocampal neurogenesis, primarily focusing on juvenile animals. We found that Ngn2 is transiently expressed by late type-2a amplifying progenitors. The Ngn2 progenies mature into hippocampal granule neurons. Interestingly, the loss of Ngn2 at early stages of development leads to a robust reduction in neurogenesis, but does not disturb granule neuron maturation per se. We found that the role of Ngn2 is to maintain progenitors in an undifferentiated state, allowing them to amplify prior to their maturation into granule neurons upon NeuroD1 induction. When we overexpressed Ngn2 and NeuroD1 in vivo, we found NeuroD1 to exhibit a more pronounced neuron-inductive effect, leading to granule neuron commitment, than that displayed by Ngn2. Finally, we observed that all markers expressed during the transcriptional control of hippocampal neurogenesis in rodents are also present in the human hippocampus. Taken together, we demonstrate a critical role of for Ngn2 and NeuroD1 in controlling neuronal commitment and hippocampal granule neuroblast formation, both during embryonic development and in post-natal hippocampal granule neurogenesis

Synthetic RNA Silencing of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.Peer reviewe

Canonical Wnt/Β-catenin signaling is required for maintenance but not activation of Pitx2 expression in neural crest during eye development

Pitx2 is a paired-like homeodomain gene that acts as a key regulator of eye development. Despite its significance, upstream regulation of Pitx2 expression during eye development remains incompletely understood. We use neural crest-specific ablation of Ctnnb1 to demonstrate that canonical Wnt signaling is not required for initial activation of Pitx2 in neural crest. However, canonical Wnt signaling is subsequently required to maintain Pitx2 expression in the neural crest. Eye development in Ctnnb1 -null mice appears grossly normal early but significant phenotypes emerge following loss of Pitx2 expression. LEF-1 and Β-catenin bind Pitx2 promoter sequences in ocular neural crest, indicating a likely direct effect of canonical Wnt signaling on Pitx2 expression. Combining our data with previous reports, we propose a model wherein a sequential code of retinoic acid followed by canonical Wnt signaling are required for activation and maintenance of Pitx2 expression, respectively. Other key transcription factors in the neural crest, including Foxc1 , do not require intact canonical Wnt signaling. Developmental Dynamics 239:3215–3225, 2010. © 2010 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78327/1/22459_ftp.pd

Maternal exposure to a Western-style diet causes differences in intestinal microbiota composition and gene expression of suckling mouse pups

Scope:The long-lasting consequences of nutritional programming during the early phase of life have become increasingly evident. The effects of maternal nutrition on the developing intestine are still underexplored. Methods and results: In this study we observed 1) altered microbiota composition of the colonic luminal content, and 2) differential gene expression in the intestinal wall in two-week-old mouse pups born from dams exposed to a Western-style (WS) diet during the perinatal period. A sexually dimorphic effect was found for the differentially expressed genes in the offspring of WS diet-exposed dams but no differences between male and female pups were found for the microbiota composition. Integrative analysis of the microbiota and gene expression data revealed that the maternal WS diet independently affected gene expression and microbiota composition. However, the abundance of bacterial families not affected by the WS diet (Bacteroidaceae, Porphyromonadaceae and Lachnospiraceae) correlated with the expression of genes playing a key role in intestinal development and functioning (e.g. Pitx2 and Ace2). Conclusion: Our data reveal that maternal consumption of a WS diet during the perinatal period alters both gene expression and microbiota composition in the intestinal tract of two-week-old offspring

Mutational analysis of the PITX2 coding region revealed no common cause for transposition of the great arteries (dTGA)

BACKGROUND: PITX2 is a bicoid-related homeodomain transcription factor that plays an important role in asymmetric cardiogenesis. Loss of function experiments in mice cause severe heart malformations, including transposition of the great arteries (TGA). TGA accounts for 5–7% of all congenital heart diseases affecting 0.2 per 1000 live births, thereby representing the most frequent cyanotic heart defect diagnosed in the neonatal period. METHODS: To address whether altered PITX2 function could also contribute to the formation of dTGA in humans, we screened 96 patients with dTGA by means of dHPLC and direct sequencing for mutations within the PITX2 gene. RESULTS: Several SNPs could be detected, but no stop or frame shift mutation. In particular, we found seven intronic and UTR variants, two silent mutations and two polymorphisms within the coding region. CONCLUSION: As most sequence variants were also found in controls we conclude that mutations in PITX2 are not a common cause of dTGA