The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping.
Left-right asymmetry in vertebrates is controlled by activities emanating from the left lateral plate. How these signals get transmitted to the forming organs is not known. A candidate mediator in mouse, frog and zebrafish embryos is the homeobox gene Pitx2. It is asymmetrically expressed in the left lateral plate mesoderm, tubular heart and early gut tube. Localized Pitx2 expression continues when these organs undergo asymmetric looping morphogenesis. Ectopic expression of Xnr1 in the right lateral plate induces Pitx2 transcription in Xenopus. Misexpression of Pitx2 affects situs and morphology of organs. These experiments suggest a role for Pitx2 in promoting looping of the linear heart and gut. (+info
The paired-domain transcription factor Pax8 binds to the upstream enhancer of the rat sodium/iodide symporter gene and participates in both thyroid-specific and cyclic-AMP-dependent transcription.
The gene encoding the Na/I symporter (NIS) is expressed at high levels only in thyroid follicular cells, where its expression is regulated by the thyroid-stimulating hormone via the second messenger, cyclic AMP (cAMP). In this study, we demonstrate the presence of an enhancer that is located between nucleotides -2264 and -2495 in the 5'-flanking region of the NIS gene and that recapitulates the most relevant aspects of NIS regulation. When fused to either its own or a heterologous promoter, the NIS upstream enhancer, which we call NUE, stimulates transcription in a thyroid-specific and cAMP-dependent manner. The activity of NUE depends on the four most relevant sites, identified by mutational analysis. The thyroid-specific transcription factor Pax8 binds at two of these sites. Mutations that interfere with Pax8 binding also decrease transcriptional activity of the NUE. Furthermore, expression of Pax8 in nonthyroid cells results in transcriptional activation of NUE, strongly suggesting that the paired-domain protein Pax8 plays an important role in NUE activity. The NUE responds to cAMP in both protein kinase A-dependent and -independent manners, indicating that this enhancer could represent a novel type of cAMP responsive element. Such a cAMP response requires Pax8 but also depends on the integrity of a cAMP responsive element (CRE)-like sequence, thus suggesting a functional interaction between Pax8 and factors binding at the CRE-like site. (+info
The highly conserved beta-hairpin of the paired DNA-binding domain is required for assembly of Pax-Ets ternary complexes.
Pax family transcription factors bind DNA through the paired domain. This domain, which is comprised of two helix-turn-helix motifs and a beta-hairpin structure, is a target of mutations in congenital disorders of mice and humans. Previously, we showed that Pax-5 (B-cell-specific activator protein) recruits proteins of the Ets proto-oncogene family to bind a composite DNA site that is essential for efficient transcription of the early-B-cell-specific mb-1 promoter. Here, evidence is provided for specific interactions between Ets-1 and the amino-terminal subdomains of Pax proteins. By tethering deletion fragments of Pax-5 to a heterologous DNA-binding domain, we show that 73 amino acids (amino acids 12 to 84) of its amino-terminal subdomain can recruit the ETS domain of Ets-1 to bind the composite site. Furthermore, an amino acid (Gln22) within the highly conserved beta-hairpin motif of Pax-5 is essential for efficient recruitment of Ets-1. The ability to recruit Ets proteins to bind DNA is a shared property of Pax proteins, as demonstrated by cooperative DNA binding of Ets-1 with sequences derived from the paired domains of Pax-2 and Pax-3. The strict conservation of sequences required for recruitment of Ets proteins suggests that Pax-Ets interactions are important for regulating transcription in diverse tissues during cellular differentiation. (+info
Involvement of poly (ADP-ribose)-polymerase in the Pax-6 gene regulation in neuroretina.
The quail Pax-6 gene is expressed from two promoters named P0 and P1. P0 promoter is under the control of a neuroretina-specific enhancer (EP). This enhancer activates the P0 promoter specifically in neuroretina cells and in a developmental stage-dependent manner. The EP enhancer binds efficiently, as revealed by southwestern experiments, to a 110 kDa protein present in neuroretina cells but not in Quail Embryos Cells and Retinal Pigmented Epithelium which do not express the P0-initiated mRNAs. To study the role of p110 in Pax-6 regulation, we have purified the p110 from neuroretina cells extracts. Based on the peptide sequence of the purified protein, we have identified the p110 as the poly(ADP-ribose) polymerase (PARP). Using bandshift experiments and footprinting studies, we present evidence that PARP is a component of protein complexes bound to the EP enhancer that increases the on rate of the protein complex formation to DNA. Using PARP inhibitors (3AB and 6.5 Hphe), we show that these products are able to inhibit EP enhancer activity in neuroretina cells. Finally, we demonstrate that these inhibitors are able to decrease the expression of the P0-initiated mRNA in the MC29-infected RPE cells which, in contrast to the RPE cells, accumulated the PARP in response to v-myc expression. Our results suggest that PARP is involved in the Pax-6 regulation. (+info
Expression of the paired-box genes Pax-1 and Pax-9 in limb skeleton development.
Vertebrate Pax genes encode a family of transcription factors that play important roles in embryonic patterning and morphogenesis. Two closely related Pax genes, Pax-1 and Pax-9, are associated with early axial and limb skeleton development. To investigate the role of these genes in cartilage formation we have examined the expression profiles of Pax-1 and Pax-9 in developing chick limb mesenchyme in vivo and in vitro. Both transcripts are detected by reverse transcription polymerase chain reaction and Northern blotting throughout chick limb development, from the early bud stages (Hamburger-Hamilton 20-23) to fully patterned appendages (stage 30). Whole-mount in situ hybridization reveals complex, nonoverlapping expression domains of these two genes. Pax-1 transcripts first appear at the anterior proximal margin of the limb buds, while Pax-9 is expressed more distally at what will be the junction of the autopod and the zeugopod. In situ hybridization to serial sections of the girdles reveals that in the pectoral region Pax-1 is expressed proximally in condensed mesenchyme surrounding the junction of the developing scapula, humerus, and coracoid. In the pelvis, Pax-1 is expressed between the femur and the developing acetabulum and along the ventral edge of the ischium; this transcript was also found in the distal hindlimb along the posterior edge of the fibula. Pax-9 transcripts were not detected in the pectoral girdle at any stage, and only weakly in the pelvis along the ventral ischial margin. In the distal parts of both wings and legs, however, Pax-9 is strongly expressed between the anterior embryonic cartilages (e.g., distal radius or tibia) and the anterior ectodermal ridge. The expression of both genes was strongest in undifferentiated cells of precartilage condensations or at the margins of differentiated cartilages, and was absent from cartilage itself. In micromass cultures of chondrifying limb bud mesenchyme expression of Pax-1 and Pax-9 is maintained for up to 3 days in vitro, most strongly at the end of the culture period during chondrogenic differentiation. As seen in vivo, transcripts are found in loose mesenchyme cells at the outer margins of developing cartilage nodules, and are absent from differentiated chondrocytes at the nodule center. Taken together, these investigations extend previous studies of Pax-1 and Pax-9 expression in embryonic limb development while validating limb bud mesenchyme culture as an accessible experimental system for the study of Pax gene function and regulation. Our in vivo and in vitro observations are discussed with reference to 1) the relationship between somitic and limb expression of these two Pax genes, 2) what regulates this expression in different regions of the embryo, and 3) the putative cellular functions of Pax-1 and Pax-9 in embryonic skeletogenesis. (+info
Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development.
Pitx1 is a Bicoid-related homeodomain factor that exhibits preferential expression in the hindlimb, as well as expression in the developing anterior pituitary gland and first branchial arch. Here, we report that Pitx1 gene-deleted mice exhibit striking abnormalities in morphogenesis and growth of the hindlimb, resulting in a limb that exhibits structural changes in tibia and fibula as well as patterning alterations in patella and proximal tarsus, to more closely resemble the corresponding forelimb structures. Deletion of the Pitx1 locus results in decreased distal expression of the hindlimb-specific marker, the T-box factor, Tbx4. On the basis of similar expression patterns in chick, targeted misexpression of chick Pitx1 in the developing wing bud causes the resulting limb to assume altered digit number and morphogenesis, with Tbx4 induction. We hypothesize that Pitx1 serves to critically modulate morphogenesis, growth, and potential patterning of a specific hindlimb region, serving as a component of the morphological and growth distinctions in forelimb and hindlimb identity. Pitx1 gene-deleted mice also exhibit reciprocal abnormalities of two ventral and one dorsal anterior pituitary cell types, presumably on the basis of its synergistic functions with other transcription factors, and defects in the derivatives of the first branchial arch, including cleft palate, suggesting a proliferative defect in these organs analogous to that observed in the hindlimb. (+info
BMP7 acts in murine lens placode development.
Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance (A. T. Dudley et al., 1995, Genes Dev. 9, 2795-2807; G. Luo et al., 1995, Genes Dev. 9, 2808-2820). Here we report that the expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and that the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, we demonstrate that addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, we examined the expression of several early lens placode-specific markers in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway which is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. We conclude that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation. (+info
A mutation in the RIEG1 gene associated with Peters' anomaly.
Mutations within the RIEG1 homeobox gene on chromosome 4q25 have previously been reported in association with Rieger syndrome. We report a 3' splice site mutation within the 3rd intron of the RIEG1 gene which is associated with unilateral Peters' anomaly. The mutation is a single base substition of A to T at the invariant -2 site of the 3' splice site. Peters' anomaly, which is characterised by ocular anterior segment dysgenesis and central corneal opacification, is distinct from Rieger anomaly. This is the first description of a RIEG1 mutation associated with Peters' anomaly. (+info