Transcriptional regulation of cardiac development: implications for congenital heart disease and DiGeorge syndrome.

In recent years, impressive advances have occurred in our understanding of transcriptional regulation of cardiac development. These insights have begun to elucidate the mystery of congenital heart disease at the molecular level. In addition, the molecular pathways emerging from the study of cardiac development are being applied to the understanding of adult cardiac disease. Preliminary results support the contention that a thorough understanding of molecular programs governing cardiac morphogenesis will provide important insights into the pathogenesis of human cardiac diseases. This review will focus on examples of transcription factors that play critical roles at various phases of cardiac development and their relevance to cardiac disease. This is an exciting and burgeoning area of investigation. It is not possible to be all-inclusive, and the reader will note important efforts in the areas of cardiomyocyte determination, left-right asymmetry, cardiac muscular dystrophies, electrophysiology and vascular disease are not covered. For a more complete discussion, the reader is referred to recent reviews including the excellent compilation of observations assembled by Harvey and Rosenthal (1).

Rnf4, a RING protein expressed in the developing nervous and reproductive systems, interacts with Gscl, a gene within the DiGeorge critical region.

A yeast 2-hybrid screen was performed to identify possible transcriptional modulators interactive with goosecoid-like (gscl), a transcription factor with suppressive activity, expressed during early brain and gonad development. The screen resulted in the identification of a RING protein known as rnf4 or snrf. Gscl/rnf4 interactions were confirmed by affinity chromatography and by immunoprecipitation. Northern analysis confirmed earlier reports of ubiquitous rnf4 expression in adult tissues. Immunohistochemical analysis of mouse embryos revealed expression primarily in the developing nervous system, with strong expression in the dorsal root ganglia and developing gonads. In contrast to previous reports, both cytoplasmic and nuclear expression of rnf4 was documented. The results reported here confirm and extend earlier reports of rnf4 expression. They suggest for the first time, that in addition to acting as a modulator of transcriptional activity, rnf4 may function, as do other RING proteins, to promote the formation of intracytoplasmic complexes involved in shuttling information between the cytoplasm and the nucleus.

p57Kip2 expression is enhanced during mid-cardiac murine development and is restricted to trabecular myocardium.

During embryonic development the heart is required to grow in size and cell number, undergo complex morphologic alterations, and function to circulate the blood. Between embryonic d 10.5 (E10.5) and E11.5, cardiac myocytes undergo rapid cell division, resulting in doubling of cardiac mass, while metabolic requirements are increased and contraction force is enhanced. Accelerated cardiomyocyte differentiation is accompanied by a significant increase in trabeculation of ventricular myocardium. Many single gene mutations in the mouse result in a "thinned myocardium" and embryonic lethality between E10.5 and E13.5 secondary to heart failure. This is the case in the Splotch mouse in which a mutation of the Pax3 gene results in neural crest and cardiac defects. Nevertheless, the molecular events governing these important developmental steps remain largely unknown. Here, we describe the use of suppression subtractive hybridization to identify mRNA transcripts whose expression is enhanced during this critical period in normal hearts. These genes encode functions related to maturation of the contractile apparatus, cardiomyocyte differentiation, altered cellular metabolism, and transcriptional regulation. One of the genes that we identified, p57Kip2, encodes a cyclin-dependent kinase inhibitor of the p21 family. We show that p57Kip2 is normally expressed in the inner trabecular layer of the developing heart. In Splotch embryos, expression of p57Kip2 is expanded to encompass the entire thickness of the myocardium. This result and further structural analysis suggests that the myocardial defect of Splotch embryos is associated with precocious cardiomyocyte differentiation.

Adenoviral-mediated expression of antisense RNA to fibroblast growth factors disrupts murine vascular development.

Fibroblast growth factors (FGFs) are expressed in the developing embryo and are postulated to regulate embryonic and vascular growth. The goal of this study was to elucidate the role of basic fibroblast growth factor (FGF-2) in early murine embryonic cardiovascular development in the mouse embryo. Gestation day 7.5 embryos were harvested and placed in culture, and 12 hr later replication-defective adenovirus (0.5 x 10(6) plaque forming units) encoding either beta-galactosidase or antisense FGF-2 RNA was injected into the sinus venosus of the cultured embryos. Embryos receiving only replication-defective adenovirus expressing the beta-galactosidase gene continued to develop normally over the next 12 hr. In contrast, those receiving adenovirus encoding antisense FGF-2 RNA displayed marked morphogenetic abnormalities, including cessation of growth and abnormal yolk sac vascular development, even though the embryonic hearts continued to beat. Abnormal development of the yolk sac vasculature was confirmed by microangiography and by histologic examination. Coinjection of virus carrying FGF-2 cDNA in the sense orientation reversed the effects of antisense FGF-2 RNA expression. These results confirm the efficacy of the replication-defective adenovirus for targeting gene expression to the developing vasculature and provide evidence for a critical role of FGF in the normal vascular assembly in the early embryo. Cessation of embryonic growth on expression of antisense FGF-2 RNA was most likely attributable to failure of efficient circulation of the early embryonic blood cells from the yolk sac into the embryo.

Adenoviral gene transfer of beta3 integrin subunit induces conversion from radial to vertical growth phase in primary human melanoma.

Expression of the beta3 subunit of the alphavbeta3 vitronectin receptor on melanoma cells is associated with tumor thickness and the ability to invade and metastasize. To address the role of alphavbeta3 in the complex process of progression from the nontumorigenic radial to the tumorigenic vertical growth phase of primary melanoma, we examined the biological consequences of overexpressing alphavbeta3 in early-stage melanoma cells using an adenoviral vector for gene transfer. Overexpression of functional alphavbeta3 in radial growth phase primary melanoma cells 1) promotes both anchorage-dependent and -independent growth, 2) initiates invasive growth from the epidermis into the dermis in three-dimensional skin reconstructs, 3) prevents apoptosis of invading cells, and 4) increases tumor growth in vivo. Thus, alphavbeta3 serves diverse biological functions during the progression from the nontumorigenic radial growth phase to the tumorigenic and-invasive vertical growth phase primary melanoma.

Immunohistochemical analysis of the expression of two serine-threonine kinases in the maturing mouse testis.

Previously we identified two intronless serine-threonine kinase genes (Tsk1 and Tsk2) located 3 kb apart on mouse chromosome 16 (Galili, N., Baldwin, H.S., Lund, J., Reeves, R., Gong, W., Wang, Z., Roe, B.A., Emanuel, B.S., Nayak, S., Mickanin, C., Budraf, M.L., Buck, C.A., 1997. A region of mouse chromosome 16 is syntenic to the DiGeorge, velocardiofacial syndrome minimal critical region. Gen. Res. 7, 17-26). Tsk1 was identical to a putative testicular kinase reported by Bielke et al. (Bielke, W., Blaschke, R.J., Miescher, G.C., Zurcher, G., Andres, A.C., Ziemiecki, A., 1994. Characterization of a novel murine testis-specific serine/threonine kinase. Gene 13, 235-239). Here we document the expression patterns of each Tsk throughout spermiogenesis showing an initial association of Tsk1 with cells in meiotic metaphase and a later association of Tsk2 with tail-like structures in the lumen of the seminiferous tubule.

Gscl, a gene within the minimal DiGeorge critical region, is expressed in primordial germ cells and the developing pons.

Gscl, a paired-type homeobox gene, has been implicated in the pathology of DGS/VCFS by virtue of its genomic location and its structural similarity to the Gsc gene family. Immunohistochemical and in situ studies were performed to examine the expression pattern of this gene during embryonic development. A polyclonal antibody, generated to the full-length protein and shown to be specific for GSCL by both Western blotting and immunofluorescence, was used for immunohistochemical localization. Both in situ and antibody staining localized GSCL expression to a cluster of cells in the pons region of the developing brain. This GSCL expression pattern showed partial overlap with that of Pax6. More detailed immunohistochemistry revealed the GSCL in primordial germ cells during migration from the epithelium of the hindgut and later as they colonize the developing gonads. GSCL was not detected in tissues affected in DGS/VCSF.

A region of mouse chromosome 16 is syntenic to the DiGeorge, velocardiofacial syndrome minimal critical region.

DGS and VCFS, haploinsufficiencies characterized by multiple craniofacial and cardiac abnormalities, are associated with a microdeletion of chromosome 22q11.2. Here we document synteny between a 150-kb region on mouse chromosome 16 and the most commonly deleted portion of 22q11.2. Seven genes, all of which are transcribed in the early mouse embryo, have been identified. Of particular interest are two serine/threonine kinase genes and a novel goosecoid-like homeobox gene (Gscl). Comparative sequence analysis of a 38-kb segment reveals similarities in gene content, order, exon composition, and transcriptional direction. Therefore, if deletion of these genes results in DGS/VCFS in humans, then haploinsufficiencies involving this region of chromosome 16 should recapitulate the developmental field defects characteristic of this syndrome.

Identification and characterization of alpha 3 beta 1 integrin on primary and transformed rat islet cells.

Dispersed rat islet cells embedded in a matrix of collagen I are known to form aggregates in vitro reminiscent of native islets. Furthermore, it appears that islet function and survival are better maintained in vitro when cells are grown in the presence of extracellular matrix. These studies suggest an important role of cell--matrix interactions in the formation and maintenance of islet structure and function. The molecular basis of these interactions is mostly unknown. In the present study, we confirm the presence of beta 1 integrins on primary and transformed (RIN-2A line) rat islet cells. Perturbation studies in vitro show that beta 1 integrins play a role in islet cell attachment and spreading on bovine extracellular matrix and on the matrix produced by A-431 cells. The alpha 3 integrin subunit is coimmunoprecipitated with beta 1 from extracts of both primary and transformed islet cells, and immunodepletion studies suggest that alpha 3 beta 1 represents nearly half of the total beta 1 integrins expressed on primary islet cells. In situ, alpha 3 and beta 1 are expressed on the surface of all islet cell types, as shown by indirect immunocytochemistry on paraformaldehyde-fixed sections of rat pancreas. In conclusion, the study demonstrates the presence of alpha 3 beta 1 on primary and transformed rat islet cells, and an important role of beta 1 integrins in islet cell attachment and spreading in vitro.

Epitopes of adhesion-perturbing monoclonal antibodies map within a predicted alpha-helical domain of the integrin beta 1 subunit.

Several recent studies have demonstrated the involvement of various domains of the beta 1 integrin subunit in ligand binding. Thus, specific amino acids have been shown to be important in divalent cation binding, and others have been implicated by peptide crosslinking to play an intimate role in integrin-ligand interactions. Added to these data are previous observations that a group of adhesion-blocking anti-chicken beta 1 antibodies mapped within the first 160 amino acid residues of the subunit. These observations suggested that this region plays a critical role in integrin ligand recognition. In order to further define the domain in which the epitopes for these antibodies are clustered, a series of mouse/chicken chimeric beta 1 constructs were examined for their reactivity with each of these antibodies. Most of the antibodies recognize a region between residues 124 to 160 of the chicken beta 1 subunit. Computer modeling predicted a possible amphipathic alpha-helical configuration for the region between residues 141 to 160. Consistent with this prediction, circular dichroism and NMR analysis revealed a tendency for a synthetic peptide containing these residues to form an alpha-helix. The significance of this structural characteristic was demonstrated by a mutation at residue 149 that disrupted the alpha-helix formation and resulted in a loss of the ability to form heterodimers with alpha subunits, localize to focal contacts, or be transported to the cell surface. The direct involvement of residues 141 to 160 in ligand binding was supported by the ability of a peptide with this sequence to elute integrins from a fibronectin affinity column. Thus, our data suggest that residues 141 to 160 of the integrin beta 1 subunit, when arranged in an alpha-helix configuration, participate in ligand binding.

Expression patterns of adhesion receptors in the developing mouse lung: functional implications.

A detailed, immunohistological study of mouse lung development from the first appearance of primary lung buds off the laryngo tracheal groove through the formation of the mature, adult lung has been carried out using monoclonal antibodies specific for endothelial cells, smooth muscle cells, adhesion receptors and markers of mature endothelial cell function. These included mAbs specific for PECAM-1, alpha-smooth muscle actin, ICAM-1, ICAM-2, VCAM-1, alpha 4 and alpha 6 integrin subunits, thrombomodulin and factor VIII. The results document a dynamic pattern of receptor expression and indicate that the expansion of the pulmonary vascular system may take place by both angiogenic and vasculogenic processes. They further document differences in receptor expression by vascular and airway smooth muscle. ICAM-1 expression was primarily extravascular during development. The expression patterns of alpha 4 integrin and its counter receptor VCAM-1 lacked the complementarity that might be expected if they were functioning as a receptor/counter-receptor pair in lung development. Thrombomodulin expression patterns support a major role for the thrombin/ thrombomodulin system in lung development. The expression of thrombomodulin only at sites of airway branching suggests that the thrombin/thrombomodulin system could play a pivotal, regulatory role in branching morphogenesis.

Alternative splicing of a specific cytoplasmic exon alters the binding characteristics of murine platelet/endothelial cell adhesion molecule-1 (PECAM-1).

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a membrane glycoprotein expressed on endothelial cells, platelets, and leukocytes. Analysis of PECAM-1 expression in the developing mouse embryo has revealed the presence of multiple isoforms of murine PECAM-1 (muPECAM-1) that appeared to result from the alternative splicing of exons encoding cytoplasmic domain sequences (exons 10-16) (Baldwin, H. S., Shen, H. M., Yan, H., DeLisser, H. M., Chung, A., Mickanin, C., Trask, T., Kirschbaum, N. E. Newman, P. J., Albelda, S., and Buck, C. A. (1994) Development 120, 2539-2553). To investigate the functional consequences of alternatively spliced muPECAM-1 cytoplasmic domains, L-cells were transfected with cDNA for each variant and their ability to promote cell aggregation was compared. In this assay, full-length muPECAM-1 and all three isoforms containing exon 14 behaved like human PECAM-1 in that they mediated calcium- and heparin-dependent heterophilic aggregation. In contrast, three muPECAM-1 variants, all missing exon 14, mediated calcium- and heparin-independent homophilic aggregation. Exon 14 thus appears to modulate the ligand and adhesive interactions of the extracellular domain of PECAM-1. These findings suggest that alternative splicing may represent a mode of regulating the adhesive function of PECAM-1 in vivo and provides direct evidence that alternative splicing involving the cytoplasmic domain affects the ligand specificity and binding properties of a cell adhesion receptor.

Monoclonal antibody 9EG7 defines a novel beta 1 integrin epitope induced by soluble ligand and manganese, but inhibited by calcium.

The monoclonal antibody 9EG7 has been previously found to recognize an epitope induced by manganese on the integrin beta 1 chain (Lenter, M., Uhlig, H., Hamann, A., Jeno, P., Imhof, B., and Vestweber, D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 9051-9055). Here we show that treatment of beta 1 integrins with manganese or soluble integrin ligands (e.g. fibronectin and RGD peptide) induced the 9EG7 epitope. This epitope was also induced upon EGTA treatment to remove calcium, and the addition of calcium inhibited 9EG7 epitope induction by manganese or by ligand. Further emphasizing the importance of the 9EG7 epitope, the 9EG7 antibody itself stimulated adhesion mediated by multiple beta 1 integrins, and conversely, ligands for alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, and alpha 5 beta 1 all stimulated 9EG7 expression. Together these results support a model whereby (i) calcium inhibits beta 1 integrin function because it prevents the appearance of a conformation favorable to ligand binding and (ii) manganese enhances beta 1 integrin function because it induces the same favorable conformation that is induced by adding ligand, or removing calcium. Notably, other beta 1-stimulating agents (magnesium and mAb TS2/16) did not induce 9EG7 expression unless ligand was also present. Thus, although 9EG7 may reliable detect the ligand-bound conformation of beta 1 integrins, its expression does not always correlate with integrin "activation". Finally, mouse/chicken beta 1 chimeric molecules were used to map the 9EG7 epitope to beta 1 residues 495-602 within the cysteine-rich region, and antibody cross-blocking studies showed that the 9EG7 epitope is distinct from all previously defined human beta 1 epitopes.

Distribution of integrin cell adhesion molecules in endometrial cancer.

Integrins are ubiquitous cell adhesion molecules that are involved in maintaining normal tissue morphology and have been implicated in the behavior of certain malignancies. We examined the expression of nine integrin subunits in 38 endometrial adenocarcinomas using immunohistochemistry. The pattern of integrin expression in the cancers was compared with that seen in the endometrium of 20 normal cycling women and 7 postmenopausal women. Integrin expression was correlated with grade, stage, nodal status, depth of invasion, steroid receptor status, and histological pattern. In endometrial cancers there was an inverse relationship between the number of integrins expressed and histological grade (P = 0.011). Of the normally expressed, constitutive endometrial epithelial integrin subunits (alpha 2, alpha 3, alpha 6, and beta 4), the least frequently seen in the cancers was the alpha 3 subunit (44.7%) and the most frequently found was alpha 6 (81.6%). The alpha 5 beta 1 integrin, a fibronectin receptor normally found only on endometrial stromal cells, was seen in 17.8% of cases of these epithelial cancers. In addition, a significant association was found between the loss of the alpha 2 beta 1 integrin and the presence of lymph node metastases (P < 0.001). These data suggest that a decline in integrin expression occurs more frequently in poorly differentiated endometrial cancers and that the loss of specific integrins may be associated with metastatic nodal spread.

Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice.

VCAM-1 is a cytokine-inducible cell surface protein capable of mediating adhesion to leukocytes expressing alpha 4 integrins. Mice deficient in VCAM-1 expression were produced by targeted homologous recombination in ES cells. VCAM-1-deficient embryos were not viable and exhibited either of two distinct phenotypes. Approximately half of the embryos died before embryonic day 11.5 and exhibited a severe defect in placental development in which the allantois failed to fuse with the chorion. The remaining VCAM-1-deficient embryos survived to embryonic day 11.5-12.5 and displayed several abnormalities in their developing hearts including a reduction of the compact layer of the ventricular myocardium and intraventricular septum. The hearts also contained significant amounts of blood in the pericardial space and lacked an epicardium. alpha 4 and VCAM-1 were found to be expressed in wild-type embryos in a reciprocal fashion in the chorion and allantois and in the epicardium and the underlying myocardium, although VCAM-1 was expressed in the intraventricular septum in the absence of adjacent alpha 4-expressing cells. These data suggest important roles for VCAM-1 and alpha 4 in the development of the placenta and the heart.

Further characterization of endometrial integrins during the menstrual cycle and in pregnancy.

OBJECTIVE: To profile the changes in integrin expression in cycling and pregnant endometrium. DESIGN: Immunohistochemistry was performed on endometrium from proliferative, early, and midsecretory phase and early pregnancy using a blinded panel of monoclonal antibodies directed against integrins. Closer examination of the cycle-dependent integrins was performed in 112 patients throughout the cycle and from the first trimester of pregnancy. SETTING: An academic teaching hospital. PATIENTS: Ovulatory women or women undergoing pregnancy termination. MAIN OUTCOME MEASURE: Staining intensity of each antibody in epithelial or stromal cells. RESULTS: Certain integrins were present constitutively throughout the menstrual cycle whereas others were expressed only during the luteal phase. Four integrins increased in the decidua of pregnancy. The timing of expression of the two cycle-dependent integrins (alpha 4 beta 1 and alpha v beta 3) framed the putative window of implantation and suggests a role in establishment of uterine receptivity. CONCLUSIONS: Our findings confirm the patterns of endometrial integrins and suggest important roles for integrins in the process of implantation and decidualization.

Aberrant integrin expression in the endometrium of women with endometriosis.

Integrins are ubiquitous cell adhesion molecules that undergo dynamic alterations during the normal menstrual cycle in the human endometrium. The alpha v beta 3 vitronectin receptor integrin is expressed in endometrium at the time of implantation, but its presence is delayed in endometrium that is assessed to be out of phase using classical histological features. To investigate the expression of this integrin in women with endometriosis, we assessed the presence of the beta 3-subunit throughout the menstrual cycle in 268 "in-phase" endometrial biopsies, using immunohistochemistry. The beta 3-subunit was expressed on endometrial epithelium after days 19-20 of the menstrual cycle. In 241 women whose biopsies were obtained after day 19, a lack of beta 3 expression was found to be closely related to the diagnosis of endometriosis (by Wilcoxon test, P = 0.02). This defect in integrin expression was associated with nulliparity, inversely related to the stage of disease, and occurred despite the presence of in-phase histological features. In a prospective double blind assessment of this integrin, we found endometrial beta 3 analysis to have a high specificity and positive predictive value as a nonsurgical diagnostic test for minimal and mild endometriosis. In conclusion, aberrant integrin expression in the native endometrium is associated with the finding of endometriosis and may identify some women with decreased cycle fecundity due to defects in uterine receptivity.