The role played by specific extracellular matrix molecules in normal endocardial cushion differentiation into valves and septa remains to be established. In this respect, type collagen VI is of particular interest because genes encoding the ␣1 and ␣2 chains are located on chromosome 21, and defects involving the atrioventricular (AV) cushions are frequent in trisomy 21. Collagen VI expression was studied in normal human embryonic and fetal hearts (5-18 weeks of development) and compared by immunohistochemistry with results from fetuses (10 -16 weeks of development) with trisomy 21. During normal endocardial cushion differentiation (5-8 weeks) there was marked collagen VI expression in the AV cushions, whereas only minor expression was seen in the outflow tract cushions. In the normal fetuses (10 -18 weeks), collagen VI in the AV cushions had condensed into a marked zone on the atrial side of the leaflets, as well as subendocardially in other regions of high shear stress. Morphological defects involving the endocardial cushion-derived structures were present in all trisomy 21 cases. An abnormally large membranous septum was observed in three cases. An AV septal defect (AVSD) was present in two, while one had a ventricular septal defect (VSD). Two cases presented with a secondary atrial septal defect (ASDII), and one had an AVSD. Mild to moderate valve dysmorphia was found in all cases. Collagen VI staining in trisomy 21 was more intense than in the normal subjects; however, there were no differences in the spatial expression patterns. We conclude that collagen VI is expressed in the AV cushions and persists during valve differentiation. Collagen VI is more prominent in fetal trisomy 21 hearts than in normal hearts. We hypothesise that collagen VI has a role in the development of heart defects involving endocardial cushion differentiation-specifically in the AV canal, the most common site of malformations affecting children with trisomy 21.

Trisomy 21 (Down syndrome) is a common genetic condition with a high incidence of congenital heart defects (CHD), particularly those involving abnormal development of the embryonic atrioventricular (AV) canal. Type VI collagen (Col VI) is expressed in the developing AV canal extracellular matrix, and has been associated with trisomy 21 AV canal defects in human genetic studies. Although the molecular mechanisms linking Col VI and trisomy 21 AV canal defects are not well understood, a computer model predicts increased cell adhesiveness is responsible for these CHD. We compared integrin-mediated cell adhesive properties for skin fibroblasts isolated from trisomy 21 and non-trisomic individuals on Col VI, fibronectin (FN) and type I collagen (Col I). Cell lines demonstrate similar adhesion profiles to FN and Col I, but all trisomy 21 cells display increased adhesive capacity for Col VI compared to non-trisomic fibroblasts. Cell adhesion to type VI collagen was shown to be GRGDS independent, but beta(1) integrin family dependent. Function-blocking antibodies identified alpha(3)beta(1) as the predominant integrin mediating trisomy 21 and non-trisomic skin fibroblast adhesion on Col VI. Trisomy 21 and non-trisomic fibroblasts display similar expression levels for each of the integrin receptors examined. A beta(1) integrin-activating antibody augments non-trisomic cell adhesion on Col VI, but has no effect upon trisomy 21 fibroblasts. These results demonstrate that beta(1) integrin family members mediate trisomy 21 and non-trisomic skin fibroblast adhesion for Col VI. Altered activation state of the beta(1) integrin is a mechanism responsible for increased trisomy 21 cell adhesion on Col VI.

SummaryThe tub61 P-tubulin gene of Glycine max (previously named spl) is highly expressed only in rapidly elongating regions of etiolated seedling hypocotyls and this expression is strongly downregulated when the seedlings are exposed to light. Primer extension demonstrated that the gene was transcribed in these tissues and contained two sites of transcriptional initiation. To determine the mechanism regulating tub61 expression, a chimeric reporter gene was constructed by fusing 5' upstream regions of tub61 to a promoterless Pglucuronidase (GUS) gene and these constructs were introduced into protoplasts by electroporation. Strong transient expression of the reporter ge?e was obtained after electroporation of chimeric constructs containing 1 kb of tub61 5' upstream sequence into tobacco protoplasts. Deletion of the distal most 300 bp from the 5' sequence of tub61 enhanced expression, suggesting the possibility of a negative transcriptional regulator in this region. Additional deletions of the 5' sequence reduced expression substantially. Constructs containing a tub61 3' terminus were expressed at much lower levels than those containing a nopaline synthase (NOS) 3' terminus. The tub61-GUS chimeric gene also was introduced into tobacco by Agrobacterium mediated Ti plasmid transformation and the organspecific expression pattern of the chimeric gene was determined in seedlings of the transgenic plants. Hypocotyls exhibited strong GUS activity when the seedlings were germinated in darkness, but lacked the GUS enzyme when the seedlings were germinated in the light. This result demonstrates that the cisacting elements within the first 2 kb of the trans-

We examined the developmental expression of a diverged soybean beta-tubulin gene (designated sb-1), which had been cloned and sequenced previously. A probe specific for the sb-1 gene was constructed from the 3' transcribed untranslated sequence. As a control, a more general probe for beta-tubulin genes and their transcripts was constructed from a highly conserved region of the third exon of another soybean beta-tubulin gene, sb-2. Poly(A)+ RNA, extracted from various soybean tissues and organs, was probed alternatively with the sb-1 gene-specific probe and with the generic beta-tubulin probe. Levels of beta-tubulin transcripts recognized by the generic probe differed by a factor of approximately 3 in the different tissues and organs and varied with the state of organ development. Highest levels were found in young, unexpanded leaves and they decreased as leaf maturation occurred. In contrast, transcripts of sb-1 were nearly undetectable in young leaves, and they increased as leaf maturation occurred. Levels of sb-1 transcript were low in all organs of the light-grown plant examined, except the hypocotyl, where they were approximately 10-fold higher. However, the highest levels of sb-1 transcripts were observed in elongating hypocotyls of etiolated seedlings. Exposure of six-day-old etiolated seedlings to light for 12 hours halted further hypocotyl elongation and brought about a dramatic, nearly 100-fold, decrease in the steady-state level of sb-1 transcripts.