Current molecular understanding of Axenfeld-Rieger syndrome.

Axenfeld-Rieger syndrome (ARS) is a rare autosomal dominant inherited disorder affecting the development of the eyes, teeth and abdomen. The syndrome is characterised by complete penetrance but variable expressivity. The ocular component of the ARS phenotype has acquired most clinical attention and has been dissected into a spectrum of developmental eye disorders, of which open-angle glaucoma represents the main challenge in terms of treatment. Mutations in several chromosomal loci have been implicated in ARS, including PITX2, FOXC1 and PAX6. Full-spectrum ARS is caused primarily by mutations in the PITX2 gene. The homeobox transcription factor PITX2 is produced as at least four different transcriptional and splicing isoforms, with different biological properties. Intriguingly, PITX2 is also involved in left-right polarity determination, although asymmetry defects are not a feature of ARS. In experimental animal models and in cell culture experiments using PITX2, abundant evidence indicates that a narrow window of expression level of this gene is vital for its correct function

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

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

Basic helix-loop-helix proteins expressed during early embryonic organogenesis.

The basic helix–loop–helix proteins form a special group of transcription factors unique for the eukaryotic organisms. They are crucial for the embryonic development of many fundamental organ systems such as muscle, heart, central nervous system, hematopoiteic system, and many others. They are very flexible in terms of regulating transcription in that they can either promote or repress transcription, and do so in many different ways. Basic helix–loop–helix proteins can form homo- or heterodimers with other members of the group, and are subject to post-transcriptional modifications. In this review, an overview of basic helix–loop–helix protein classification, biochemical function, and examples of past and recent advances in our understanding of embryonic development are presented, with emphasis on the vertebrate muscle, heart, brain, and eye

Nulp1, a novel basic helix-loop-helix protein expressed broadly during early embryonic organogenesis and prominently in developing dorsal root ganglia.

The basic helix-loop-helix (bHLH) proteins control differentiation and development of a variety of organs. We have isolated the complementary DNA (cDNA) of a novel class of bHLH transcription factors. The previously uncharacterized bHLH messenger RNA (mRNA) was identified by RNA fingerprinting by comparing embryonic and adult mRNA. The reading frame sequence predicts a new class of bHLH family. Northern blotting of embryonic stages demonstrated a 3.2-kb transcript present in several embryonic tissues, including kidney, brain, heart, and lung, in a fashion confirmatory with the RNA-fingerprinting data. In situ hybridization of cryosections detected strong signals in the dorsal root ganglia of 14-day-old mouse embryos (E14). Transient transfection of human embryonic kidney cells with Nulp1-EGFP demonstrated nuclear localization. The complex expression pattern and unique protein sequence, including an acidic amino terminal and putative transcription activation domain, suggests that Nulp1 may have a distinct role in embryonic development of many organs, including the adult brain

Gene expression profiling of differentiating embryonic stem cells expressing dominant negative fibroblast growth factor receptor 2.

Embryonic stein (ES) cells are derived from the inner cell mass of the blastocyst and can be cultured as three-dimensional embryoid bodies (EBs) in which embryonic pregastrulation stages are faithfully mimicked. Fibroblast growth factor receptors (mainly FGFR2) are involved in the first differentiation events during early mammalian embryogenesis. It has been demonstrated that the presence of FGFR2 is a prerequisite for laminin-111 and collagen type IV synthesis and subsequently basement membrane formation in EBs. To identify genes that are influenced by FGFR signalling, we performed global gene expression profiling of differentiating EBs expressing dominant negative FGFR2 (dnFGFR2), acquiring an extensive catalogue of down- and up-regulated genes. We show a strong down-regulation of endodermal and basement membrane related genes, which strengthen the view that the FGFR signalling pathway is a main stimulator of basement membrane synthesis in EBs. We further present down-regulation of genes previously not linked to FGFR signalling, and in addition an active transcription of some mesodermal related genes in differentiating dnFGFR2 EBs