bozozok and squint act in parallel to specify dorsal mesoderm and anterior neuroectoderm in zebrafish

In vertebrate embryos, maternal (beta)-catenin protein activates the expression of zygotic genes that establish the dorsal axial structures. Among the zygotically acting genes with key roles in the specification of dorsal axial structures are the homeobox gene bozozok (boz) and the nodal-related (TGF-(beta) family) gene squint (sqt). Both genes are expressed in the dorsal yolk syncytial layer, a source of dorsal mesoderm inducing signals, and mutational analysis has indicated that boz and sqt are required for dorsal mesoderm development. Here we examine the regulatory interactions among boz, sqt and a second nodal-related gene, cyclops (cyc). Three lines of evidence indicate that boz and sqt act in parallel to specify dorsal mesoderm and anterior neuroectoderm. First, boz requires sqt function to induce high levels of ectopic dorsal mesoderm, consistent with sqt acting either downstream or in parallel to boz. Second, sqt mRNA is expressed in blastula stage boz mutants, indicating that boz is not essential for activation of sqt transcription, and conversely, boz mRNA is expressed in blastula stage sqt mutants. Third, boz;sqt double mutants have a much more severe phenotype than boz and sqt single mutants. Double mutants consistently lack the anterior neural tube and axial mesoderm, and ventral fates are markedly expanded. Expression of chordin and noggin1 is greatly reduced in boz;sqt mutants, indicating that the boz and sqt pathways have overlapping roles in activating secreted BMP antagonists. In striking contrast to boz;sqt double mutants, anterior neural fates are specified in boz;sqt;cyc triple mutants. This indicates that cyc represses anterior neural development, and that boz and sqt counteract this repressive function. Our results support a model in which boz and sqt act in parallel to induce dorsalizing BMP-antagonists and to counteract the repressive function of cyc in neural patterning.

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Screening of the duplication 24 pb of ARX gene in Moroccan patients with X-linked Intellectual Disability

BMC Research Notes ◽

10.1186/s13104-021-05526-7 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yousra Benmakhlouf ◽

Renaud Touraine ◽

Ines Harzallah ◽

Zeineb Zian ◽

Kaoutar Ben Makhlouf ◽

...

Keyword(s):

Intellectual Disability ◽

Sample Size ◽

Mutational Analysis ◽

Homeobox Gene ◽

Recurrent Mutation ◽

Neuropsychiatric Disorder ◽

Good Tool ◽

Genetic Studies ◽

Exon 2 ◽

Moroccan Patients

Abstract Objective Intellectual Disability (ID) represents a neuropsychiatric disorder, which its etiopathogenesis remains insufficiently understood. Mutations in the Aristaless Related Homeobox gene (ARX) have been identified to cause syndromic and nonsyndromic (NS-ID). The most recurrent mutation of this gene is a duplication of 24pb, c.428-451dup. Epidemiological and genetic studies about ID in the Moroccan population remain very scarce, and none study is carried out on the ARX gene. This work aimed to study c.428–451dup (24 bp) mutation in the exon 2 of the ARX gene in 118 males’ Moroccan patients with milder NS-ID to evaluate if the gene screening is a good tool for identifying NS-ID. Results Our mutational analysis did not show any dup(24pb) in our patients. This is because based on findings from previous studies that found ARX mutations in 70% of families with NS-ID, and in most cases, 1.5–6.1% of individuals with NS-ID have this duplication. Since 1/118 = 0.0084 (0.84%) is not much different from 1.5%, then it is reasonable that this could a sample size artifact. A complete screening of the entire ARX gene, including the five exons, should be fulfilled. Further investigations are required to confirm these results.

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EXPRESSION STUDIES AND MUTATIONAL ANALYSIS OF THE ANDROGEN REGULATED HOMEOBOX GENE NKX3.1 IN BENIGN AND MALIGNANT PROSTATE EPITHELIUM

The Journal of Urology ◽

10.1016/s0022-5347(01)69894-x ◽

2001 ◽

Vol 165 (4) ◽

pp. 1329-1334 ◽

Cited By ~ 56

Author(s):

DAVID K. ORNSTEIN ◽

MARIO CINQUANTA ◽

SOLLY WEILER ◽

PAUL H. DURAY ◽

MICHAEL R. EMMERT-BUCK ◽

...

Keyword(s):

Mutational Analysis ◽

Homeobox Gene ◽

Expression Studies ◽

Prostate Epithelium ◽

Malignant Prostate

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Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants

Development ◽

10.1242/dev.119.2.419 ◽

1993 ◽

Vol 119 (2) ◽

pp. 419-431 ◽

Cited By ~ 87

Author(s):

T.J. Lints ◽

L.M. Parsons ◽

L. Hartley ◽

I. Lyons ◽

R.P. Harvey

Keyword(s):

Progenitor Cells ◽

Myogenic Differentiation ◽

Homeobox Genes ◽

Homeobox Gene ◽

Heart Tube ◽

New Members ◽

Mesoderm Development ◽

Early Entry ◽

Developing Heart ◽

Valuable Marker

We have isolated two murine homeobox genes, Nkx-2.5 and Nkx-2.6, that are new members of a sp sub-family of homeobox genes related to Drosophila NK2, NK3 and NK4/msh-2. In this paper, we focus on the Nkx-2.5 gene and its expression pattern during post-implantation development. Nkx-2.5 transcripts are first detected at early headfold stages in myocardiogenic progenitor cells. Expression preceeds the onset of myogenic differentiation, and continues in cardiomyocytes of embryonic, foetal and adult hearts. Transcripts are also detected in future pharyngeal endoderm, the tissue believed to produce the heart inducer. Expression in endoderm is only found laterally, where it is in direct apposition to promyocardium, suggesting an interaction between the two tissues. After foregut closure, Nkx-2.5 expression in endoderm is limited to the pharyngeal floor, dorsal to the developing heart tube. The thyroid primordium, a derivative of the pharyngeal floor, continues to express Nkx-2.5 after transcript levels diminish in the rest of the pharynx. Nkx-2.5 transcripts are also detected in lingual muscle, spleen and stomach. The expression data implicate Nkx-2.5 in commitment to and/or differentiation of the myocardial lineage. The data further demonstrate that cardiogenic progenitors can be distinguished at a molecular level by late gastrulation. Nkx-2.5 expression will therefore be a valuable marker in the analysis of mesoderm development and an early entry point for dissection of the molecular basis of myogenesis in the heart.

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Regulation of the twist target gene tinman by modular cis-regulatory elements during early mesoderm development

Development ◽

10.1242/dev.124.24.4971 ◽

1997 ◽

Vol 124 (24) ◽

pp. 4971-4982 ◽

Cited By ~ 12

Author(s):

Z. Yin ◽

X.L. Xu ◽

M. Frasch

Keyword(s):

Homeobox Gene ◽

Regulatory Elements ◽

Bhlh Protein ◽

Expression Domain ◽

Enhancer Activity ◽

Enhancer Element ◽

Mesoderm Development ◽

Visceral Mesoderm

The Drosophila tinman homeobox gene has a major role in early mesoderm patterning and determines the formation of visceral mesoderm, heart progenitors, specific somatic muscle precursors and glia-like mesodermal cells. These functions of tinman are reflected in its dynamic pattern of expression, which is characterized by initial widespread expression in the trunk mesoderm, then refinement to a broad dorsal mesodermal domain, and finally restricted expression in heart progenitors. Here we show that each of these phases of expression is driven by a discrete enhancer element, the first being active in the early mesoderm, the second in the dorsal mesoderm and the third in cardioblasts. We provide evidence that the early-active enhancer element is a direct target of twist, a gene encoding a basic helix-loop-helix (bHLH) protein, which is necessary for tinman activation. This 180 bp enhancer includes three E-box sequences which bind Twist protein in vitro and are essential for enhancer activity in vivo. Ectodermal misexpression of twist causes ectopic activation of this enhancer in ectodermal cells, indicating that twist is the only mesoderm-specific activator of early tinman expression. We further show that the 180 bp enhancer also includes negatively acting sequences. Binding of Even-skipped to these sequences appears to reduce twist-dependent activation in a periodic fashion, thus producing a striped tinman pattern in the early mesoderm. In addition, these sequences prevent activation of tinman by twist in a defined portion of the head mesoderm that gives rise to hemocytes. We find that this repression requires the function of buttonhead, a head-patterning gene, and that buttonhead is necessary for normal activation of the hematopoietic differentiation gene serpent in the same area. Together, our results show that tinman is controlled by an array of discrete enhancer elements that are activated successively by differential genetic inputs, as well as by closely linked activator and repressor binding sites within an early-acting enhancer, which restrict twist activity to specific areas within the twist expression domain.

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Acceleration of mesoderm development and expansion of hematopoietic progenitors in differentiating ES cells by the mouse Mix-like homeodomain transcription factor

Blood ◽

10.1182/blood-2005-10-4120 ◽

2006 ◽

Vol 107 (8) ◽

pp. 3122-3130 ◽

Cited By ~ 29

Author(s):

Stephen Willey ◽

Angel Ayuso-Sacido ◽

Hailan Zhang ◽

Stuart T. Fraser ◽

Kenneth E. Sahr ◽

...

Keyword(s):

Es Cells ◽

Critical Role ◽

Embryonic Stem ◽

Homeobox Gene ◽

Embryoid Bodies ◽

Hematopoietic Progenitors ◽

Adult Type ◽

Mesoderm Development ◽

Molecular Events ◽

Organ Systems

Abstract The cellular and molecular events underlying the formation and differentiation of mesoderm to derivatives such as blood are critical to our understanding of the development and function of many tissues and organ systems. How different mesodermal populations are set aside to form specific lineages is not well understood. Although previous genetic studies in the mouse embryo have pointed to a critical role for the homeobox gene Mix-like (mMix) in gastrulation, its function in mesoderm development remains unclear. Hematopoietic defects have been identified in differentiating embryonic stem cells in which mMix was genetically inactivated. Here we show that conditional induction of mMix in embryonic stem cell–derived embryoid bodies results in the early activation of mesodermal markers prior to expression of Brachyury/T and acceleration of the mesodermal developmental program. Strikingly, increased numbers of mesodermal, hemangioblastic, and hematopoietic progenitors form in response to premature activation of mMix. Differentiation to primitive (embryonic) and definitive (adult type) blood cells proceeds normally and without an apparent bias in the representation of different hematopoietic cell fates. Therefore, the mouse Mix gene functions early in the recruitment and/or expansion of mesodermal progenitors to the hemangioblastic and hematopoietic lineages.

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Antimorphic goosecoids

Development ◽

10.1242/dev.125.8.1347 ◽

1998 ◽

Vol 125 (8) ◽

pp. 1347-1359 ◽

Cited By ~ 7

Author(s):

B. Ferreiro ◽

M. Artinger ◽

K. Cho ◽

C. Niehrs

Keyword(s):

Transcriptional Activation ◽

Low Dose ◽

Marginal Zone ◽

Homeobox Gene ◽

Axis Formation ◽

Wild Type ◽

Transcriptional Activation Domain ◽

Spemann Organizer ◽

Axial Defects ◽

Dorsal Mesoderm

goosecoid (gsc) is a homeobox gene expressed in the Spemann organizer that has been implicated in vertebrate axis formation. Here antimorphic gscs are described. One antimorphic gsc (MTgsc) was fortuitously created by adding 5 myc epitopes to the N terminus of gsc. The other antimorph (VP16gsc) contains the transcriptional activation domain of VP16. mRNA injection of either antimorph inhibits dorsal gastrulation movements and leads to embryos with severe axial defects. They upregulate ventral gene expression in the dorsal marginal zone and inhibit dorsal mesoderm differentiation. Like the VP16 domain, the N-terminal myc tags act by converting wild-type gsc from a transcriptional repressor into an activator. However, unlike MTgsc, VP16gsc is able at low dose to uncouple head from trunk formation, indicating that different antimorphs may elicit distinct phenotypes. The experiments reveal that gsc and/or gsc-related genes function in axis formation and gastrulation. Moreover, this work warns against using myc tags indiscriminately for labeling DNA-binding proteins.

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Mutational analysis of mouse Wnt-1 identifies two temperature-sensitive alleles and attributes of Wnt-1 protein essential for transformation of a mammary cell line.

Molecular Biology of the Cell ◽

10.1091/mbc.3.5.521 ◽

1992 ◽

Vol 3 (5) ◽

pp. 521-533 ◽

Cited By ~ 60

Author(s):

J O Mason ◽

J Kitajewski ◽

H E Varmus

Keyword(s):

Cell Line ◽

Neural Development ◽

Mutational Analysis ◽

Proteolytic Processing ◽

Site Directed Mutagenesis ◽

Signal Peptidase ◽

Epithelial Cell Line ◽

Temperature Sensitive ◽

Glycosylation Sites ◽

Transforming Activity

The proto-oncogene Wnt-1 encodes a cysteine-rich, secretory glycoprotein implicated in virus-induced mouse mammary cancer and intercellular signaling during vertebrate neural development. To attempt to correlate structural motifs of Wnt-1 protein with its function, 12 mutations were introduced singly and in several combinations into the coding sequence of Wnt-1 cDNA by site-directed mutagenesis. Mutant alleles in a retroviral vector were tested for their ability to transform the mouse mammary epithelial cell line C57MG in two ways: by direct infection of C57MG cells and by infection of NIH3T3 cells that serve as donors of Wnt-1 protein to adjacent C57MG cells in a secretion-dependent (paracrine) assay. In addition, the synthesis and secretion of mutant proteins were monitored in multiple cell types by immunological assays. Deletion of the signal peptide demonstrated that transformation in both direct and paracrine assays depends upon entry of Wnt-1 protein into the endoplasmic reticulum. Changes in potential proteolytic processing sites (two basic dipeptides and a probable signal peptidase cleavage site) did not adversely impair biological activity or protein processing and uncovered a second site for cleavage by signal peptidase. Replacement of each of the four asparagine-linked glycosylation sites did not affect transforming activity at normal temperatures, but one glycosylation site mutant was found to be temperature-sensitive for transformation. An allele encoding a protein that lacks all four glycosylation sites was also transformation competent. In two of four cases, substitution of serine for a cysteine residue impaired transforming activity at the usual temperature, and transformation was temperature sensitive in a third case, implying that at least some of the highly conserved cysteine residues are important for Wnt-1 function.

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Endodermal Nodal-related signals and mesoderm induction in Xenopus

Development ◽

10.1242/dev.127.6.1173 ◽

2000 ◽

Vol 127 (6) ◽

pp. 1173-1183 ◽

Cited By ~ 21

Author(s):

E. Agius ◽

M. Oelgeschlager ◽

O. Wessely ◽

C. Kemp ◽

E.M. De Robertis

Keyword(s):

Dose Response ◽

Mesoderm Induction ◽

Beta Catenin ◽

Blastula Stage ◽

Modified Model ◽

Terminal Fragment ◽

Endogenous Activity ◽

Carboxy Terminal ◽

Endodermal Cells ◽

Molecular Nature

In Xenopus, mesoderm induction by endoderm at the blastula stage is well documented, but the molecular nature of the endogenous inductive signals remains unknown. The carboxy-terminal fragment of Cerberus, designated Cer-S, provides a specific secreted antagonist of mesoderm-inducing Xenopus Nodal-Related (Xnr) factors. Cer-S does not inhibit signalling by other mesoderm inducers such as Activin, Derriere, Vg1 and BMP4, nor by the neural inducer Xnr3. In the present study we show that Cer-S blocks the induction of both dorsal and ventral mesoderm in animal-vegetal Nieuwkoop-type recombinants. During blastula stages Xnr1, Xnr2 and Xnr4 are expressed in a dorsal to ventral gradient in endodermal cells. Dose-response experiments using cer-S mRNA injections support the existence of an endogenous activity gradient of Xnrs. Xnr expression at blastula can be activated by the vegetal determinants VegT and Vg1 acting in synergy with dorsal (beta)-catenin. The data support a modified model for mesoderm induction in Xenopus, in which mesoderm induction is mediated by a gradient of multiple Nodal-related signals released by endoderm at the blastula stage.

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The homeobox gene bozozok promotes anterior neuroectoderm formation in zebrafish through negative regulation of BMP2/4 and Wnt pathways

Development ◽

10.1242/dev.127.11.2333 ◽

2000 ◽

Vol 127 (11) ◽

pp. 2333-2345 ◽

Cited By ~ 2

Author(s):

K. Fekany-Lee ◽

E. Gonzalez ◽

V. Miller-Bertoglio ◽

L. Solnica-Krezel

Keyword(s):

Wnt Signaling ◽

Homeobox Gene ◽

Neural Induction ◽

Negative Regulation ◽

Neural Patterning ◽

Step Process ◽

Head Formation ◽

Simultaneous Inhibition ◽

Wnt Pathways

The neuroectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be regionalized into forebrain, midbrain, hindbrain and spinal cord by a two-step process. In the activation step, the Spemann gastrula organizer induces neuroectoderm with anterior character, followed by posteriorization by a transforming signal. Recently, simultaneous inhibition of BMP and Wnt signaling was shown to induce head formation in frog embryos. However, how the inhibition of BMP and Wnt signaling pathways specify a properly patterned head, and how they are regulated in vivo, is not understood. Here we demonstrate that the loss of anterior neural fates observed in zebrafish bozozok (boz) mutants occurs during gastrulation due to a reduction and subsequent posteriorization of neuroectoderm. The neural induction defect was correlated with decreased chordino expression and consequent increases in bmp2b/4 expression, and was suppressed by overexpression of BMP antagonists. Whereas expression of anterior neural markers was restored by ectopic BMP inhibition in early boz gastrulae, it was not maintained during later gastrulation. The posteriorization of neuroectoderm in boz was correlated with ectopic dorsal wnt8 expression. Overexpression of a Wnt antagonist rescued formation of the organizer and anterior neural fates in boz mutants. We propose that boz specifies formation of anterior neuroectoderm by regulating BMP and Wnt pathways in a fashion consistent with Nieuwkoop's two-step neural patterning model. boz promotes neural induction by positively regulating organizer-derived chordino and limiting the antineuralizing activity of BMP2b/4 morphogens. In addition, by negative regulation of Wnt signaling, boz promotes organizer formation and limits posteriorization of neuroectoderm in the late gastrula.

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The armadillo repeat region targets ARVCF to cadherin-based cellular junctions

Journal of Cell Science ◽

10.1242/jcs.113.22.4121 ◽

2000 ◽

Vol 113 (22) ◽

pp. 4121-4135 ◽

Cited By ~ 3

Author(s):

U. Kaufmann ◽

C. Zuppinger ◽

Z. Waibler ◽

M. Rudiger ◽

C. Urbich ◽

...

Keyword(s):

Mammalian Cells ◽

Mutational Analysis ◽

Transmembrane Protein ◽

Cytoplasmic Domain ◽

Cell Contact ◽

Beta Catenin ◽

Repeat Region ◽

Mcf7 Cells ◽

Armadillo Repeat

The cytoplasmic domain of the transmembrane protein M-cadherin is involved in anchoring cytoskeletal elements to the plasma membrane at cell-cell contact sites. Several members of the armadillo repeat protein family mediate this linkage. We show here that ARVCF, a member of the p120 (ctn) subfamily, is a ligand for the cytoplasmic domain of M-cadherin, and characterize the regions involved in this interaction in detail. Complex formation in an in vivo environment was demonstrated in (1) yeast two-hybrid screens, using a cDNA library from differentiating skeletal muscle and part of the cytoplasmic M-cadherin tail as a bait, and (2) mammalian cells, using a novel experimental system, the MOM recruitment assay. Immunoprecipitation and in vitro binding assays confirmed this interaction. Ectopically expressed EGFP-ARVCF-C11, an N-terminal truncated fragment, targets to junctional structures in epithelial MCF7 cells and cardiomyocytes, where it colocalizes with the respective cadherins, beta-catenin and p120 (ctn). Hence, the N terminus of ARVCF is not required for junctional localization. In contrast, deletion of the four N-terminal armadillo repeats abolishes this ability in cardiomyocytes. Detailed mutational analysis revealed the armadillo repeat region of ARVCF as sufficient and necessary for interaction with the 55 membrane-proximal amino acids of the M-cadherin tail.

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