Loss of ninein interferes with osteoclast formation and causes premature ossification

  1. Thierry Gilbert
  2. Merlin Barbier
  3. Benjamin Duployer
  4. Ophélie Dufrançais
  5. Laure-Elene Martet
  6. Elisa Dalbard
  7. Loelia Segot
  8. Christophe Tenailleau
  9. Christel Vérollet
  10. Christiane Bierkamp
  11. Andreas Merdes

  • Curated by eLife

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    This valuable study offers new insight into the role of centrosome protein ninein in skeletal development through an analysis of the skeletal phenotype of ninein-deficient mice. While there is solid evidence supporting the conclusion that the absence of ninein leads to transient skeletal abnormalities and a lasting reduction in osteoclastogenesis, the evidence to substantiate the claim that enhanced ossification is attributed to reduced osteoclast formation/activity is insufficient. This work will be of interest to scientists in bone biology and skeletal development field.

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Abstract

Ninein is a centrosome protein that has been implicated in microtubule anchorage and centrosome cohesion. Mutations in the human ninein gene have been linked to Seckel syndrome and to a rare form of skeletal dysplasia. However, the role of ninein in skeletal development remains unknown. Here, we describe a ninein knockout mouse with advanced endochondral ossification during embryonic development. Although the long bones maintain a regular size, the absence of ninein delays the formation of the bone marrow cavity in the prenatal tibia. Likewise, intramembranous ossification in the skull is more developed, leading to a premature closure of the interfrontal suture. We demonstrate that ninein is strongly expressed in osteoclasts of control mice, and that its absence reduces the fusion of precursor cells into syncytial osteoclasts. As a consequence, ninein-deficient osteoclasts have a reduced capacity to resorb bone. At the cellular level, the absence of ninein interferes with centrosomal microtubule organization, reduces centrosome cohesion, and provokes the loss of centrosome clustering in multinucleated mature osteoclasts. We propose that centrosomal ninein is important for osteoclast fusion, to enable a functional balance between bone-forming osteoblasts and bone-resorbing osteoclasts during skeletal development.

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  1. eLife

    Author response:

    Reviewer #1 (Public Review):

    “… it remains unclear how ninein reduction causes bone defects …”

    We have added several control experiments that permit us to conclude that osteoblast numbers remain unaltered in the ninein-knockout embryos, and that bone abnormalities in vivo are caused by fusion defects of osteoclast precursor cells, whereas the proliferation, viability, or the adhesion of these precursor cells remain unaffected. For details, please see our comments below.

    “Discussion includes several unfounded potential mechanisms that really need to be thoroughly analyzed to gain a mechanistic understanding of the bone defects…”

    The new data back up our claim of fusion defects as a cause for limited osteoclast function. We have re-written parts of the discussion, to take into account our new findings.

    “Data showing normal osteoblasts in ninein-null mice was qualitative and requires further in-depth analysis and quantification of osteoblast …”

    To address this point, quantification of osteoblast numbers in tibiae at E16.5 and E18.5 was performed in control and ninein-deleted mouse embryos. The data are presented in the new Figures 3G and J.

    “In ninein knock-out mice, reduced TRAP+ve multinuclear cells were observed (Figure 6A and 6B). However, the magnitude of difference (about 5% decrease in multinucleated cells) is not consistent with the skeletal deformities reported in Figures 2-4, potentially suggesting the contribution of additional mechanisms.”

    We agree that the difference appears to be small at first glance, but nevertheless it remains statistically significant (a more than three-fold difference). We would like to recall that these observations (Fig. 6A) were performed at E14.5, i.e. at a stage when no ossification has occurred yet. We are looking at the first fusion events of myeloid precursors, likely derived from the fetal liver, that colonize the area of the first bone to form, and small differences in the number of functional osteoclasts may account for different timing of ossification. We think that differences in osteoclast fusion also account for the premature appearance of ossification centers for other skeletal elements, at later time points during development.

    “The fusion assay in Figure 6C needs further clarification. How was the syncytia perimeter defined to measure cell surface? The x-axis suggests that there are syncytia that contain up to 160 nuclei at day 3. How were the nuclei differentially stained and quantified?”

    We provide now additional information on the experimental approach in the revised manuscript, on pages 16-17 (Materials and Methods). For information: high numbers of syncytial nuclei in cultures were also observed by other groups in the past (Tiedemann et al., 2017, Front Cell Dev Biol. 5:54). In addition, we performed new experiments and quantified the fusion of osteoclast precursors by staining for actin and nuclei (new Figure 7C). This allowed us to quantify several additional parameters related to cell fusion (as initially performed in Raynaud-Messina et al., 2018, PNAS, 115:E2556-E2565).

    “Some text needs clarification. … What is the definition of "large syncytia"? Is the fusion index increase by day 5 diminished in later days? A graph of the syncytia size/ nuclei number or fusion index in the above-mentioned days will be helpful.”

    Information on the definition of “large syncytia” is now provided on page 10 (1st paragraph). We added further experimental details on osteoclast size for days 3, 4, and 5 in the supplemental Figures 7A and B. Most importantly, we performed additional assays of the fusion index by quantifying syncytial versus non-syncytial nuclei in a semi-automated manner. The new data are presented in Figure 7C, and the methods are explained on page 17. Together with our new analysis of cell proliferation, cell viability, and cell adhesion (Figure 7C, D, suppl. Fig. 7C-G), we provide now solid evidence for a fusion defect at the origin of impaired formation of ninein del/del osteoclasts.

    “Assessment of resorption was qualitative in Figure 6E and since the fusion deficiencies are transient, quantification of a corresponding resorption activity is needed. This should be described in the Materials and Methods section.”

    Quantifications of the bone resorption activities are now provided in the new Figure 7E, and a reference for the methods is provided on page 16.

    “Further experiments are needed to show connections between reduced centrosome clustering and reduced osteoclast formation as there is no evidence to date that suggest centrosome clustering is required for cell fusion. Multi-color live imaging and dynamic analysis can be used to determine if the ninein deficient cells show defective movement/migration/ fusion dynamics.”

    We agree that it is an important question, and studying potential links between centrosomal microtubule organization and osteoclast fusion is an ongoing project of the team. However, we estimate that in order to obtain conclusive results this will require 1-2 additional years of research activity, and we intend to present this as a separate project in the future. At the current point of our investigation, we think that providing a solid link between ninein, osteoclast fusion, and controlled timing of ossification, as shown in this manuscript, represents valuable progress to understand previously published bone abnormalities in patients with ninein mutations.

    “Quantification of the % of multinucleated osteoclasts that contain clustered and dispersed centrosomes is needed.”

    New quantification experiments on centrosome clustering are now provided in Figure 8H. These quantifications demonstrate that the potential of centrosome clustering is almost completely lost in osteoclasts without ninein.

    Reviewer #2 (Public Review):

    “Based on the decrease in the number of osteoclasts (Fig 5E, G, and also per coverslip after 2 days in culture), the authors suggest that the loss of ninein impacts osteoclast proliferation. First, proliferation can be directly quantified using Ki67 staining or EdU incorporation. Second, other interpretations are also plausible and can also be experimentally tested. These include less adhesion and attachment of the mutants to the coverslips, but perhaps more relevant in vivo is cell death of the ninein mutant osteoclasts. It has been established that the loss of centrosome function activates p53- dependent cell death and osteoclasts might be a vulnerable cell population. Quantifying p53 immunoreactivity and/or cell death in osteoclasts might help clarify the phenotype of osteoclast reduction.”

    In response to the reviewers, we have performed a series of new experiments that include

    1. A careful analysis of the fusion index, using a semi-automated approach, indicating significant differences in the fusion of precursor cells into osteoclasts (Fig. 7C).

    2. We have repeated the quantification of cell numbers prior to fusion and find variations between samples from different mice (also among mice of the same genotype), but we see on average comparable cell adhesion between samples from control mice and ninein-del/del mice. The data are provided in the supplemental Figure 7F. Moreover, we have quantified the expression of three main beta-integrins at the surface of control and ninein del/del osteoclast precursors (suppl. Fig. 7G), without detecting significant differences. Altogether, these data suggest the cell adhesion is comparable for the two genotypes.

    3. We have addressed the question of altered cell proliferation, by performing flow cytometry experiments and by quantifying the different cell cycle stages (Fig. 7D), and by quantifying Ki67 expression (suppl. Fig. 7C). We see no significant differences between samples from control and ninein-del/del mice.

    4. We have addressed the question of cell death, by performing Annexin V staining and flow cytometry (suppl. Fig. 7D), and by immunoblotting for cleaved caspase 3 and PARP (suppl. Fig. 7E). These experiments reveal no significant differences between the control and ninein del/del samples. Our data permit us to exclude cell death as a likely cause for the reduction of fused osteoclasts in the absence of ninein.

    Overall, the new experiments show that the defects in osteoclast formation from ninein-deleted samples are due to defects in cell fusion, but not in cell proliferation, cell adhesion or viability.

    Reviewer #3 (Public Review):

    “The authors put much emphasis on the centrosome in the Introduction session. However, it was not until Figure 7 did they show abnormal centriole clustering in osteoclasts. The introduction should include more background on osteoclast and osteoblast balance during skeletal development.”

    To address this, we included more background on the role of osteoclasts and osteoblasts in the revised introduction (page 4).

  2. eLife

    eLife assessment

    This valuable study offers new insight into the role of centrosome protein ninein in skeletal development through an analysis of the skeletal phenotype of ninein-deficient mice. While there is solid evidence supporting the conclusion that the absence of ninein leads to transient skeletal abnormalities and a lasting reduction in osteoclastogenesis, the evidence to substantiate the claim that enhanced ossification is attributed to reduced osteoclast formation/activity is insufficient. This work will be of interest to scientists in bone biology and skeletal development field.

  3. eLife

    Reviewer #1 (Public Review):

    The impact of this paper is that it shows conclusively the bone defects caused by ninein depletion, albeit transient defects, which has been indirectly deduced in past studies. The paper is largely descriptive including the cytoskeletal analysis of osteoclasts thus it remains unclear how ninein reduction causes bone defects and why this defect is transient. The Discussion includes several unfounded potential mechanisms that really need to be thoroughly analyzed to gain a mechanistic understanding of the bone defects in ninein-null mice.

    Other points:
    Data showing normal osteoblasts in ninein-null mice was qualitative and requires further in-depth analysis and quantification of osteoblast and osteocyte presence and activity in ninein del/del mice to strengthen the study.

    In ninein knock-out mice, reduced TRAP+ve multinuclear cells were observed (Figure 6A and 6B). However, the magnitude of difference (about 5% decrease in multinucleated cells) is not consistent with the skeletal deformities reported in Figures 2-4, potentially suggesting the contribution of additional mechanisms.

    The fusion assay in Figure 6C needs further clarification. How was the syncytia perimeter defined to measure cell surface? The x-axis suggests that there are syncytia that contain up to 160 nuclei at day 3. How were the nuclei differentially stained and quantified?

    Some text needs clarification. For instance, "On days 3 and 4, we found only about half as many large syncytia in cultures from ninein-deleted mice, compared to controls, but on day 5 large syncytia lacking ninein exceeded 90% of control levels. Altogether, this suggests that fusion deficiencies are a transient phenomenon in in vitro-induced adult osteoclasts. On later days of culture, fusion efficiency started to diminish." What is the definition of "large syncytia"? Is the fusion index increase by day 5 diminished in later days? A graph of the syncytia size/ nuclei number or fusion index in the above-mentioned days will be helpful.

    Assessment of resorption was qualitative in Figure 6E and since the fusion deficiencies are transient, quantification of a corresponding resorption activity is needed. This should be described in the Materials and Methods section.

    Further experiments are needed to show connections between reduced centrosome clustering and reduced osteoclast formation as there is no evidence to date that suggest centrosome clustering is required for cell fusion. Multi-color live imaging and dynamic analysis can be used to determine if the ninein deficient cells show defective movement/migration/ fusion dynamics.

    Quantification of the % of multinucleated osteoclasts that contain clustered and dispersed centrosomes is needed.

  4. eLife

    Reviewer #2 (Public Review):

    The paper by Gilbert et al. is well-written in a detailed format and the authors are candid in their data interpretation by acknowledging that the described ninein bone defects are mild, transient, and do not lead to major long-lasting defects in adulthood.

    The main strength of the study is presenting a novel link between a centrosomal protein and osteoclasts in the mouse. However, the majority of the work is dedicated to describing the premature ossification phenotype and less attention is paid to how a centrosomal protein affects osteoclast proliferation, survival, and/or differentiation into mature osteoclasts.

    Based on the decrease in the number of osteoclasts (Fig 5E, G, and also per coverslip after 2 days in culture), the authors suggest that the loss of ninein impacts osteoclast proliferation. First, proliferation can be directly quantified using Ki67 staining or EdU incorporation. Second, other interpretations are also plausible and can also be experimentally tested. These include less adhesion and attachment of the mutants to the coverslips, but perhaps more relevant in vivo is cell death of the ninein mutant osteoclasts. It has been established that the loss of centrosome function activates p53-dependent cell death and osteoclasts might be a vulnerable cell population. Quantifying p53 immunoreactivity and/or cell death in osteoclasts might help clarify the phenotype of osteoclast reduction.

  5. eLife

    Reviewer #3 (Public Review):

    Ninein is a centrosome protein that has been implicated in microtubule anchorage and centrosome cohesion. Mutations in the human ninein gene have been linked to Seckel syndrome and a rare form of skeletal dysplasia. However, the role of ninein in skeletal development remains unknown. Here, we describe a ninein knockout mouse with advanced endochondral ossification during embryonic development. Although the long bones maintain a regular size, the absence of ninein delays the formation of the bone marrow cavity in the prenatal tibia. Likewise, intramembranous ossification in the skull is more developed, leading to a premature closure of the interfrontal suture. We demonstrate that ninein is strongly expressed in osteoclasts of control mice and that its absence reduces the fusion of precursor cells into syncytial osteoclasts. As a consequence, ninein-deficient osteoclasts have a reduced capacity to resorb bone. At the cellular level, the absence of ninein interferes with
    centrosomal microtubule organization, reduces centrosome cohesion, and provokes the loss of centrosome clustering in multinucleated mature osteoclasts. We propose that centrosomal ninein is important for osteoclast fusion, to enable a functional balance between bone-forming osteoblasts and bone-resorbing osteoclasts during skeletal development.

  6. Version published to 10.1101/2023.11.03.565572v1 on bioRxiv