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During mitosis and meiosis in the majority of eukaryotes, centromeric chromatin comprised of CENP-A nucleosomes and their reader CENP-C recruits components of the outer kinetochore to build an interface with spindle microtubules(1, 2). One exception is C. elegans oocyte meiosis, where outer kinetochore proteins form cup-like structures on chromosomes independently of centromeric chromatin(3). Here we show that the nucleoporin MEL-28 (ortholog of human ELYS) and CENP-C(HCP−4) act in parallel to recruit outer kinetochore components to oocyte meiotic chromosomes. Unexpectedly, co-inhibition of MEL-28 and CENP-C(HCP−4) resulted in chromosomes being expelled from the meiotic spindle prior to anaphase onset, a more severe phenotype than what was observed following ablation of the outer kinetochore(4, 5). This observation suggested that MEL-28 and the outer kinetochore independently link chromosomes to spindle microtubules. Consistent with this, the chromosome expulsion defect was observed following co-inhibition of MEL-28 and the microtubule-coupling KNL-1/MIS-12/NDC-80 (KMN) network of the outer kinetochore. Use of engineered mutants showed that MEL-28 acts in conjunction with the microtubule-binding NDC-80 complex to keep chromosomes within the oocyte meiotic spindle, and that this function likely involves the Y-complex of nucleoporins that associate with MEL-28; by contrast, the ability to dock protein phosphatase 1, shared by MEL-28 and KNL-1, is not involved. These results highlight nuclear pore-independent functions for a conserved nucleoporin and explain two unusual features of oocyte meiotic chromosome segregation in C. elegans – centromeric chromatin-independent outer kinetochore assembly, and dispensability of the outer kinetochore for constraining chromosomes in the acentrosomal meiotic spindle.

During meiosis, diploid organisms reduce their chromosome number by half to generate haploid gametes. This process depends on the repair of double strand DNA breaks as crossover recombination events between homologous chromosomes, which hold homologs together to ensure their proper segregation to opposite spindle poles during the first meiotic division. Although most organisms are limited in the number of crossovers between homologs by a phenomenon called crossover interference, the consequences of excess interfering crossovers on meiotic chromosome segregation are not well known. Here we show that extra interfering crossovers lead to a range of meiotic defects and we uncover mechanisms that counteract these errors. Using chromosomes that exhibit a high frequency of supernumerary crossovers in Caenorhabditis elegans, we find that essential chromosomal structures are mispatterned in the presence of multiple crossovers, subjecting chromosomes to improper spindle forces and leading to defects in metaphase alignment. Additionally, the chromosomes with extra interfering crossovers often exhibited segregation defects in anaphase I, with a high incidence of chromatin bridges that sometimes created a tether between the chromosome and the first polar body. However, these anaphase I bridges were often able to resolve in a LEM-3 nuclease dependent manner, and chromosome tethers that persisted were frequently resolved during Meiosis II by a second mechanism that preferentially segregates the tethered sister chromatid into the polar body. Altogether these findings demonstrate that excess interfering crossovers can severely impact chromosome patterning and segregation, highlighting the importance of limiting the number of recombination events between homologous chromosomes for the proper execution of meiosis., Author summary Meiosis is a process that ensures developing eggs and sperm contain the correct number of chromosomes. Failure to accurately segregate chromosomes during meiosis is one of the leading causes of birth defects and miscarriages. During meiosis, crossover events must form between the homologous chromosomes to ensure proper chromosome segregation. Although crossover events are required for proper chromosome segregation in most organisms, crossover numbers are limited even when the meiotic cell is overloaded with DNA breaks, the initiating events for crossovers. This stringent limitation of crossovers in multiple organisms suggests that there are negative consequences to having too many crossovers, but this has not been formally tested. In this study, we find that increasing crossover number negatively impacts chromosome segregation during meiosis by altering chromosome-associated structures, which leads to misalignment of the chromosomes on the meiotic spindle. Moreover, chromosomes with excess crossovers often have large chromatin bridges during the chromosome segregation process, but we find that these bridges can be corrected by at least two mechanisms. Our results thus highlight the importance of limiting crossover numbers to enable faithful chromosome segregation during sperm and egg development.

The processes that govern fracture repair rely on many mechanisms that recapitulate embryonic skeletal development. Hox genes are transcription factors that perform critical patterning functions in regional domains along the axial and limb skeleton during development. Much less is known about roles for these genes in the adult skeleton. We recently reported that Hox11 genes, which function in zeugopod development (radius/ulna and tibia/fibula), are also expressed in the adult zeugopod skeleton exclusively in PDGFRα + /CD51 + /LepR+ mesenchymal stem/stromal cells (MSCs). In this study, we use a Hoxa11eGFP reporter allele and loss-of-function Hox11 alleles, and we show that Hox11 expression expands after zeugopod fracture injury, and that loss of Hox11 function results in defects in endochondral ossification and in the bone remodeling phase of repair. In Hox11 compound mutant fractures, early chondrocytes are specified but show defects in differentiation, leading to an overall deficit in the cartilage production. In the later stages of the repair process, the hard callus remains incompletely remodeled in mutants due, at least in part, to abnormal bone matrix organization. Overall, our data supports multiple roles for Hox11 genes following fracture injury in the adult skeleton.This article is protected by copyright. All rights reserved

Posterior Hox genes (Hox9-13) are critical for patterning the limb skeleton along the proximodistal axis during embryonic development. Here we show that Hox11 paralogous genes, which developmentally pattern the zeugopod (radius/ulna and tibia/fibula), remain regionally expressed in the adult skeleton. Using Hoxa11EGFP reporter mice, we demonstrate expression exclusively in multipotent mesenchymal stromal cells (MSCs) in the bone marrow of the adult zeugopod. Hox-positive cells express PDGFRα and CD51, are marked by LepR-Cre, and exhibit colony-forming unit fibroblast activity and tri-lineage differentiation in vitro. Loss of Hox11 function leads to fracture repair defects, including reduced cartilage formation and delayed ossification. Hox mutant cells are defective in osteoblastic and chondrogenic differentiation in tri-lineage differentiation experiments, and these defects are zeugopod specific. In the stylopod (humerus and femur) and sternum, bone marrow MSCs express other regionally restricted Hox genes, and femur fractures heal normally in Hox11 mutants. Together, our data support regional Hox expression and function in skeletal MSCs.

The requirements for oocyte meiotic cytokinesis during polar body extrusion are not well understood. In particular, the relationship between the oocyte meiotic spindle and polar body contractile ring dynamics remains largely unknown. We have used live cell imaging and spindle assembly defective mutants lacking the function of CLASP/CLS-2, kinesin-12/KLP-18, or katanin/MEI-1 to investigate the relationship between meiotic spindle structure and polar body extrusion in C. elegans oocytes. We show that spindle bipolarity and chromosome segregation are not required for polar body contractile ring formation and chromosome extrusion in klp-18 mutants. In contrast, oocytes with similarly severe spindle assembly defects due to loss of CLS-2 or MEI-1 have penetrant and distinct polar body extrusion defects: CLS-2 is required early for contractile ring assembly or stability, while MEI-1 is required later for contractile ring constriction. We also show that CLS-2 both negatively regulates membrane ingression throughout the oocyte cortex during meiosis I, and influences the dynamics of the central spindle-associated proteins Aurora B/AIR-2 and MgcRacGAP/CYK-4. We suggest that proper regulation by CLS-2 of both oocyte cortical stiffness and central spindle protein dynamics may influence contractile ring assembly during polar body extrusion in C. elegans oocytes., Author summary The precursor cells that produce gametes—sperm and eggs in animals—have two copies of each chromosome, one from each parent. These precursors undergo specialized cell divisions that leave each gamete with only one copy of each chromosome; defects that produce incorrect chromosome number cause severe developmental abnormalities. In oocytes, these cell divisions are highly asymmetric, with extra chromosomes discarded into small membrane bound polar bodies, leaving one chromosome set within the much larger oocyte. How oocytes assemble the contractile apparatus that pinches off polar bodies remains poorly understood. To better understand this process, we have used the nematode Caenorhabditis elegans to investigate the relationship between the bipolar structure that separates oocyte chromosomes, called the spindle, and assembly of the contractile apparatus that pinches off polar bodies. We used a comparative approach, examining this relationship in three spindle assembly defective mutants. Bipolar spindle assembly and chromosome separation were not required for polar body extrusion, as it occurred normally in mutants lacking a protein called KLP-18. However, mutants lacking the protein CLS-2 failed to assemble the contractile apparatus, while mutants lacking the protein MEI-1 assembled a contractile apparatus that failed to fully constrict. We also found that CLS-2 down-regulates membrane ingression throughout the oocyte surface, and we discuss the relationship between oocyte membrane stiffness and polar body extrusion.

Development of the musculoskeletal system requires precise integration of muscles, tendons and bones. The molecular mechanisms involved in the differentiation of each of these tissues have been the focus of significant research; however, much less is known about how these tissues are integrated into a functional unit appropriate for each body position and role. Previous reports have demonstrated crucial roles for Hox genes in patterning the axial and limb skeleton. Loss of Hox11 paralogous gene function results in dramatic malformation of limb zeugopod skeletal elements, the radius/ulna and tibia/fibula, as well as transformation of the sacral region to a lumbar phenotype. Utilizing a Hoxa11eGFP knock-in allele, we show that Hox11 genes are expressed in the connective tissue fibroblasts of the outer perichondrium, tendons and muscle connective tissue of the zeugopod region throughout all stages of development. Hox11 genes are not expressed in differentiated cartilage or bone, or in vascular or muscle cells in these regions. Loss of Hox11 genes disrupts regional muscle and tendon patterning of the limb in addition to affecting skeletal patterning. The tendon and muscle defects in Hox11 mutants are independent of skeletal patterning events as disruption of tendon and muscle patterning is observed in Hox11 compound mutants that do not have a skeletal phenotype. Thus, Hox genes are not simply regulators of skeletal morphology as previously thought, but are key factors that regulate regional patterning and integration of the musculoskeletal system.

Anterior cruciate ligament (ACL) ruptures rank among the most prevalent and costly sports-related injuries. Current tendon grafts used for ACL reconstruction are limited by suboptimal biomechanical properties. We have addressed these issues by engineering multiphasic bone-ligament-bone (BLB) constructs that develop structural and mechanical properties similar to native ACL. The purpose of this study was to examine the acute remodeling process that occurs as the BLB grafts advance toward the adult ligament phenotype in vivo. Thus, we implanted BLB constructs fabricated from male cells into female host sheep and allowed 3, 7, 14, or 28 days (n = 4 at each time point) for recovery. To address whether or not graft-derived cells were even necessary, a subset of BLB constructs (n = 3) were acellularized, implanted, and allowed 28 days for recovery. At each recovery time point, the following histological analyses were performed: picrosirius red staining to assess collagen alignment and immunohistochemistry to assess both graft development and host immune response. Polymerase chain reaction (PCR) analysis, performed on every explanted BLB, was used to detect the presence of graft-derived male cells remaining in the constructs and/or migration into surrounding host tissue. The analysis of the PCR and histology samples revealed a rapid migration of host-derived macrophages and neutrophils into the graft at 3 days, followed by increased collagen density and alignment, vascularization, innervation, and near complete repopulation of the graft with host cells within 28 days. This study provides a greater understanding of the processes of ligament regeneration in our BLB constructs as they remodel toward the adult ligament phenotype.