Overall and allele-specific expression of the SMC1A gene in female Cornelia de Lange syndrome patients and healthy controls

Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder characterized by facial dysmorphisms, limb anomalies, and growth and cognitive deficits. Mutations in genes encoding subunits (SMC1A, SMC3, RAD21) or regulators (NIPBL, HDAC8) of the cohesin complex account for approximately 65% of clinically diagnosed CdLS cases. The SMC1A gene (Xp11.22), responsible for 5% of CdLS cases, partially escapes X chromosome inactivation in humans and the allele on the inactive X chromosome is variably expressed. In this study, we evaluated overall and allele-specific SMC1A expression. Real-time PCR analysis conducted on 17 controls showed that SMC1A expression in females is 50% higher than in males. Immunoblotting experiments confirmed a 44% higher protein level in healthy females than in males, and showed no significant differences in SMC1A protein levels between controls and patients. Pyrosequencing was used to assess the reciprocal level of allelic expression in six female carriers of different SMC1A mutations and 15 controls who were heterozygous at a polymorphic transcribed SMC1A locus. The two alleles were expressed at a 1:1 ratio in the control group and at a 2:1 ratio in favor of the wild type allele in the test group. Since a dominant negative effect is considered the pathogenic mechanism in SMC1A-defective female patients, the level of allelic preferential expression might be one of the factors contributing to the wide phenotypic variability observed in these patients. An extension of this study to a larger cohort containing mild to borderline cases could enhance our understanding of the clinical spectrum of SMC1A-linked CdLS.     

Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation

In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos. A T7 phage display cDNA library was constructed from zebrafish tailbud embryos and used to identify the antigens potentially recognized by scFv1, which showed the highest frequency and strongest binding against the tailbud embryos. We acquired 4 candidate epitopes using scFv1 and the corresponding genes showed significantly higher expression levels at tailbud stage than at 30%-epiboly. The most potent epitope of scFv1 was the clone scFv1-2, which showed strong homology to zebrafish myristoylated alanine-rich C-kinase substrate b (Marcksb). Western blot analysis confirmed the high expression of marcksb in the post-gastrula embryos, and the endogenous expression of Marcksb was interfered by injection of scFv1. Zebrafish marcksb showed dynamic expression patterns during embryonic development. Knockdown of marcksb strongly affected gastrulation movements. Moreover, we revealed that zebrafish marcksb is required for cell membrane protrusion and F-actin alignment. Thus, our study uncovered 4 types of scFvs binding to zebrafish post-gastrula embryos, and the epitope of scFv1 was found to be required for normal gastrulation of zebrafish. To our knowledge, this was the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates, and we were able to identify zebrafish marcksb that was required for gastrulation.     

Interaction of Wnt and caudal-related genes in zebrafish posterior body formation

Although Wnt signaling plays an important role in body patterning during early vertebrate embryogenesis, the mechanisms by which Wnts control the individual processes of body patterning are largely unknown. In zebrafish, wnt3a and wnt8 are expressed in overlapping domains in the blastoderm margin and later in the tailbud. The combined inhibition of Wnt3a and Wnt8 by antisense morpholino oligonucleotides led to anteriorization of the neuroectoderm, expansion of the dorsal organizer, and loss of the posterior body structure-a more severe phenotype than with inhibition of each Wnt alone-indicating a redundant role for Wnt3a and Wnt8. The ventrally expressed homeobox genes vox, vent, and ved mediated Wnt3a/Wnt8 signaling to restrict the organizer domain. Of posterior body-formation genes, expression of the caudal-related cdx1a and cdx4/kugelig, but not bmps or cyclops, was strongly reduced in the wnt3a/wnt8 morphant embryos. Like the wnt3a/wnt8 morphant embryos, cdx1a/cdx4 morphant embryos displayed complete loss of the tail structure, suggesting that Cdx1a and Cdx4 mediate Wnt-dependent posterior body formation. We also found that cdx1a and cdx4 expression is dependent on Fgf signaling. hoxa9a and hoxb7a expression was down-regulated in the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos, and in embryos with defects in Fgf signaling. Fgf signaling was required for Cdx-mediated hoxa9a expression. Both the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos failed to promote somitogenesis during mid-segmentation. These data indicate that the cdx genes mediate Wnt signaling and play essential roles in the morphogenesis of the posterior body in zebrafish.     

Mutant-specific gene expression profiling identifies SRY-related HMG box 11b (SOX11b) as a novel regulator of vascular development in zebrafish

Previous studies have identified two zebrafish mutants, cloche and groom of cloche, which lack the majority of the endothelial lineage at early developmental stages. However, at later stages, these avascular mutant embryos generate rudimentary vessels, indicating that they retain the ability to generate endothelial cells despite this initial lack of endothelial progenitors. To further investigate molecular mechanisms that allow the emergence of the endothelial lineage in these avascular mutant embryos, we analyzed the gene expression profile using microarray analysis on isolated endothelial cells. We find that the expression of the genes characteristic of the mesodermal lineages are substantially elevated in the kdrl ⁺ cells isolated from avascular mutant embryos. Subsequent validation and analyses of the microarray data identifies Sox11b, a zebrafish ortholog of SRY-related HMG box 11 (SOX11), which have not previously implicated in vascular development. We further define the function sox11b during vascular development, and find that Sox11b function is essential for developmental angiogenesis in zebrafish embryos, specifically regulating sprouting angiogenesis. Taken together, our analyses illustrate a complex regulation of endothelial specification and differentiation during vertebrate development.     

Smooth muscle cell differentiation from human bone marrow: Variations in cell type specific markers and Id gene expression in a new model of cell culture

Stromal cells follow a vascular smooth muscle differentiation pathway. However, cell culture models performed from human bone marrow do not allow the obtention of a large proportion of highly differentiated smooth muscle cells (SMC) and their differentiation pathways remain unclear. We have characterized a new model of SMC differentiation from human bone marrow stromal cells by using different factors (bFGF, EGF, insulin and BMP-4). A relative homogeneous population of differentiated SMC was reproducibly obtained in short-term culture with high expression of SMC markers. Id gene expression was investigated and showed that (1) Id2 mRNA expression was upregulated during SMC differentiation without change of Id1 mRNA and (2) Id1 gene expression highly increased concomitantly with a decrease of SMC markers while Id2 mRNA was slightly modulated. Our data suggested that Id genes are potentially implicated in the differentiation pathway of human SMC from bone marrow.     

PV.1 Suppresses the Expression of FoxD5b during Neural Induction in Xenopus Embryos

Suppression of bone morphogenetic protein (BMP) signaling induces neural induction in the ectoderm of developing embryos. BMP signaling inhibits eural induction via the expression of various neural suppressors. Previous research has demonstrated that the ectopic expression of dominant negative BMP receptors (DNBR) reduces the expression of target genes down-stream of BMP and leads to neural induction. Additionally, gain-of-function experiments have shown that BMP downstream target genes such as MSX1, GATA1b and Vent are involved in the suppression of neural induction. For example, the Vent1/2 genes are involved in the suppression of Geminin and Sox3 expression in the neural ectodermal region of embryos. In this paper, we investigated whether PV.1, a BMP downstream target gene, negatively regulates the expression of FoxD5b, which plays a role in maintaining a neural progenitor population. A promoter assay and a cyclohexamide experiment demonstrated that PV.1 negatively regulates FoxD5b expression.     

colgate/hdac1 Repression of foxd3 expression is required to permit mitfa-dependent melanogenesis

Neural crest-derived pigment cell development has been used extensively to study cell fate specification, migration, proliferation, survival and differentiation. Many of the genes and regulatory mechanisms required for pigment cell development are conserved across vertebrates. The zebrafish mutant colgate (col)/histone deacetylase1 (hdac1) has reduced numbers, delayed differentiation and decreased migration of neural crest-derived melanophores and their precursors. In hdac1^col mutants normal numbers of premigratory neural crest cells are induced. Later, while there is only a slight reduction in the number of neural crest cells in hdac1^col mutants, there is a severe reduction in the number of mitfa-positive melanoblasts suggesting that hdac1 is required for melanoblast specification. Concomitantly, there is a significant increase in and prolonged expression of foxd3 in neural crest cells in hdac1^col mutants. We found that partially reducing Foxd3 expression in hdac1^col mutants rescues mitfa expression and the melanophore defects in hdac1^col mutants. Furthermore, we demonstrate the ability of Foxd3 to physically interact at the mitfa promoter. Because mitfa is required for melanoblast specification and development, our results suggest that hdac1 is normally required to suppress neural crest foxd3 expression thus de-repressing mitfa resulting in melanogenesis by a subset of neural crest-derived cells.     

Wnt Signaling Interacts with Bmp and Edn1 to Regulate Dorsal-Ventral Patterning and Growth of the Craniofacial Skeleton

Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC) cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D) and ventral (V) elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3), (Tg(hsp70I:tcf3-GFP), or the canonical Wnt inhibitor dickkopf1 (dkk1), (Tg(hsp70i:dkk1-GFP) after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe), and ventral cartilage differentiation (e.g. lower jaws). These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.     

Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis

Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.