Coordinated production and remodeling of the extracellular matrix is essential during development. It is of particular importance for skeletogenesis, as the ability of cartilage and bone to provide structural support is determined by the composition and organization of the extracellular matrix. Connective tissue growth factor (CTGF, CCN2) is a secreted protein containing several domains that mediate interactions with growth factors,integrins and extracellular matrix components. A role for CTGF in extracellular matrix production is suggested by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. To test whether CTGF is required for extracellular matrix remodeling and/or angiogenesis during development, we examined the pattern of Ctgf expression and generated Ctgf-deficient mice. Ctgf is expressed in a variety of tissues in midgestation embryos, with highest levels in vascular tissues and maturing chondrocytes. We confirmed that CTGF is a crucial regulator of cartilage extracellular matrix remodeling by generating Ctgf-/- mice. Ctgf deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte proliferation and extracellular matrix composition within the hypertrophic zone. Decreased expression of specific extracellular matrix components and matrix metalloproteinases suggests that matrix remodeling within the hypertrophic zones in Ctgf mutants is defective. The mutant phenotype also revealed a role for Ctgf in growth plate angiogenesis. Hypertrophic zones of Ctgf mutant growth plates are expanded, and endochondral ossification is impaired. These defects are linked to decreased expression of vascular endothelial growth factor (VEGF) in the hypertrophic zones of Ctgf mutants. These results demonstrate that CTGF is important for cell proliferation and matrix remodeling during chondrogenesis, and is a key regulator coupling extracellular matrix remodeling to angiogenesis at the growth plate.

For most organisms, chromosome segregation during meiosis relies on deliberate induction of DNA double-strand breaks (DSBs) and repair of a subset of these DSBs as inter-homolog crossovers (COs). However, timing and levels of DSB formation must be tightly controlled to avoid jeopardizing genome integrity. Here we identify the DSB-2 protein, which is required for efficient DSB formation during C. elegans meiosis but is dispensable for later steps of meiotic recombination. DSB-2 localizes to chromatin during the time of DSB formation, and its disappearance coincides with a decline in RAD-51 foci marking early recombination intermediates and precedes appearance of COSA-1 foci marking CO-designated sites. These and other data suggest that DSB-2 and its paralog DSB-1 promote competence for DSB formation. Further, immunofluorescence analyses of wild-type gonads and various meiotic mutants reveal that association of DSB-2 with chromatin is coordinated with multiple distinct aspects of the meiotic program, including the phosphorylation state of nuclear envelope protein SUN-1 and dependence on RAD-50 to load the RAD-51 recombinase at DSB sites. Moreover, association of DSB-2 with chromatin is prolonged in mutants impaired for either DSB formation or formation of downstream CO intermediates. These and other data suggest that association of DSB-2 with chromatin is an indicator of competence for DSB formation, and that cells respond to a deficit of CO-competent recombination intermediates by prolonging the DSB-competent state. In the context of this model, we propose that formation of sufficient CO-competent intermediates engages a negative feedback response that leads to cessation of DSB formation as part of a major coordinated transition in meiotic prophase progression. The proposed negative feedback regulation of DSB formation simultaneously (1) ensures that sufficient DSBs are made to guarantee CO formation and (2) prevents excessive DSB levels that could have deleterious effects.

Crossover (CO) recombination events between homologous chromosomes are required to form chiasmata, temporary connections between homologs that ensure their proper segregation at meiosis I1. Despite this requirement for COs and an excess of the double-strand DNA breaks (DSBs) that are the initiating events for meiotic recombination, most organisms make very few COs per chromosome pair2. Moreover, COs tend to inhibit the formation of other COs nearby on the same chromosome pair, a poorly understood phenomenon known as CO interference3,4. Here we show that the synaptonemal complex (SC), a meiosis-specific structure that assembles between aligned homologous chromosomes, both constrains and is altered by CO recombination events. Utilizing a cytological marker of CO sites in Caenorhabditis elegans5, we demonstrate that partial depletion of the SC central region proteins (SYPs) attenuates CO interference, elevating COs and reducing the effective distance over which interference operates, indicating that SYPs limit COs. Moreover, we show that COs are associated with a local 0.4-0.5 μm increase in chromosome axis length. We propose that meiotic CO regulation operates as a self-limiting system in which meiotic chromosome structures establish an environment that promotes CO formation, which in turn alters chromosome structure to inhibit other COs at additional sites.

Most organisms rely on interhomolog crossovers (COs) to ensure proper meiotic chromosome segregation but make few COs per chromosome pair. By monitoring repair events at a defined double-strand break (DSB) site during Caenorhabditis elegans meiosis, we reveal mechanisms that ensure formation of the obligate CO while limiting CO number. We find that CO is the preferred DSB repair outcome in the absence of inhibitory effects of other (nascent) recombination events. Thus, a single DSB per chromosome pair is largely sufficient to ensure CO formation. Further, we show that access to the homolog as a repair template is regulated, shutting down simultaneously for both CO and noncrossover (NCO) pathways. We propose that regulation of interhomolog access limits CO number and contributes to CO interference.

Proper histone levels are critical for transcription, chromosome segregation, and other chromatin-mediated processes(1-7). In Saccharomyces cerevisiae, the histones H2A and H2B are encoded by two gene pairs, named HTA1-HTB1 and HTA2-HTB2 (ref. 8). Previous studies have demonstrated that when HTA2-HTB2 is deleted, HTA1-HTB1 dosage compensates at the transcriptional level(4,9). Here we show that a different mechanism of dosage compensation, at the level of gene copy number, can occur when HTA1-HTB1 is deleted. In this case, HTA2-HTB2 amplifies via creation of a new, small, circular chromosome. This duplication, which contains 39 kb of chromosome II, includes HTA2-HTB2, the histone H3-H4 locus HHT1-HHF1, a centromere and origins of replication. Formation of the new chromosome occurs by recombination between two Ty1 retrotransposon elements that flank this region. Following meiosis, recombination between these two particular Ty1 elements occurs at a greatly elevated level in hta1-htb1Delta mutants, suggesting that a decreased level of histones H2A and H2B specifically stimulates this amplification of histone genes. Our results demonstrate another mechanism by which histone gene dosage is controlled to maintain genomic integrity.