Transcription Factors of the Alx Family: Evolutionarily Conserved Regulators of Deuterostome Skeletogenesis

Members of the alx gene family encode transcription factors that contain a highly conserved Paired-class, DNA-binding homeodomain, and a C-terminal OAR/Aristaless domain. Phylogenetic and comparative genomic studies have revealed complex patterns of alx gene duplications during deuterostome evolution. Remarkably, alx genes have been implicated in skeletogenesis in both echinoderms and vertebrates. In this review, we provide an overview of current knowledge concerning alx genes in deuterostomes. We highlight their evolutionarily conserved role in skeletogenesis and draw parallels and distinctions between the skeletogenic gene regulatory circuitries of diverse groups within the superphylum.

Algorithm Study of Face Recognition on Improved 2DLDA

Linear Discriminant Analysis (LDA) [1] is a well-known method for face recognition in feature extraction and dimension reduction. To solve the “small sample” effect of LDA, Two-Dimensional Linear Discriminant Analysis (2DLDA) [2] has been used for face recognition recently，but its could hardly take use of the relationship between the adjacent scatter matrix. In this paper, I improved the between-class scatter matrix, proposed paired-class scatter matrix for face representation and recognition. In this new method, a paired between-class scatter matrix distance metric is used to measure the distance between random paired between-class scatter matrix. To test this new method, ORL face database is used and the results show that the paired between-class scatter matrix based 2DLDA method （N2DLDA） outperforms the 2DLDA method and achieves higher classification accuracy than the 2DLDA algorithm.

Five novel single nucleotide polymorphisms (SNPs) of the prophet of PIT1 (<i>PROP1</i>) gene in bovine (Brief report)

The prophet of Pit1 (PROP1) gene encodes a paired class homeodomain transcription factor of 226 amino acids and is organized of 3 exons. PROP1 is necessary for the specification, differentiation and proliferation of cells. Its function is essential for anterior pituitary organogenesis, and heritable mutations in this gene are associated with combined pituitary hormone deficiency (CPHD) in human patients and animals (SAVAGE et al., 2003; CARVALHO et al., 2006). To date, no polymorphisms of the bovine PROP1 gene were described. In the present experiment, the 5' flanking region, the coding region and partial introns of bovine PROP1 were scanned for single nucleotide polymorphism (SNPs) in five cattle breeds of China.

The Hematopoietic Regulator Mouse Mix Functions as a Transcriptional Activator in Embryonic Stem Cells.

During mammalian embryogenesis, hemangioblastic and hematopoietic progenitors are differentiated from mesoderm, but the underlying mechanisms are poorly understood. Mouse Mixl (also known as Mixl1 or Mml) is a member of the Mix/Bix family of Paired-class homeobox genes regulated by Nodal/activin/BMP signaling. Targeted disruption of mMix results in mesodermal and endodermal defects in the embryo and deficient hematopoiesis in embryonic stem (ES) cell-derived embryoid bodies (EBs). Conditional induction of mMix (i-Mix) in EBs results in accelerated mesodermal development and increased numbers of mesodermal, hemangioblastic, and hematopoietic progenitors, suggesting that mMix promotes the recruitment and/or expansion of mesodermal progenitors to these lineages (Willey et al., Blood107:3122, 2006). Conditional activation of mMix in differentiating i-Mix ES cells led to rapid upregulation of goosecoid (gsc). In the frog, gsc is a direct target of XMix.1 and represses transcription of the Brachuyry/T homologue, Xbra. XMix.1 is, therefore, thought to function indirectly in the negative regulation of Xbra. Genetic ablation of mMix in the mouse resulted in an expanded domain of T expression, also suggesting direct or indirect suppression. However, induction of mMix in differentiating i-Mix EBs was followed by early activation of T. Like all known vertebrate members of the Mix/Bix family, mMix contains a highly conserved C-terminal region of polar and acidic residues that have the potential to form an amphipathic helix (a characteristic of many transcriptional activators) and is part of a larger region that can activate transcription in yeast two-hybrid assays. Therefore, mMix may function as an activator or a repressor depending on the cellular context, e.g. through differential recruitment of coactivators and corepressors. Indeed, microarray analysis of induced and uninduced i-Mix EBs revealed dynamic changes in gene expression (both up- and down-regulation) within 24 hr of addition of DOX (unpublished data). To understand the molecular mechanisms by which mMix regulates mesodermal differentiation and hematopoiesis, we have identified the optimal target DNA sequence of the mouse Mix protein in vitro using a PCR-assisted binding site selection assay and a GST-mMix homeodomain fusion protein. The consensus target sequence consists of two 4 base pair half sites separated by a 3 base pair spacer. This consensus DNA recognition sequence is different from the consensus target sequence identified for the Drosophila Paired protein, the founder member of the Paired-class homeodomain protein family. We found that mMix bound to the bipartite target sequence primarily as a dimer. Importantly, mMix protein activates the transcription of a luciferase reporter gene linked to multimerized copies of the target sequence in both NIH3T3 cells and in ES cells in which mMix is conditionally expressed. Mutations of critical DNA contacting residues in the homeodomain of mMix abolish transcriptional activation, suggesting that transactivation by mMix is dependent on homeodomain-DNA interactions. In summary, these findings indicate that mMix can function as a sequence-specific transcriptional activator. We propose that mMix may regulate mesodermal and hematopoietic development, at least in part, through activation of essential target genes. To test this hypothesis, we are currently identifying potential mMix target genes using microarray analysis and chromatin immunoprecipitation assays.

Otx1 Enhances Erythroid Differenciation through Activation of the SCL Gene.

Members of the paired class of homeobox proteins are critical determinants of left-right asymmetry and establish the antero-posterior axis, suggesting that they could also be involved in asymmetric determination within the hematopoietic system. We have previously shown that mice lacking Otx1, a bicoid homeodomain-containing gene, exhibit an impairment of the erythroid compartment, associated with decreased SCL levels. In the present study, we show that Otx1 is coexpressed with SCL in yolk sac during embryonic development; in differentiating embryonic stem cells, Otx1 is upregulated with SCL in both primitive and definitive erythroid colonies, while Otx expression is absent in cardiomyocytes and skeletomyocytes. To address the role of Otx1 in hematopoiesis, we overexpressed Otx1 in primary hematopoietic cells using the MSCV retrovirus. The gain of Otx1 function gives rise to a 6-fold increase in endogenous SCL levels together with an increase in TER119-positive erythroid cells. Strikingly, the generation of CD11b-positive myeloid cells was almost abrogated by ectopic Otx1 expression, suggesting that Otx1 favours the erythroid lineage at the expense of the myeloid lineage. Furthermore, we took several approaches to provide molecular and functional evidence that SCL is a direct transcriptional target of Otx1. Indeed, Otx1 synergizes with GATA-1 to activate transcription from the SCL proximal promoter and this activity is dependent on the proximal GATA site of the SCL promoter. Next, we show by chromatin immunoprecipitation that Otx1 and GATA-1 occupy the SCL proximal promoter in vivo in erythroid cells. At the molecular level, we show that Otx1 physically interacts with GATA-1 in erythroid cells, and the homeodomain of Otx1 is sufficient for this interaction. Finally, a gain of function of SCL rescues the erythroid deficiency of Otx1−/− mice, consistent with the model in which SCL operates downstream of Otx1. Taken together, our observations indicate that Otx1, GATA-1 and SCL are involved in the same genetic pathway to specify the erythroid fate during hematopoiesis.

Role of Otx1 in the Differentiation of Myelo-Monocytic Precursors.

Homeobox-containing genes encode transcription factors implicated in the development and differentiation of multiple cell systems, including hematopoiesis. Several lines of evidence, obtained in our laboratory, indicate that Otx1, a homeobox gene of the paired class strictly required for brain morphogenesis, plays an important role also in blood cell production. It is differentially expressed in hematopoietic progenitors, particularly within the erythroid lineage and loss of Otx1 function results in a cell-autonomous erythroid impairment. Furthermore, Otx1 contributes to the control of erythropoiesis through a direct action on Scl/Tal1, a major hematopoietic regulator. In this study we have investigated whether Otx1 is also implicated in the regulation of myelo-monocytic differentiation. Analysis of Otx1 expression indicated that the gene is transcriptionally active in myelo-monocytic precursors. Moreover, using clonogenic assays we observed that Otx1−/− mice display abnormal frequencies of bone marrow myeloid subpopulations, although the total number of myeloid precursors is normal, as compared to wild type animals. Inactivation of Otx1 leads to a significant decrease of myelo-monocytic (CFU-GM) and monocytic (CFU-M) progenitors, and a concomitant increase of granulocytic precursors (CFU-G). In addition, morphological analysis of Otx1−/− bone marrow cells confirmed a high percentage of cells of the granulocytic lineage. Furthermore, at the molecular level, we detected in null mutant cells the upregulation of the gene encoding the G-CSF receptor and the modulation of other key myeloid regulators. Finally, to understand whether the action of Otx1 in myeloid cells is, like in erythoid cells, mediated by Scl we tested whether expression of a Scl transgene in Otx1−/− mice can re-establish normal ratios between the myeloid subpopulations. Results showed that, in contrast to the findings within the erythroid compartment, constitutive Scl expression does not fully rescue the alterations within the myelo-monocytic lineage. Taken together our data indicate that the brain morphogenetic gene Otx1 is an important regulator of the blood system, as it plays a relevant role in erythropoiesis, and contributes to control also myelo-monocytic differentiation, likely at the level of the commitment events of bipotential progenitor cells.

X-Linked Lissencephaly With Abnormal Genitalia as a Tangential Migration Disorder Causing Intractable Epilepsy: Proposal for a New Term, “Interneuronopathy”

X-linked lissencephaly with abnormal genitalia is the first human disorder in which deficient tangential migration in the brain has been demonstrated. Male patients with X-linked lissencephaly with abnormal genitalia show intractable seizures, especially clonic convulsions or myoclonus from the first day of life, but neither infantile spasms nor hypsarrhythmia on electroencephalograms so far. Brain magnetic resonance imaging shows anterior pachygyria and posterior agyria with a mildly thick cortex, agenesis of the corpus callosum, and dysplastic basal ganglia. ARX, a paired-class homeobox gene with four polyalanine sequences, is a responsible gene for X-linked lissencephaly with abnormal genitalia. The brain of Arx knockout mice shows aberrant tangential migration and differentiation of γ-aminobutyric acid (GABA)ergic interneurons. In human X-linked lissencephaly with abnormal genitalia, a neuropathologic study has suggested a loss of interneurons. Meanwhile, polyalanine expansion of ARX causes symptomatic or nonsymptomatic West's syndrome and nonsyndromic mental retardation. The striking epileptogenicity of X-linked lissencephaly with abnormal genitalia and West's syndrome associated with ARX mutations is considered to be caused by a disorder of interneurons involving a tangential migration disorder. We propose “interneuronopathy” as a term for this. ( J Child Neurol 2005;20:392—397).

The zebrafish bonnie and clyde gene encodes a Mix family homeodomain protein that regulates the generation of endodermal precursors

Vertebrate endoderm development has recently become the focus of intense investigation. In this report, we first show that the zebrafishbonnie and clyde (bon) gene plays a critical early role in endoderm formation. bon mutants exhibit a profound reduction in the number of sox17-expressing endodermal precursors formed during gastrulation, and, consequently, a profound reduction in gut tissue at later stages. The endodermal precursors that do form inbon mutants, however, appear to differentiate normally indicating that bon is not required at later steps of endoderm development. We further demonstrate that bon encodes a paired-class homeodomain protein of the Mix family that is expressed transiently before and during early gastrulation in both mesodermal and endodermal progenitors. Overexpression of bon can rescue endodermal gene expression and the formation of a gut tube inbon mutants. Analysis of a newly identified mutant allele reveals that a single amino acid substitution in the DNA recognition helix of the homeodomain creates a dominant interfering form of Bon when overexpressed. We also show through loss- and gain-of-function analyses that Bon functions exclusively downstream of cyclopsand squint signaling. Together, our data demonstrate that Bon is a critical transcriptional regulator of early endoderm formation.