Distinct requirements of wls, wnt9a, wnt5b and gpc4 in regulating chondrocyte maturation and timing of endochondral ossification

Formation of the mandible requires progressive morphologic change, proliferation, differentiation and organization of chondrocytes preceding osteogenesis. The Wnt signaling pathway is involved in regulating bone development and maintenance. Chondrocytes that are fated to become bone require Wnt to polarize and orientate appropriately to initiate the endochondral ossification program. Although the canonical Wnt signaling has been well studied in the context of bone development, the effects of non-canonical Wnt signaling in regulating the timing of cartilage maturation and subsequent bone formation in shaping ventral craniofacial structure is not fully understood.. Here we examined the role of the non-canonical Wnt signaling pathway (wls, gpc4, wnt5b and wnt9a) in regulating zebrafish Meckel's cartilage maturation to the onset of osteogenic differentiation. We found that disruption of wls resulted in a significant loss of craniofacial bone, whereas lack of gpc4, wnt5b and wnt9a resulted in severely delayed endochondral ossification. This study demonstrates the importance of the non-canonical Wnt pathway in regulating coordinated ventral cartilage morphogenesis and ossification

Calcium sensitivity and myofilament lattice structure in titin N2B KO mice

The cellular basis of the Frank-Starling "Law of the Heart" is the length-dependence of activation, but the mechanisms by which the sarcomere detects length changes and converts this information to altered calcium sensitivity has remained elusive. Here the effect of titin-based passive tension on the length-dependence of activation (LDA) was studied by measuring the tension-pCa relation in skinned mouse LV muscle at two sarcomere lengths (SLs). N2B KO myocardium, where the N2B spring element in titin is deleted and passive tension is elevated, was compared to WT myocardium. Myofilament lattice structure was studied with low-angle X-ray diffraction; the myofilament lattice spacing (d(10)) was measured as well as the ratio of the intensities of the 1,1 and 1,0 diffraction peaks (I(11)/I(10)) as an estimate of the degree of association of myosin heads with the thin filaments. Experiments were carried out in skinned muscle in which the lattice spacing was reduced with Dextran-T500. Experiments with and without lattice compression were also carried out following PKA phosphorylation of the skinned muscle. Under all conditions that were tested, LDA was significantly larger in N2B KO myocardium compared to WT myocardium, with the largest differences following PKA phosphorylation. A positive correlation between passive tension and LDA was found that persisted when the myofilament lattice was compressed with Dextran and that was enhanced following PKA phosphorylation. Low-angle X-ray diffraction revealed a shift in mass from thin filaments to thick filaments as sarcomere length was increased. Furthermore, a positive correlation was obtained between myofilament lattice spacing and passive tension and the change in I(11)/I(10) and passive tension and these provide possible explanations for how titin-based passive tension might regulate calcium sensitivity

Functional genomics of chicken, mouse, and human titin supports splice diversity as an important mechanism for regulating biomechanics of striated muscle

Titin is a giant filamentous elastic protein that spans from the Z-disk to M-band regions of the sarcomere. The I-band region of titin is extensible and develops passive force in stretched sarcomeres. This force has been implicated as a factor involved in regulating cardiac contraction. To better understand the adaptation in the extensible region of titin we report the sequence and annotation of the chicken and mouse titin genes and compare them to the human titin gene. Our results reveal a high degree of conservation within the genomic region encoding the A-band segment of titin, consistent with the structural similarity of vertebrate A-bands. In contrast the genomic region encoding the Z-disk and I-band segments is highly divergent. This is most prominent within the central I-band segment, where chicken titin has fewer but larger PEVK exons (up to 1992 bp). Furthermore, in mouse titin we found two LINE-repeats that are inserted in the Z-disk and I-band regions - the regions that account for most of the splice isoform diversity. Transcript studies show that a group of 55 I-band exons is differentially expressed in chicken titin. Consistent with a large degree of titin isoform plasticity and variation in PEVK content, chicken skeletal titins range in size from ~3000 to ~3700 kDa, and vary greatly in passive mechanical properties. Low angle X-ray diffraction experiments reveal significant differences in myofilament lattice spacing that correlate with titin isoform expression. We conclude that titin splice diversity regulates structure and biomechanics of the sarcomere