Biomedical subjects

Danny Huylebroeck

Publications and source records attributed to Danny Huylebroeck.

At least 19 recordsLinked to original sources

PubMed2006

Sesn1 is a novel gene for left-right asymmetry and mediating nodal signaling.

Remarkable progress has been made in understanding the molecular mechanisms underlying left-right asymmetry in vertebrate animal models but little is known on left-right axis formation in humans. Previously, we identified SESN1 (also known as PA26) as a candidate gene for heterotaxia by positional cloning of the breakpoint regions of a de novo translocation in a heterotaxia patient. In this study, we show by means of a zebrafish sesn1-knockdown model that Sesn1 is required for normal embryonic left-right determination. In this model, developmental defects and expression data of genes implicated in vertebrate left-right asymmetry indicate a role for Sesn1 in mediating Nodal signaling. In the lateral plate mesoderm, Nodal signaling plays a central role in left-right axis formation in vertebrates and is mediated by FoxH1 transcriptional induction. In line with this, we show that Sesn1 physically interacts with FoxH1 or a FoxH1-containing complex. Mutation analysis in a panel of 234 patients with isolated heterotaxia did not reveal mutations, indicating that these are only exceptional causes of human heterotaxia. In this study, we identify SESN1 as an indispensable gene for vertebrate left-right asymmetry and a new player in mediating Nodal signaling.

Animals↗

PubMed2006

Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels.

Smad5 is an intracellular mediator of bone morphogenetic protein (Bmp) signalling. It is essential for primordial germ cell (PGC) development, for the development of the allantois and for amnion closure, as demonstrated by loss of Bmp signalling. By contrast, the appearance of ectopic PGC-like cells and regionalized ectopic vasculogenesis and haematopoiesis in thickened Smad5(m1/m1) amnion are amnion defects that have not been associated with loss of Bmp signalling components. We show that defects in amnion and allantois can already be detected at embryonic day (E) 7.5 in Smad5 mutant mice. However, ectopic Oct4-positive (Oct4(+)) and alkaline phosphatase-positive (AP(+)) cells appear suddenly in thickened amnion at E8.5, and at a remote distance from the allantois and posterior primitive streak, suggesting a change of fate in situ. These ectopic Oct4(+), AP(+) cells appear to be Stella negative and hence cannot be called bona fide PGCs. We demonstrate a robust upregulation of Bmp2 and Bmp4 expression, as well as of Erk and Smad activity, in the Smad5 mutant amnion. The ectopic expression of several Bmp target genes in different domains and the regionalized presence of cells of several Bmp-sensitive lineages in the mutant amnion suggest that different levels of Bmp signalling may determine cell fate. Injection of rBMP4 in the exocoelom of wild-type embryos can induce thickening of amnion, mimicking the early amnion phenotype in Smad5 mutants. These results support a model in which loss of Smad5 results paradoxically in gain of Bmp function defects in the amnion.

Alleles↗

PubMed2006

deltaEF1 and SIP1 are differentially expressed and have overlapping activities during Xenopus embryogenesis.

The zinc finger/homeo-domain transcription factor (zfh x 1) family in vertebrates consists of two members, deltaEF1 and SIP1. They have been proposed to display antagonistic activities in the interpretation of Smad-dependent TGFbeta signaling during mesoderm formation. We cloned Xenopus deltaEF1 cDNA, analyzed the expression profile of the gene, and compared the inducing and interacting properties of the protein to that of XSIP1. Whereas XSIP1 RNA is selectively expressed in the early developing nervous system, we show that XdeltaEF1 gene transcription is only activated during neurulation and that its expression is restricted to the paraxial mesoderm. From early tail bud stage, XdeltaEF1 and XSIP1 are coexpressed in migratory cranial neural crest, in the retina, and in the neural tube. Overproduction of XdeltaEF1 in RNA-injected embryos, like that of XSIP1, reduced the expression of BMP-dependent genes but only XSIP1 has the ability to induce neural markers. We find that XdeltaEF1 and XSIP1 can both form complexes, although with different efficiency, with Smad3, with the coactivators p300 and pCAF, and with the corepressor CtBP1. Together, these results indicate that deltaEF1 and SIP1 do not function as antagonists during Xenopus early embryogenesis but do display different repression efficiencies and interaction properties.

Amino Acid Sequence↗

PubMed2006

Complementary expression pattern of Zfhx1 genes Sip1 and deltaEF1 in the mouse embryo and their genetic interaction revealed by compound mutants.

In mouse embryos, the Zfhx1 transcription factor genes, Sip1 and deltaEF1, are expressed in complementary domains in many tissues. Their possible synergism in embryogenesis was investigated by comparing the phenotype of Sip1-/-;deltaEF1-/- double homozygotes with single homozygous embryos. Unexpectedly, in Sip1-/- embryos deltaEF1 was ectopically activated, suggesting a negative regulation of deltaEF1 expression by Sip1. Sip1-/-;deltaEF1-/- embryos were similar to Sip1-/- embryos in short somite production and developmental arrest around E8.5, but showed more severe defects in dorsal neural tube morphogenesis accompanied by a larger reduction of Sox2 expression, ascribable to the loss of the ectopic deltaEF1 expression. Sip1+/-;deltaEF1-/- embryos develop various morphological defects after E10 that were absent in deltaEF1-/- embryos even in tissues without significant overlap of Sip1 and deltaEF1 expression, and arrested during mid gestation earlier than deltaEF1-/- embryos. These findings indicate that complex synergistic interactions occur between Zfhx1 transcription factor genes during mouse embryogenesis.

Animals↗

PubMed2005

Synaptopodin and 4 novel genes identified in primary sensory neurons.

We performed differential gene expression profiling in the peripheral nervous system by comparing the transcriptome of sensory neurons with the transcriptome of lower motor neurons. Using suppression subtractive cDNA hybridization, we identified 5 anonymous transcripts with a predominant expression in sensory neurons. We determined the gene structures and predicted the functional protein domains. The 4930579P15Rik gene encodes for a novel inhibitor of protein phosphatase-1 and 9030217H17Rik was found to be the mouse gene synaptopodin. We performed in situ hybridization for all genes in mouse embryos, and found expression predominantly in the primary class of sensory neurons. Expression of 4930579P15Rik and synaptopodin was restricted to craniospinal sensory ganglia. Neither synaptopodin, nor any known family member of 4930579P15Rik, has ever been described in sensory neurons. The identification of protein domains and expression patterns allows further functional analysis of these novel genes in relation to the development and biology of sensory neurons.

Animals↗

PubMed2005

Alk3/Bmpr1a receptor is required for development of the atrioventricular canal into valves and annulus fibrosus.

Endocardial cushions are precursors of mature atrioventricular (AV) valves. Their formation is induced by signaling molecules originating from the AV myocardium, including bone morphogenetic proteins (BMPs). Here, we hypothesized that BMP signaling plays an important role in the AV myocardium during the maturation of AV valves from the cushions. To test our hypothesis, we used a unique Cre/lox system to target the deletion of a floxed Alk3 allele, the type IA receptor for BMPs, to cardiac myocytes of the AV canal (AVC). Lineage analysis indicated that cardiac myocytes of the AVC contributed to the tricuspid mural and posterior leaflets, the mitral septal leaflet, and the atrial border of the annulus fibrosus. When Alk3 was deleted in these cells, defects were seen in the same leaflets, ie, the tricuspid mural leaflet and mitral septal leaflet were longer, the tricuspid posterior leaflet was displaced and adherent to the ventricular wall, and the annulus fibrosus was disrupted resulting in ventricular preexcitation. The defects seen in mice with AVC-targeted deletion of Alk3 provide strong support for a role of Alk3 in human congenital heart diseases, such as Ebstein's anomaly. In conclusion, our mouse model demonstrated critical roles for Alk3 signaling in the AV myocardium during the development of AV valves and the annulus fibrosus.

Animals↗

PubMed2005

Transforming growth factor-beta-activated kinase-1 (TAK1), a MAP3K, interacts with Smad proteins and interferes with osteogenesis in murine mesenchymal progenitors.

TAK1 (transforming growth factor-beta-activated kinase-1), a MAP3K with considerable sequence similarity to Raf-1 and MEKK-1, has been identified as a transforming growth factor-beta/bone morphogenetic protein (BMP)-activated cytosolic component of the MAPK pathways. In this investigation, the molecular interactions between TAK1 and Smad proteins were characterized as well as their influence on BMP-mediated mesenchymal cell differentiation along the osteogenic/chondrogenic pathway. In co-immunoprecipitations we found an interaction of TAK1 with all Smads tested, R-Smads Smads1-5, the co-Smad Smad4, and the inhibitory Smads (I-Smad6 and I-Smad7). Smad interaction with TAK1 takes place through their MH2 domain. This interaction is dependent on the presence of an active kinase domain in TAK1. TAK1 dramatically interferes with R-Smad transactivation in reporter assays and affects subcellular distribution of Smad proteins. Activated TAK1 also interferes with BMP-dependent osteogenic development in murine mesenchymal progenitor cells (C3H10T 1/2). A potential TAK1-mediated apoptosis process could be excluded for these cells. Both synergistic and interfering influences of TAK1 on BMP-mediated Smad-signaling have been reported previously. We suggest that TAK1 is a factor that is involved in the fine-tuning of BMP effects during osteogenic development.

Active Transport, Cell Nucleus↗

PubMed2005

Involvement of SIP1 in positioning of somite boundaries in the mouse embryo.

Periodical production of somites provides an excellent model system for understanding genesis of metameric structures underlying embryonic development. This study reports production of somites with roughly half rostro-caudal length in homozygous Sip1 (Smad-interacting protein 1) knockout mouse embryos. This altered periodicity of somitogenesis is caused by the rostral expansion of the expression domain of genes involved in the maintenance of unsegmented state of paraxial mesoderm, e.g., Fgf8, Wnt3a, Dll3, and Tbx6. This is accompanied by the rostral extension of oscillatory gene expression such as L-fng, Hes7, and Dll1, and the rostrally shifted termination of Raldh2 expression that continues from the anterior embryonic side. The phenotype of Sip1-/- embryo introduces a new molecular component SIP1 in positioning of somite boundaries, and provides support for the current "clock and wavefront" model.

Aldehyde Oxidoreductases↗

PubMed2005

Smicl is a novel Smad interacting protein and cleavage and polyadenylation specificity factor associated protein.

Ligand-bound receptors of the Transforming Growth Factor-beta (TGF-beta) family promote the formation of complexes between Smad proteins that subsequently accumulate in the nucleus and interact there with other transcriptional regulators, leading to modulation of target gene expression. We identified a novel nuclear protein, Smicl, which binds to Smad proteins. Smicl and Smads cooperate and enhance TGF-beta mediated activation of a Smad-responsive reporter gene. A domain with five CCCH-type zinc fingers in Smicl is structurally and functionally, at least in vitro, similar to a domain in CPSF-30, the 30 kDa subunit of Cleavage and Polyadenylation Specificity Factor (CPSF). Like CPSF-30, Smicl can associate with some other CPSF subunits characterized previously. Its effect on the induction of a reporter gene for TGF-beta requires the cleavage/polyadenylation signal downstream of the coding sequence of that gene. Thus, Smicl is a novel protein that displays CPSF-30-like activities, interacts in the nucleus with activated Smads, and potentiates in TGF-beta stimulated cells Smad-dependent transcriptional responses, possibly in conjunction with the activity of CPSF complexes.

Activin Receptors, Type I↗

PubMed2005

The novel Smad-interacting protein Smicl regulates Chordin expression in the Xenopus embryo.

In this paper, we investigate the function of Smicl, a zinc-finger Smad-interacting protein that is expressed maternally in the Xenopus embryo. Inhibition of Smicl function by means of antisense morpholino oligonucleotides causes the specific downregulation of Chordin, a dorsally expressed gene encoding a secreted BMP inhibitor that is involved in mesodermal patterning and neural induction. Chordin is activated by Nodal-related signalling in an indirect manner, and we show here that Smicl is involved in a two-step process that is necessary for this activation. In the first, Smad3 (but not Smad2) activates expression of Xlim1 in a direct fashion. In the second, a complex containing Smicl and the newly induced Xlim1 induces expression of Chordin. As well as revealing the function of Smicl in the early embryo, our work yields important new insight in the regulation of Chordin and identifies functional differences between the activities of Smad2 and Smad3 in the Xenopus embryo.

Amino Acid Sequence↗

PubMed2004

Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis.

Osteopoikilosis, Buschke-Ollendorff syndrome (BOS) and melorheostosis are disorders characterized by increased bone density. The occurrence of one or more of these phenotypes in the same individual or family suggests that these entities might be allelic. We collected data from three families in which affected individuals had osteopoikilosis with or without manifestations of BOS or melorheostosis. A genome-wide linkage analysis in these families, followed by the identification of a microdeletion in an unrelated individual with these diseases, allowed us to map the gene that is mutated in osteopoikilosis. All the affected individuals that we investigated were heterozygous with respect to a loss-of-function mutation in LEMD3 (also called MAN1), which encodes an inner nuclear membrane protein. A somatic mutation in the second allele of LEMD3 could not be identified in fibroblasts from affected skin of an individual with BOS and an individual with melorheostosis. XMAN1, the Xenopus laevis ortholog, antagonizes BMP signaling during embryogenesis. In this study, LEMD3 interacted with BMP and activin-TGFbeta receptor-activated Smads and antagonized both signaling pathways in human cells.

Base Sequence↗

PubMed2004

Direct regulation of the Nrarp gene promoter by the Notch signaling pathway.

Nrarp encodes for an evolutionarily conserved small ankyrin repeat-containing protein that functions as a negative regulator of Notch signaling. Interestingly, increased Nrarp transcription was observed following induction of Notch signaling, suggesting the existence of a negative feedback loop. We show here that both mouse and human promoter regions of Nrarp share two conserved regions located approximately 2 and approximately 3 kb upstream of the transcription start site each containing a perfect putative binding site for the Notch-dependent transcription factor Su(H). A 4.4 kb genomic fragment of the mouse Nrarp locus containing those conserved regions and fused to a luciferase reporter gene showed basal promoter activity in 293T cells and this activity was strongly increased by the intracellular domain of Notch, NICD. NICD-dependent stimulation was attenuated by a dominant negative mutant of Su(H), Su(H)DBM, and was not observed in Su(H)-deficient cells (OT-11). Promoter bashing and gel shift assays revealed that the most distal putative Su(H) binding site located within the -3 kb conserved element plays a crucial role in this induction. Collectively, these results provide definitive support for direct regulation of the Nrarp gene by the Notch pathway.

Animals↗

PubMed2003

Slowed conduction and thin myelination of peripheral nerves associated with mutant rho Guanine-nucleotide exchange factor 10.

Slowed nerve-conduction velocities (NCVs) are a biological endophenotype in the majority of the hereditary motor and sensory neuropathies (HMSN). Here, we identified a family with autosomal dominant segregation of slowed NCVs without the clinical phenotype of HMSN. Peripheral-nerve biopsy showed predominantly thinly myelinated axons. We identified a locus at 8p23 and a Thr109Ile mutation in ARHGEF10, encoding a guanine-nucleotide exchange factor (GEF) for the Rho family of GTPase proteins (RhoGTPases). Rho GEFs are implicated in neural morphogenesis and connectivity and regulate the activity of small RhoGTPases by catalyzing the exchange of bound GDP by GTP. Expression analysis of ARHGEF10, by use of its mouse orthologue Gef10, showed that it is highly expressed in the peripheral nervous system. Our data support a role for ARHGEF10 in developmental myelination of peripheral nerves.

Animals↗

PubMed2003

Heteromeric MAPPIT: a novel strategy to study modification-dependent protein-protein interactions in mammalian cells.

We recently reported a two-hybrid trap for detecting protein-protein interactions in intact mammalian cells (MAPPIT). The bait protein was fused to a STAT recruitment-deficient, homodimeric cytokine receptor and the prey protein to functional STAT recruitment sites. In such a configuration, STAT-dependent responses can be used to monitor a given bait-prey interaction. Using this system, we were able to demonstrate both modification-independent and tyrosine phosphorylation- dependent interactions. Protein modification in this approach is, however, strictly dependent on the receptor-associated JAK tyrosine kinases. We have now extended this concept by using extracellular domains of the heteromeric granulocyte/macrophage colony-stimulating factor receptor (GM-CSFR). Herein, the bait was fused to the (beta)c chain and its modifying enzyme to the GM-CSFRalpha chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFbeta/Smad pathway using the catalytic domains of the ALK4 or ALK6 serine/threonine kinase receptors. In all cases tested, STAT-dependent signaling was completely abolished when mutant baits were used wherein critical serine residues were replaced by alanines. This approach operates both in transient and stable expression systems and may not be limited to serine phosphorylation but has the potential for studying various different types of protein modification-dependent interactions in intact cells.

Animals↗

PubMed2003

New intracellular components of bone morphogenetic protein/Smad signaling cascades.

Bone morphogenetic proteins (BMPs) regulate many processes in the embryo, including cell type specification, patterning, apoptosis, and epithelial-mesenchymal interaction. They also act in soft and hard tissues in adult life. Their signals are transduced from the plasma membrane to the nucleus through a limited number of Smad proteins. The list of Smad-interacting proteins is however growing and it is clear that these partners determine the outcome of the signal. We summarize the present status in BMP/Smad signaling, with emphasis on recently identified Smad partners and how these proteins may cooperate in the regulation of the expression of BMP target genes.

Animals↗

PubMed2003

Interaction between Smad-interacting protein-1 and the corepressor C-terminal binding protein is dispensable for transcriptional repression of E-cadherin.

deltaEF1 and SIP1 (or Zfhx1a and Zfhx1b, respectively) are the only known members of the vertebrate Zfh1 family of homeodomain/zinc finger-containing proteins. Similar to other transcription factors, both Smad-interacting protein-1 (SIP1) and deltaEF1 are capable of repressing E-cadherin transcription through binding to the E2 boxes located in its promoter. In the case of deltaEF1, this repression has been proposed to occur via interaction with the corepressor C-terminal binding protein (CtBP). In this study, we show by coimmunoprecipitation that SIP1 and CtBP interact in vivo and that an isolated CtBP-binding SIP1 fragment depends on CtBP for transcriptional repression. However, and most importantly, full-length SIP1 and deltaEF1 proteins do not depend on their interaction with CtBP to repress transcription from the E-cadherin promoter. Furthermore, in E-cadherin-positive kidney epithelial cells, the conditional synthesis of mutant SIP1 that cannot bind to CtBP abrogates endogenous E-cadherin expression in a similar way as wild-type SIP1. Our results indicate that full-length SIP1 can repress E-cadherin in a CtBP-independent manner.

Alcohol Oxidoreductases↗

PubMed2003

Alzheimer-associated C allele of the promoter polymorphism -22C>T causes a critical neuron-specific decrease of presenilin 1 expression.

We, amongst others, have shown that CC homozygosity at the -22C>T promoter polymorphism in presenilin 1 (PSEN1) is associated with increased risk for Alzheimer's disease (AD). Also, studies in AD brains suggested that CC homozygosity increased the risk for AD by increasing the Abeta load. We characterized the PSEN1 promoter by deletion mapping, and analysed the effect of the -22C and -22T alleles on the transcriptional activity of PSEN1 in a transient transfection system. We showed a neuron-specific 2-fold decrease in promoter activity for the -22C risk allele, which in homozygous individuals would lead to a critical decrease in PSEN1 expression. The deletion mapping suggested that the 13 bp region (-33/-20) spanning the -22C>T polymorphism harbours a binding site for a negative regulatory factor. This factor has a higher affinity for the -22C risk allele and is strongly dependent on downstream sequences for cell-type-specific expression differences. Together, these studies provide evidence that the increased risk for AD associated with PSEN1 may result from genetic variations in the regulatory region, leading to altered expression levels of PSEN1 in neurons.

Alleles↗

PubMed2003

Mice lacking ZFHX1B, the gene that codes for Smad-interacting protein-1, reveal a role for multiple neural crest cell defects in the etiology of Hirschsprung disease-mental retardation syndrome.

Recently, mutations in ZFHX1B, the gene that encodes Smad-interacting protein-1 (SIP1), were found to be implicated in the etiology of a dominant form of Hirschsprung disease-mental retardation syndrome in humans. To clarify the molecular mechanisms underlying the clinical features of SIP1 deficiency, we generated mice that bear a mutation comparable to those found in several human patients. Here, we show that Zfhx1b-knockout mice do not develop postotic vagal neural crest cells, the precursors of the enteric nervous system that is affected in patients with Hirschsprung disease, and they display a delamination arrest of cranial neural crest cells, which form the skeletomuscular elements of the vertebrate head. This suggests that Sip1 is essential for the development of vagal neural crest precursors and the migratory behavior of cranial neural crest in the mouse. Furthermore, we show that Sip1 is involved in the specification of neuroepithelium.

Animals↗