The heart defect (hdf) mouse is a recessive lethal that arose from a transgene insertional mutation on chromosome 13. Embryos homozygous for the transgene die in utero by embryonic day 10.5 postcoitus and exhibit specific defects along the anterior-posterior cardiac axis. The future right ventricle and conus/truncus of the single heart tube fail to form and the endocardial cushions in the atrioventricular and conus/truncus regions are absent. Because the hdf mouse mutation provided the opportunity to identify a gene required for endocardial cushion formation and for specification or maintenance of the anterior most segments of the heart, we initiated studies to further characterize the phenotype, clone the insertion site, and identify the gene disrupted. Chromosome mapping studies first identified the gene, Cspg2 (versican), as a candidate hdf gene. In addition, an antibody recognizing a glycosaminoglycan epitope on versican was found to be positive by immunohistochemistry in the extracellular matrix of normal wild-type embryonic hearts, but absent in homozygous hearts. Expression analysis of the Cspg2 gene showed that the 6/8, 6/9, and 7/9 Cspg2 exon boundaries were present in mRNA of normal wild-type embryonic hearts but absent in the homozygous mutant embryos. DNA sequence flanking the transgene was used to isolate from a normal mouse library overlapping genomic DNA segments that span the transgene insertion site. The contiguous genomic DNA segment was found to contain exon 7 of the Cspg2 in a position 3' to the transgene insertion site. These four separate lines of evidence support the hypothesis that Cspg2 is the gene disrupted by the transgene insertion in the hdf mouse line. The findings of this study and our previous studies of the hdf insertional mutant mouse have shown that normal expression of the Cspg2 gene is required for the successful development of the endocardial cushion swellings and the embryonic heart segments that give rise to the right ventricle and conus/truncus in the outlet of the looped heart.

Somatolactin (SL) is a pituitary hormone belonging to the growth hormone/prolactin superfamily, with recognizable homologues in all fish taxa examined to date. Although sequences from most fish share reasonably high sequence identity, several more highly divergent SLs have been reported. Goldfish SL and a second SL protein found in rainbow trout (rtSLP) are remarkably different from each other and also dissimilar to other SLs. It has been unclear whether rtSLP is a recent paralogue restricted to rainbow trout, or reflects a more ancient duplication of the SL gene, and whether it is related to the goldfish sequence. Here we report the cloning of two different zebrafish SL cDNAs, which share only 57·5% nucleotide and 47·7% deduced amino acid identities. One copy, designated zebrafish SL (zfSL ), displays a typical range of sequence similarity to most other SLs. The other copy, zebrafish SL (zfSL ), shows low identity to most other SLs; surprisingly, it is most similar to the divergent SL sequence from goldfish. The mRNAs of zfSL and zfSL were expressed specifically in two distinct regions of the pars intermedia in zebrafish. Cells expressing zfSL are located at the posterior pars intermedia, bordering the neurohypophysis, whereas zfSL is expressed in the anterior part of the pars intermedia, bordering the pars distalis. Phylogenetic analyses indicate that zfSL , goldfish SL and rtSLP all belong to the SL hormone family; however, along with the genes from eel and catfish, these divergent sequences form a group that is clearly distinct from all other SLs. These results suggest the presence of two distinct SL families, SL and SL , which may trace back to a teleost genome duplication prior to divergence of the cyprinids and salmonids.

Versican, an extracellular matrix proteoglycan, has been implicated in limb development and is expressed in precartilage mesenchymal condensations. However, studies have lacked precise spatial and temporal investigation of versican localization during skeletogenesis and its relationship to patterning of muscle and nerve during mammalian limb development. The transgenic mouse line hdf (heart defect), which bears a lacZ reporter construct disrupting Cspg2 encoding versican, allowed ready detection of hdf transgene expression through histochemical analysis. Hdf transgene expression in whole mount heterozygous embryos and localization of versican relative to cartilage, muscle, and nerve tissues in paraffin-embedded limb sections of wild-type embryos from 10.5-14 days postcoitum were evaluated by lacZ histochemistry, immunohistochemistry, and in situ hybridization. Versican was localized within precartilage condensations and nascent cartilages with expression diminishing during maturation of the cartilage model at later time points. Interestingly, versican remained highly expressed in developing synovial joint interzones, suggesting potential function for versican in joint morphogenesis. Isolated myoblasts, incipient skeletal muscle masses, and neurites were not present in areas of strong versican expression within the developing limb. Versican-expressing tissues may reserve space for the future limb skeleton and developing joints and may aid in patterning of muscle and nerve by deterring muscle migration and innervation into these regions.

No abstract

TC2 is a novel monoclonal antibody produced by in vitro immunization of splenocytes with a peanut agglutinin-positive fraction from extracts of prechondrogenic micromass cultures of chick limb mesenchyme. ELISA results demonstrated TC2 reactivity with a native epitope on a glycosaminoglycan (GAG) enriched in chondroitin-4-sulfate and with multiple intact proteoglycans, but not with other GAGs tested. TC2 immunohistochemical reactivity was abolished by pretreatment of sections with chondroitinase AC or preadsorption with chondroitin-4-sulfate GAG. Strong TC2 localization occurred throughout the developing heart at stage 9. As looping ensued, a graded reactivity was observed from lowest in the atrium to highest in the conotruncus that correlated well with versican localization. The superior atrioventricular cushion stained preferentially with TC2 as compared to the inferior cushion at stages 16-18. At these later stages TC2 patterns did not agree completely with anti-versican reactivity. By stage 23 there was a marked reduction in TC2 localization in the heart, however, strong reactivity remained at certain sites, including the conotruncus and in subcompartments of both atrioventricular cushions. A heterogeneous distribution of other native chondroitin sulfate glycosaminoglycan epitopes recognized by monoclonal antibodies d1C4 and CS-56 was observed as well. The distribution of the TC2 epitope usually did not overlap with d1C4 or CS-56 localization at the stages examined. Overall, the spatiotemporal characteristics of TC2 reactivity in the developing chick heart appear to correlate with subdomains of the endocardial cushions as well as with trabecular and atrial septal formation.

The extracellular matrix (ECM) plays a critical role in governing cell behavior and phenotype during limb skeletogenesis. Chondroitin sulfate proteoglycans (Cspgs) are highly expressed in the ECM of precartilage mesenchymal condensations and are important to limb chondrogenesis and cartilage structure, but little is known regarding their involvement in formation of synovial joints in the embryonic limb. Matrix versican Cspg expression has previously been reported in the epiphysis of developing long bones and presumptive joint; however, detailed analysis has not yet been conducted. In the present study we immunolocalized versican and aggrecan Cspgs during chick elbow joint morphogenesis between HH st25-41 of development. In this study we show that versican and aggrecan expression initially overlapped in the incipient cartilage model of long bones in the wing, but versican was also highly expressed in the perichondrium and presumptive joint interzone during early stages of morphogenesis (HH st25-34). By HH st36-41 versican localization was restricted to the future articular surfaces of the developing joint and surrounding joint capsule while aggrecan localized in an immediately adjacent and predominately non-overlapping region of chondrogenic cells at the epiphyses. These results suggest a potential role for versican proteoglycan in development and maintenance of the synovial joint interzone.

No abstract

Mesenchymal cell aggregation is critical for cartilage formation in the vertebrate limb. The extracellular matrix (ECM) plays a critical role in governing cell behavior and cell phenotype in this tissue, and the hyalectin versican is highly expressed in the ECM of precartilage mesenchymal cells and developing synovial joints. Although several in vitro studies have been conducted in an attempt to address versican's role during limb mesenchymal condensation, factors such as differences in cell density in culture, variations between chondrogenic cell lines, and the inability to prolong the viability of limb explants have led to conflicting data, mandating an in vivo analysis. By using a morpholino directed strategy in ovo, we performed knock-down of versican expression in the presumptive ulnar region of the developing chick wing at time points critical to skeletogenesis. These data indicate that in ovo misexpression of versican compromised mesenchymal condensation with resulting ulnar cartilages reduced in length distally by an average of 53% relative to contralateral control limbs. In select versican morphants the olecranon process was also reduced in size proximally and failed to cup the humerus, likely impairing joint morphogenesis. This study represents the first report assessing the role of versican in the developing chick limb in ovo, further demonstrating the importance of versican proteoglycan expression during chondrogenesis and extending previous findings to suggest a role for versican during synovial joint development.