ABSTRACT

   In mammals, osteoclasts differentiate from macrophages in the monocyte
   lineage. Although many factors driving osteoclast formation are known,
   the detailed processes underlying precursor recruitment,
   differentiation, and interaction of macrophages with other cell types
   involved in bone remodeling are poorly understood. Using live imaging
   in a transgenic medaka osteoporosis model, where ectopic osteoclasts
   are induced by RANKL expression, we show that a subset of macrophages
   is recruited to bone matrix to physically interact with bone‐forming
   osteoblast progenitors. These macrophages subsequently differentiate
   into cathepsin K‐ (ctsk‐) positive osteoclasts. One day later, other
   macrophages are recruited to clear dying osteoclasts from resorbed bone
   by phagocytosis. To better understand the molecular changes underlying
   these dynamic processes, we performed transcriptome profiling of
   activated macrophages upon RANKL induction. This revealed an
   upregulation of several bone‐related transcripts. Besides osteoclast
   markers, we unexpectedly also found expression of osteoblast‐promoting
   signals in activated macrophages, suggesting a possible non‐cell
   autonomous role in osteogenesis. Finally, we show that macrophage
   differentiation into osteoclasts is dependent on inflammatory signals.
   Medaka deficient for TNFα or treated with the TNFα‐inhibitor
   pentoxifylline exhibited impaired macrophage recruitment and osteoclast
   differentiation. These results show the involvement of inflammatory
   signals and the dynamics of a distinct subset of macrophages during
   osteoclast formation. © 2020 The Authors. JBMR Plus published by Wiley
   Periodicals LLC on behalf of American Society for Bone and Mineral
   Research.

   Keywords: MACROPHAGES, OSTEOCLASTS, RANKL, TNFα, BONE RESORPTION, BONE
   HOMEOSTASIS
     __________________________________________________________________

   Using live imaging in a medaka fish osteoporosis model, we report the
   dynamic recruitment of macrophages to bone matrix, their close
   interaction with osteoblasts and subsequent differentiation into
   osteoclasts. These processes require TNFa and involve significant
   transcriptomic changes as activated macrophages switch from an immune
   to a bone‐modulatory signature. Based on this signature, we propose a
   non‐cell autonomous role of RANKL‐activated macrophages in promoting
   cartilage and bone formation to achieve bone homeostasis.

   graphic file with name JBM4-4-e10409-g007.jpg

Introduction

   Since their initial description in 1883,^( [36]^1 ^) macrophages have
   been intensively studied for their involvement in various physiological
   processes including embryogenesis, infection, tissue regeneration, and
   bone homeostasis (reviewed in Wynn and colleagues^( [37]^2 ^)). Bone
   marrow‐derived monocytes and macrophages have been identified as
   precursors of cathepsin K‐ (ctsk‐) positive osteoclasts, which digest
   mineralized matrix and thereby function in bone resorption.^( [38]^3 ,
   [39]^4 ^) On the other hand, osteal macrophages, a subpopulation of
   specialized bone‐homing macrophages, were shown to have essential roles
   in bone formation during regeneration and fracture healing.^( [40]^5 ,
   [41]^6 ^) This cell type also modulates cytokine expression and
   performs clearance of apoptotic cells in a process known as
   efferocytosis.^( [42]^7 ^) During an inflammation episode, the
   activation of macrophages often includes dynamic regulation of
   proinflammatory cytokines, especially IL1β and TNFα. These cytokines
   trigger an immune response and recruit other immune cells to
   inflammation sites.^( [43]^7 , [44]^8 ^) TNFα, a member of the tumor
   necrosis factor superfamily, is produced by different cell types such
   as monocytes, neutrophils, and adipocytes.^( [45]^9 , [46]^10 ^) It
   plays important roles in cell proliferation, wound healing, and cancer
   progression.^( [47]^8 , [48]^11 ^) TNFα is also found predominantly
   expressed in activated macrophages in inflammatory diseases such as
   rheumatoid arthritis.^( [49]^12 ^) Inhibition of this cytokine has been
   an effective therapy to alleviate bone inflammatory symptoms.^( [50]^13
   ^) Patients with osteoporosis, who also have elevated serum levels of
   IL1β and TNFα, often experience severe bone loss and fractures that
   result from hyperactivity of osteoclasts.^( [51]^14 ^) Several studies
   have shown that in the presence of macrophage colony stimulating factor
   (M‐CSF), TNFα and RANKL synergistically or independently stimulate
   osteoclast formation via their respective receptors.^( [52]^15 ,
   [53]^16 , [54]^17 ^) However, how these cytokines control the dynamics
   of osteoclast precursor recruitment and differentiation in vivo remains
   unclear, mostly because commonly used animal models have limited
   accessibility for live imaging.

   In this study, we used a transgenic medaka osteoporosis model, where
   excessive osteoclast formation is triggered by inducible RANKL
   expression.^( [55]^18 ^) Upon RANKL induction, macrophages migrate to
   the vertebral column where they eventually mature into ctsk‐positive
   osteoclasts.^( [56]^19 ^) In the present study, we describe the
   dynamics of macrophage migration and their close interaction with
   bone‐lining osteoblasts. We report that tnfa is upregulated in
   activated macrophages during recruitment and that deletion of tnfa or
   treatment with the tnfa transcription inhibitor pentoxifylline (PTX)
   blocks macrophage recruitment and osteoclast differentiation.
   Transcriptome profiling further revealed that the cytokine receptor
   gene il22ra2b was downregulated when macrophages differentiate into
   osteoclasts. Together, these findings suggest that inflammatory signals
   are initially required for macrophage recruitment towards bone matrix
   under osteoporotic conditions. Subsequently, these signals are
   downregulated to allow differentiation of recruited cells into
   osteoclasts. Interestingly, we also find that upon RANKL induction,
   macrophages upregulate genes that are known to control chondrocyte and
   osteoblast differentiation in a non‐cell autonomous manner. This
   suggests an involvement of activated macrophages in bone remodeling and
   homeostasis under pathological conditions.

Materials and Methods

   All experiments were performed according to protocols approved by the
   Institutional Animal Care and Use Committee (IACUC) of the National
   University of Singapore (NUS; protocol numbers R14‐293, R18‐0562, and
   BR15‐0119).

Transgenic and mutant medaka lines

   Transgenic rankl:HSE:cfp/ctsk:GFP medaka embryos were grown and
   subjected to RANKL induction by heat‐shock as previously described.^(
   [57]^18 ^) Generation of the mpeg1:mCherry‐F macrophage reporter line
   was described before.^( [58]^19 ^) Briefly, a 2.1‐kb sequence of the
   mpeg1 promoter including the endogenous ATG was amplified from genomic
   DNA of WT medaka using primers mpeg1.2F and mpeg1.2R (Supplementary
   Table [59]S1). The PCR product was digested with NotI and BamHI and
   then ligated in‐frame to a farnesylated mCherry in a pI‐SceI plasmid.
   The plasmid was microinjected into one‐cell stage medaka embryos
   together with meganuclease I‐SceI enzyme as previously described.^(
   [60]^20 ^) Injected embryos were screened for mCherry‐F transgene
   expression in macrophages, and stable lines were established and
   verified by in situ hybridization. For the generation of tnfa mutants,
   guide RNAs (gRNAs) targeting exons 1, 3, and 4 of tnfa were designed
   using CCTop^( [61]^21 ^) and synthesized by IDT (Singapore; Fig.
   [62]S8; Supplementary Table [63]S1). The three gRNAs were mixed with
   tracrRNA and HiFi Cas9 protein (IDT) and injected into one‐cell stage
   medaka embryos. Injected fish and their offspring were genotyped using
   primers listed in Supplementary Table [64]S1, and stable mutant lines
   were established.

Macrophage depletion and drug treatment

   For macrophage depletion, rankl:HSE:cfp/mpeg1:mCherry‐F/ctsk:GFP
   embryos at 9 days post fertilization (dpf) were injected intravenously
   with 15‐nL lipo‐clodronate (5 mg/mL) (Lipo‐Clo) or Lipo‐PBS (Encapsula
   NanoSciences, Brentwood, TN, USA). Macrophage‐ablated embryos were
   selected at 10 dpf based on the reduced fluorescence signal of
   mCherry‐F reporter. For inhibition of tnfa expression, RANKL‐induced
   embryos after heat‐shock were immediately transferred to fish medium
   (30% Danieau's solution containing 19.3mM NaCl, 0.23mM KCl, 0.13mM
   MgSO[4], 0.2mM Ca(NO[3])[2], 1.7mM HEPES, pH 7.0) supplemented with
   200μM PTX (Sigma‐Aldrich, St. Louis, MO, USA) or 0.05% DMSO as control.
   The medium was changed daily with fresh drug added for the whole course
   of the experiments.

In situ hybridization

   Sense and antisense riboprobes spanning 790 nucleotides of the mpeg1
   cDNA (nt1029‐nt1818; ENSORLT00000006101.2; the Ensembl database
   project, [65]http://www.ensembl.org/) were synthesized using a DIG RNA
   labeling kit (Roche Diagnostics, Mannheim, Germany). In situ
   hybridization was performed as previously described.^( [66]^18 ^)

Bone staining

   Alizarin Red staining of mineralized bone matrix in fixed embryos was
   performed as previously described.^( [67]^22 ^) Briefly, larvae at
   3 days post heat‐shock (dphs) were fixed with 4% paraformaldehyde,
   washed three times with PBS solution plus Tween‐20 (PBST), incubated
   for 15 min in 0.5% potassium hydroxide (KOH), then transferred to
   0.001% Alizarin Red solution in 0.5% KOH for at least 4 hours with
   agitation. Samples were washed for 2 hours with 0.5% KOH, followed by
   depigmentation with 2% H[2]O[2] in 0.5% KOH for 1 hour. Embryos were
   washed two times with 0.5% KOH and mounted in 100% glycerol for
   imaging. For live bone staining, embryos were incubated in 0.01%
   calcein (Sigma‐Aldrich) and kept in the dark for at least 1 hour at
   30°C. Embryos were washed twice with fish medium and mounted in
   low‐melting agarose for imaging.

Cryosectioning and immunostaining

   rankl:HSE:cfp/ctsk:GFP transgenic embryos were fixed in 4%
   paraformaldehyde, washed with PBST, and mounted in 1.5% low‐melting
   agarose in 5% sucrose. The set agar was kept overnight at 4°C in 30%
   sucrose before cryosectioning using a Cryostat 1850 (Leica, Wetzlar,
   Germany). Sections of 20 μm were collected on SuperFrost Plus slides
   (Thermo Fisher Scientific, Waltham, MA, USA) and dried at room
   temperature. For immunostaining, sections were incubated in 10% goat
   serum in PBST (3.2mM Na[2]HPO[4], 0.5mM KH[2]PO[4], 1.3mM KCl, 135mM
   NaCl, and 0.05% Tween 20) for 1 hour at room temperature and incubated
   overnight at 4°C with a 1:100 dilution of cleaved‐caspase‐3 antibody
   (Asp175; Cell Signaling Technology, Beverly, MA, USA) in blocking
   buffer. Alexa Fluor 633 IgG goat anti‐rabbit antibody (Invitrogen,
   Carlsbad, CA, USA) was used as secondary antibody at a 1:500 dilution.
   Samples were washed with PBS, stained with
   4,6‐diamidino‐2‐phenylindole, and kept in Mowiol (Sigma‐Aldrich) until
   imaging.

Imaging of live and fixed samples

   For live imaging, embryos were anesthetized in 0.016% tricaine (MS‐222;
   Sigma‐Aldrich) and embedded in 1.2% low melting agarose (Bio‐Rad, USA)
   in 35 mm glass‐bottom dishes (Eppendorf, Hamburg, Germany), and covered
   with 2‐mL fish medium containing tricaine. A Nikon SMZ18
   epi‐fluorescence microscope was used for capturing whole‐embryo images.
   Z‐stack images and time‐lapse movies were recorded using a Nikon FV3000
   confocal microscope (Nikon, Minato City, Tokyo, Japan) equipped with a
   ×30/1.05, WD 0.8 silicon oil objective. Time‐lapse files were processed
   using Imaris (Bitplane AG, Zurich, Switzerland) and Fiji (ImageJ; NIH,
   Bethesda, MD, USA; [68]https://imagej.nih.gov/ij/) software to generate
   movies. Z‐stack images were processed into maximum intensity
   projections and channels merged using Fiji. Alizarin Red‐stained bone
   samples were imaged using a Nikon Eclipse 90i upright microscope
   equipped with NIS‐Elements version BR 3.0.

Cell quantification and statistics

   Fluorescent images of whole embryos and confocal z‐stacks were used for
   the quantification of macrophage and osteoclast numbers as previously
   described^( [69]^23 ^) with slight modifications. Using Fiji,
   fluorescent images were converted to greyscale, thresholded, and the
   area of a cell (averaged from 15 cells from three embryos), as well as
   the total area of cells in a selected region of interest, were derived.
   Cell numbers were determined by dividing the total area of cells with
   the average cell area. For statistical analysis, Student's t test
   (two‐tailed, unpaired) was performed using GraphPad Prism 8.0 (GraphPad
   Software, La Jolla, CA, USA).

Fluorescence‐activated cell sorting, RNA sequencing, and bioinformatics
analysis

   FACS and RNA extraction were performed using previously described
   protocols^( [70]^24 ^) with some modifications. RNA library
   construction, Illumina sequencing, and bioinformatics analysis were
   performed by Novogene (Singapore) following company procedures.
   Briefly, RANKL expression was induced in
   rankl:HSE:cfp/mpeg1:mCherry‐F/ctsk:GFP transgenic medaka embryos at 9
   dpf. At 1 day post heat shock (dphs), 23 larvae per sample were
   dissociated in 500 μL of 0.25% trypsin EDTA (HyClone; HyClone
   Laboratories, South Logan, UT, USA) plus collagenase (0.2 mg/mL) at
   30°C and pipetted every 10 min for 40 min. The digestion was stopped by
   adding 50‐μL sterile‐filtered FBS. Cells were washed with PBS
   supplemented with 2% FBS and resuspended in 400‐μL Leibovitz's L‐15
   medium (Gibco; Thermo Fisher Scientific) for FACS using a BD FACSAria
   II platform (BD Biosciences, San Jose, CA, USA). Fluorescent
   macrophages and osteoclasts were separated and collected into 400‐μL
   TRIzol (Thermo Fisher Scientific). RNA extraction was done from three
   independent biological replicates using PureLink RNA Micro kit (Thermo
   Fisher Scientific). The RNA quality was determined with an Agilent
   Bioanalyzer 2100 using Agilent RNA 6000 Pico kit (Agilent Technologies,
   Santa Clara, CA, USA). Samples with RNA integrity number (RIN) values
   >7 were submitted for sequencing. Clean reads were aligned to the
   reference genome for Japanese medaka HdrR, version ASM223467v1
   ([71]http://ensembl.org/) using TopHat v2.0.12. Differential expression
   analysis of two biological replicates per condition was performed using
   Deseq R package (1.18.0). P values were adjusted using Benjamini and
   Hochberg's approach and genes with P values <0.05 were assigned as
   differentially expressed. Gene ontology (GO) enrichment of
   differentially expressed genes was analyzed using GOseq R package and
   only GO terms with a P value <0.05 were considered significantly
   enriched. ShinyGO V0.61 was used to analyze networks of functional
   groups of up‐ and downregulated genes. Input data were matched to human
   genes, the P‐value cutoff was 0.05, and the 30 most significant terms
   were used ([72]http://bioinformatics.sdstate.edu/go/).

Phylogeny and synteny analysis

   Amino acid sequences were retrieved from the National Center for
   Biotechnology Information (NCBI; [73]www.ncbi.nlm.nih.gov/) and ENSEMBL
   ([74]www.ensembl.org/Multi/Tools/Blast) databases using BLAST searches
   with default parameters. Sequences were aligned using Clustal W^(
   [75]^25 ^) and trees were constructed using the maximum likelihood
   method in MEGA version X^( [76]^26 ^) with the JTT model for aa
   substitutions and 1000 bootstrap iterations. Conserved synteny analysis
   was done using GENOMICUS version 100.01
   ([77]https://www.genomicus.biologie.ens.fr/genomicus-100.01/cgi-bin/sea
   rch.pl).^( [78]^27 ^)

Quantitative polymerase chain reaction

   For whole embryos, RNA was isolated from 10 embryos per sample using
   NucleoSpin RNA kit (Macherey‐Nagel, Duren, Germany), and total mRNA was
   reverse transcribed using the RevertAid First Strand cDNA Synthesis kit
   (Thermo Fisher Scientific). For RNA isolation from FAC‐sorted
   macrophages and osteoclasts, the NucleoSpin RNA XS kit (Macherey‐Nagel)
   was used. RNA isolated from three independent biological samples was
   converted into cDNA and preamplified using Fluidigm Reverse
   Transcription Master Mix and Preamp Master Mix, respectively (Fluidigm,
   South San Francisco, CA, USA). All steps were performed following the
   manufacturers' standard protocols. PowerUp SYBR Green Master Mix
   (Applied Biosystems, Foster City, CA, USA) was used for qPCR conducted
   in a CFX96 Touch system (Bio‐Rad Laboratories, Hercules, CA, USA). Data
   analysis was performed using Bio‐Rad's CFX Maestro 1.0 software with
   β‐actin as the loading control for normalization. Two‐tailed Student's
   t tests were performed for statistical analysis.

Results

RANKL triggers macrophage recruitment, proliferation, and differentiation at
bone matrix

   To study the dynamic behavior of macrophages by live imaging in medaka,
   we generated a reporter line expressing farnesylated mCherry under
   control of the medaka mpeg1 promoter (Fig. [79]1A ). At 10 dpf,
   mpeg1:mCherry‐F positive macrophages were found throughout the body
   with increased numbers in heart, liver, aorta‐gonad‐mesonephros (AGM)
   and caudal fin (Fig. [80]1B ). The mpeg1 transgenic line faithfully
   recapitulated endogenous mpeg1 transcription as confirmed by RNA in
   situ hybridization (Fig. [81]1C–G ). Next, mpeg1:mCherry‐F reporter
   fish were crossed with rankl:HSE:cfp/ctsk:GFP transgenic fish, which
   harbor a RANKL transgene under control of a bidirectional heat‐shock
   promoter that also drives cyan fluorescent protein (CFP) expression,
   and express green fluorescent protein (GFP) in osteoclasts under
   control of the ctsk promoter (for details, see To and colleagues^(
   [82]^18 ^)). Confocal time‐lapse imaging showed that in the absence of
   ectopic RANKL expression (–RANKL), the majority of
   mpeg1:mCherry‐F‐positive macrophages reside within the AGM with only
   few migratory cells found in other regions of the body, including the
   vertebral column (Supplementary Fig. [83]S1 A; [84]Movie S1). Shortly
   after RANKL induction (4 hours post heat‐shock [hphs]), mpeg1‐positive
   macrophages started to accumulate in the vertebral column (Fig.
   [85]2A,B ). Time‐lapse analysis revealed that the majority of mpeg1
   cells in the vertebral column originated from the AGM, but also cells
   from other regions were recruited (Supplementary Fig. [86]S1).
   Macrophages were almost exclusively found at the mineralized matrix of
   vertebral bodies, but not in the nonmineralized intervertebral discs
   (Fig. [87]2B ; Supplementary Fig. [88]S1 A; [89]Movie S2 ). In
   vertebral bodies, macrophages were in close contact with osteoblasts
   and osteoblast progenitors, which line the bone surface (Supplementary
   [90]Movie S7). At 30 to 64 hphs, the recruited macrophages gradually
   differentiated into ctsk‐positive osteoclasts (Fig. [91]2C,D ;
   Supplementary Movies [92]S3 and [93]S4).

Fig 1.

   Fig 1
   [94]Open in a new tab

   Generation of the mpeg1:mCherry‐F reporter line. (A) A farnesylated
   mCherry reporter is driven by a 2.1 kb mpeg1 promoter and flanked by
   I‐SceI sites. (B) At 10 days postfertilization (dpf), mCherry‐F–labeled
   macrophages are distributed throughout the body except for the
   notochord and spinal cord. (C–F) In situ hybridizations of 10 dpf
   embryos with a mpeg1 antisense probe on the transverse section of the
   anterior region (blue dotted line in B) (C), and a longitudinal section
   around the AGM‐CHT area (F). D shows high magnification of liver tissue
   boxed in C, whole‐mount fin region (E) arrows point to Mpeg1 positive
   signals; patterns are similar to the mCherry reporter signals. (G) In
   situ hybridizations with a mpeg1 sense probe on a control whole‐mount
   fin. NA = neural arch; NC = notochord; AGM = aorta–gonad–mesonephros;
   CHT = caudal hematopoietic tissue; CF = caudal fin; H = heart. Scale
   bar: 1 mm (in B); 200 μm (in C); 100 μm (in E–G).

Fig 2.

   Fig 2
   [95]Open in a new tab

   Macrophage recruitment and differentiation into osteoclasts in the
   medaka vertebral column. (A) Experimental scheme.
   rankl:HSE:cfp/mpeg1:mCherry‐F/ctsk:GFP transgenic medaka embryos were
   heat‐shocked to induce RANKL expression and subjected to confocal
   time‐lapse imaging. (B–D) Still images extracted from movies recorded
   during the indicated time frame (hours post heat‐shock, hphs).
   Macrophages are recruited towards the vertebral column as early as 2
   hphs and begin to differentiate into ctsk osteoclasts around 18 hphs,
   starting at the anterior region of the vertebral column. At 40 hphs,
   most of the recruited macrophages have differentiated into osteoclasts,
   while later recruited macrophages are located around the vertebral
   column to perform phagocytosis of dying cells. Blue dotted box in (A)
   shows imaged area shown in (B–D); white dotted boxes in (B) depict
   vertebral bodies. Mφ = macrophage; OC = osteoclast. Time: hh:mm:ss.
   Scale bar: 50 μm.

   Starting from 35 hphs, the first osteoclasts started to undergo cell
   death and became cleared by newly recruited phagocytic macrophages
   (Supplementary Fig. [96]S2; Movies [97]S3 and [98]S4). Quantification
   of mpeg1:mCherry‐F and ctsk:GFP cells showed high numbers of recruited
   macrophages and ectopic osteoclasts along the vertebral column of
   RANKL‐induced embryos compared with controls (Supplementary Fig.
   [99]S3). Osteoclast numbers gradually declined after 2 dphs, while new
   macrophages continued to migrate towards the vertebral column over the
   course of 1 week (data not shown). The total number of macrophages
   increased partly by steady‐state hematopoiesis, but also because of
   cell proliferation that occurred both in the AGM and around the
   vertebral column (Supplementary Fig. [100]S3; [101]Movie S7). Our
   findings suggest that in medaka ectopic RANKL expression triggers the
   activation of macrophages, stimulates their recruitment towards
   mineralized matrix in a directed manner, and promotes their
   differentiation into osteoclasts.

Macrophages reduce their dynamics during osteoclast maturation

   To quantitate the dynamics of macrophages during osteoclast
   differentiation, we analyzed time‐lapse movies of RANKL‐induced
   macrophages and osteoclasts recorded before and after differentiation.
   In an early phase at 4 hphs, macrophages in RANKL‐induced embryos
   exhibited higher speed and displacement when compared with –RANKL
   controls. A more angular and less spherical cell morphology was
   observed in +RANKL macrophages, indicating their motile state
   (Supplementary Fig. [102]S1 A). However, at 34 hphs, the motility of
   differentiating macrophages became reduced as they started to express
   ctsk:GFP, while undifferentiated macrophages remained highly mobile
   (Supplementary Fig. [103]S1 B; [104]Movie S4). This suggests that the
   reduction of macrophage dynamics is a prerequisite for osteoclast
   differentiation, possibly to allow tighter attachment of forming
   osteoclasts to bone matrix for subsequent cell fusion to generate
   multinucleated osteoclasts.

Depletion of macrophages prevents osteoclast formation

   As in mammals^( [105]^3 ^), medaka macrophages also differentiate into
   osteoclasts. We next determined whether osteoclasts also form in the
   absence of macrophages, indicating possible alternative cellular
   sources. We depleted macrophages by Lipo‐Clo injection into medaka
   embryos at 9 dpf and induced RANKL expression by heat‐shock at 10 dpf.
   In the absence of RANKL induction, Lipo‐Clo treatment did not cause any
   obvious changes to bone development (Fig. [106]3A–D ). After RANKL
   induction, at 1 and 2 dphs, Lipo‐PBS control‐injected embryos showed
   abundant macrophage recruitment and osteoclast formation along the
   vertebral column as expected (Fig. [107]3C–F ). In contrast, Lipo‐Clo
   treatment resulted in a 75% ablation of macrophages (Fig. [108]3C–E ).
   Only a few macrophages were recruited to the vertebral column, and
   osteoclast formation was strongly impaired (Fig. [109]3C–F ;
   Supplementary Movies [110]S5 and [111]S6). The effect of macrophage
   depletion on bone protection was then assessed using Alizarin Red
   staining at 3 dphs. After RANKL induction, the bone matrix of
   Lipo‐Clo–injected embryos was efficiently protected with only few minor
   defects, which was in stark contrast to the severe lesions formed along
   the vertebral columns of Lipo‐PBS–injected embryos (Fig. [112]3D ).
   This observation indicates that the integrity of a macrophage
   population is important for osteoclast formation.

Fig 3.

   Fig 3
   [113]Open in a new tab

   Macrophage depletion prevents osteoclast formation. (A) Experimental
   scheme. rankl:HSE:cfp/mpeg1:mCherry‐F/ctsk:GFP transgenic embryos are
   injected with lipo‐clodronate or Lipo‐PBS at 9 days postfertilization
   (dpf). At 10 dpf, embryos with efficient macrophage depletion were
   selected based on the quantity and morphology of mCherry signals, and
   then subjected to RANKL induction. (B) Lipo‐clodronate (Lipo‐Clo)
   injection causes macrophage death at 1 day after injection (round cells
   pointed by cyan arrowheads), while Lipo‐PBS injected embryos show
   normal macrophage morphology and distribution (white arrowheads). (C)
   Lipo‐PBS injected control embryos show normal macrophage recruitment
   and osteoclast differentiation at 1 day post heat‐shock (dphs; white
   arrowheads). In macrophage‐ablated embryos, osteoclast formation along
   the trunk is notably inhibited (cyan arrowheads). (D) At 2 dphs,
   abundant osteoclasts have formed in Lipo‐PBS–injected embryos (white
   arrowheads), while only single osteoclasts are seen in the anterior
   region of Lipo‐Clo–injected embryos (white arrows). Alizarin Red
   staining of mineralized bone matrix at 3 dphs reveals normal bone
   development in both Lipo‐PBS and Lipo‐Clo–treated embryos in the
   absence of RANKL induction. Upon RANKL induction, control larvae have
   severe lesions in the mineralized matrix (black arrows), while
   Lipo‐Clo–injected embryos show minor or no defects (blue arrows).
   Insets show individual vertebral bodies at higher magnification. (E,F)
   Quantification of cell numbers shows efficient depletion of macrophages
   and inhibition of osteoclast formation in Lipo‐Clo–treated embryos.
   N[Lipo‐PBS larvae] = 5–14, N[Lipo‐Clo larvae] = 6–19, from three
   independent experiments. Mφ = macrophage; OC = osteoclast. Scale bar:
   200 μm.

Transcriptome profiling reveals similarities between medaka and human
osteoclasts

   The behavior and dynamics of macrophages change rapidly during
   differentiation into osteoclasts ([114][Link], [115][Link]). To
   identify molecular signatures underlying these changes, we performed
   RNAseq analysis and compared transcriptome profiles of FACS‐purified
   macrophages (mpeg1 ^+/ctsk ^−cells) with those of osteoclasts (mpeg
   ^+/ctsk ^+ double‐positive cells) at 1 dphs. Analysis of differentially
   expressed genes (DEGs) revealed gene sets that are similarly regulated
   in human and mouse osteoclastogenesis. Conventional markers of
   osteoclastogenesis in mammals such as trap, ctsk, siglec15, nfatc1,
   tgfb1, and dap12 were highly upregulated in mpeg ^+/ctsk ^+
   double‐positive medaka cells, similar to the situation in human and
   mouse osteoclasts (Table [116]1). Our RNAseq datasets also indicated
   that genes encoding the medaka chemokine/cytokine receptors Cx3cr1,
   Il22ra2a, and Il22ra2b were downregulated in medaka osteoclasts, but
   significance could only be validated by qPCR analysis for il22ra2b
   (Supplementary Figs. [117][Link], [118][Link]; Table [119]1). The
   downregulation of cytokine receptor genes in our medaka model suggests
   a possible reduction of the motility of RANKL‐induced macrophages as
   they differentiate into osteoclasts.

Table 1.

   Selected Regulated Medaka Genes With Orthologs Involved in Mammalian
   Osteoporosis and Bone Loss
   Gene Description Reads Mφ Reads OC Log2 FC p adj value Known function
   in mammalian bone Ref
   trap Acid phosphatase 5a, tartrate resistant 1766 62181 5.1 0.000
   Modulation of osteoclast adhesion and resorption ^( [120]^65 ^)
   siglec15 Sialic acid binding Ig like lectin 15 131 3833 4.9 0.000
   Osteoclast differentiation ^( [121]^66 ^)
   nfatc1 Nuclear factor of activated T cells 1 139 3218 4.5 0.001
   Osteoclast fusion ^( [122]^67 , [123]^68 ^)
   a‐sdf1a/cxcl12 Stromal cell‐derived factor 1a 61 1233 4.3 0.001
   Osteoclast precursor recruitment ^( [124]^69 ^)
   mcoln3a Mucolipin 3a 72 1276 4.2 0.050 Triggering Ca^2+ release and
   influx during osteoclastogenesis and bone remodeling ^( [125]^70 ^)
   angptl7 Angiopoietin‐like 7 176 3074 4.1 0.000 Osteoclast stimulation.
   ^( [126]^71 , [127]^72 ^)
   tspan5 Tetraspanin 5 137 1213 3.2 0.043 Control of osteoclast fusion ^(
   [128]^73 ^)
   dap12/ tyrobp dap12/TYRO protein tyrosine kinase binding protein 642
   4642 2.9 0.000 Osteoclast differentiation and function ^( [129]^74 ^)
   LOC101158504 C‐X‐C motif chemokine 9 520 2681 2.3 0.01 Produced by
   osteoblasts and osteoclasts; induces osteoclast migration and adhesion.
   ^( [130]^75 ^)
   ctsk Cathepsin K 53392 164567 1.6 0.000 Protease for osteoclast
   resorptive activity ^( [131]^76 ^)
   ckba Creatine kinase B 4260 10737 1.3 0.025 Osteoclast resorption;
   crucial for Actin ring formation and bone resorption. ^( [132]^77 ^)
   gsna Gelsolin a 7076 16649 1.2 0.016 Assembly/disassembly of Actin
   filaments in osteoclast podosomes; cell migration ^( [133]^78 ^)
   LOC101163603 C‐C chemokine receptor type 1 341 0 inf 0.020 Osteoclast
   recruitment, cell motility ^( [134]^79 ^)
   socs1b Suppressor of cytokine signaling 1b 348 1 −9.4 0.020 Osteoclast
   formation by cytokine modulation ^( [135]^80 ^)
   sh2dp1 SH2 domain‐containing protein 1B 511 3 −7.4 0.010 Control of
   osteoclast resorptive activity and fusion ^( [136]^81 ^)
   fes FES proto‐oncogene, tyrosine kinase 823 20 −5.4 0.000 Osteoclast
   differentiation ^( [137]^82 ^)
   LOC105355996 Interleukin‐20 receptor subunit alpha 3043 205 −3.9 0.000
   Osteoclast differentiation ^( [138]^28 ^)
   sbno2 Strawberry notch homolog 2 1559 188 −3.1 0.020 Osteoclast fusion
   ^( [139]^83 ^)
   pip5k1ba Phosphatidylinositol‐4‐phosphate 5‐kinase, type I, beta a 2591
   427 −2.6 0.000 Modulation of osteoblast and osteoclast differentiation
   ^( [140]^84 ^)
   grna Granulin a 17770 7107 −1.3 0.010 Osteoclast formation ^( [141]^85
   ^)
   coro1a Coronin, Actin binding protein, 1A 29530 13890 −1.1 0.020
   Regulation of lysosomal secretion of cathepsin K ^( [142]^86 ^)
   [143]Open in a new tab

   Abbreviations: Mφ: macrophage, OC: osteoclast, FC: fold change.

RANKL‐induced macrophages acquire a bone‐remodeling signature

   Our DEG analysis identified more than 1,000 genes that were either
   commonly or uniquely expressed in macrophages before and after
   differentiation into osteoclasts (Fig. [144]4A ). Of the commonly
   expressed genes, 88 genes were significantly upregulated and 39 genes
   downregulated with adjusted P values of <0.05 (Fig. [145]4A,C ). A KEGG
   analysis of DEGs revealed an enrichment of the terms phagosome, focal
   adhesion, extracellular membrane‐receptor interaction, and
   cytokine‐cytokine receptor interaction (Fig. [146]4B ). Apart from
   genes for which the corresponding human orthologs have important
   functions in osteoclastogenesis, such as fes, sh2dp1, socs1b, tspan5,
   and mcoln3a (Fig. [147]4D ; Table [148]1), we observed other highly
   regulated genes with so far uncharacterized functions in
   osteoclastogenesis. This includes upregulation of emilin2a, which is
   known to block cancer cell proliferation;^( [149]^29 ^) and sfrp2,
   which controls normal osteoblast differentiation and exerts an
   antiapoptotic effect.^( [150]^30 , [151]^31 ^) Among downregulated
   genes, we identified gimap1‐like, which is essential for development of
   T and B lymphocytes; rad18, which is a key player in controlling DNA
   damage tolerance^( [152]^32 , [153]^33 ^); and ogdh, which is involved
   in glucose oxidation and cancer cell viability^( [154]^34 , [155]^35 ^)
   (Fig. [156]4D ). Next, we performed a GO analysis using ShinyGO v0.61.
   Individual lists of up‐ and downregulated genes were converted into
   human gene IDs and UniProtKB IDs using BioMart ([157]Ensembl.org).
   UniProtKB IDs were later used as input for the analysis.

Fig 4.

   Fig 4
   [158]Open in a new tab

   Transcriptome profiles of medaka macrophages and osteoclasts. (A) Venn
   diagram showing the total number of genes expressed in macrophages and
   osteoclasts at 1 day post heat‐shock (dphs), with the overlapping
   region showing commonly expressed genes. (B) KEGG pathway enrichment
   analysis of differentially expressed genes (DEGs) showing significantly
   enriched categories including phagosome, focal adhesion,
   cytokine–cytokine receptor interaction, and extracellular
   matrix‐receptor interaction. (C) Volcano plot showing up‐ and
   downregulated genes in osteoclasts compared with macrophages with
   adjusted p values <0.05 (p adj value <0.05). Insignificantly regulated
   genes are shown as blue dots. (D) List of genes with the highest
   regulation indicated by log[2]‐fold change; all genes have adjusted p
   values <0.05.

   The analysis of upregulated genes surprisingly showed a strong
   enrichment of pathways involved in chondrocyte and cartilage
   development, bone remodeling, and osteoblast differentiation. This
   included the collagen‐coding genes col6a2, col12a1, as well as the
   secreted osteoblast differentiation inducers tgfb1 and sfrp2
   (Supplementary Fig. [159]S4, Table [160]2).^( [161]^36 ^) Consistent
   also with earlier studies that reported expression of collagen genes in
   macrophages,^( [162]^37 , [163]^38 , [164]^39 ^) qPCR analysis
   confirmed the upregulation of col6a2 and col12a1, as well as integrin
   subunit beta 3 (itgb3) in RANKL‐induced macrophages and validated our
   RNAseq analysis. Interestingly, also genes encoding the respective
   complexing subunits, ie, col6a1, col6a3, itga5, and itga2.2, were
   upregulated (Fig. [165]S5 B).

Table 2.

   Upregulation of Osteoblast/Chondrocyte Promoting Genes in Medaka
   Osteoclasts
   Gene Description Reads Mφ Reads OC Log2 FC p adj value Protein
   localization Role in bone/cartilage formation Ref #
   itgb3 Integrin subunit beta 3 0 3016 inf[166] ^a 0.000 Transmembrane
   Corticalization for bone growth ^( [167]^87 ^)
   col6a2 Collagen alpha‐2(VI) chain 0 880 inf 0.000 Secreted Chondrocyte
   proliferation and cartilage generation ^( [168]^88 ^)
   foxc1 Forkhead box C1‐A 0 362 inf 0.007 Nuclear Osteoprogenitor
   proliferation; osteogenesis ^( [169]^89 ^)
   sfrp2 Secreted frizzled related protein 2 3 854 8 0.000 Secreted
   Osteogenic differentiation ^( [170]^90 ^)
   cthrc1 Collagen triple helix repeat containing 1 90 6684 6.21 0.000
   Secreted Osteoblast proliferation, bone cell coupling ^( [171]^91 ^)
   myoc Myocilin 61 1070 4.14 0.009 Secreted Osteoblast differentiation;
   bone remodeling ^( [172]^92 ^)
   col12a1 Collagen alpha‐1(XII) chain 83 1038 3.64 0.027 Secreted
   Osteoblast polarity, bone matrix ^( [173]^93 ^)
   tgfb1 Transforming growth factor beta 1 367 3291 3.16 0.000 Secreted
   Mesenchymal progenitor differentiation; chondrogenesis ^( [174]^94 ^)
   thbs1 Thrombospondin 1 426 2689 2.66 0.003 Secreted Mesenchymal
   progenitor differentiation; chondrogenesis ^( [175]^95 ^)
   ccn1 Cellular communication network factor 1 2434 14444 2.57 0.000
   Secreted Chondrocyte maturation ^( [176]^96 ^)
   cldn11 Claudin‐11 440 2538 2.53 0.006 Transmembrane Osteoblast
   differentiation via Notch ^( [177]^97 ^)
   [178]Open in a new tab
   ^^a

   Infinite.

   A larger fraction of downregulated genes was immune‐related and is
   known to be involved in the activation of the immune response of
   macrophages (Supplementary Fig. [179]S4 A). Our data suggest that
   macrophages are highly dynamic in regulating their gene expression, not
   only to favor bone‐resorbing activity, but also potentially to promote
   bone formation by secreting factors that trigger osteoblast
   differentiation.

Macrophage recruitment and differentiation are impaired in tnfa mutants

   Treatment of mouse bone marrow‐derived macrophages with the
   proinflammatory cytokine TNFα in the presence of M‐CSF and RANKL
   stimulates osteoclast differentiation in vitro.^( [180]^15 , [181]^16 ,
   [182]^17 ^) We therefore investigated the requirement of this
   proinflammatory cytokine for macrophage recruitment and osteoclast
   differentiation in vivo in the medaka osteoporosis model. First, we
   performed quantitative PCR analysis to test whether tnfa transcription
   is affected by heat‐shock induction and RANKL overexpression. Compared
   with non‐heat shocked controls, heat shocked −RANKL embryos showed a
   significant reduction of tnfa expression at 1, 2, and 3 dphs
   (Supplementary Fig. [183]S8 A). This is consistent with a
   temperature‐induced reduction of Tnfa transcription in mouse
   macrophages.^( [184]^40 ^) Importantly, however, RANKL induction
   (+RANKL) resulted in a significant upregulation of tnfa transcription
   suggesting that tnfa is involved in the osteoporotic response. To test
   this, we generated a tnfa medaka mutant using CRISPR/Cas9
   (Supplementary Fig. [185]S8 B–D). Similar to Tnfa mouse mutants, which
   show normal development,^( [186]^41 ^) stable CRISPR/Cas9 medaka tnfa
   mutants were viable and developed normally with no obvious alterations
   (data not shown). Mutants were crossed with
   rankl:HSE:cfp/mpeg1:mCherry‐F/ctsk:GFP transgenic fish, and macrophage
   behavior and osteoclast formation were tracked by live imaging as
   described above. In the absence of RANKL induction (–RANKL), tnfa
   mutants exhibited normal distribution and density of macrophages when
   compared with nonmutant siblings (Fig. [187]5A–C ). Upon RANKL
   induction (+RANKL), tnfa ^−/− mutant macrophages were recruited towards
   the vertebral column at 1 dphs, similar to WT macrophages. Also, the
   total number of macrophages was initially similar in both WT and mutant
   embryos at 1 dphs. At 2 dphs, however, both total and recruited
   macrophage numbers were significantly reduced in homozygous mutants
   compared with that of WT siblings (Fig. [188]5B,C ). Furthermore, tnfa
   ^−/− mutants exhibited significantly reduced osteoclast numbers, while
   osteoclasts were abundant in RANKL‐induced nonmutant siblings, (Fig.
   [189]5A,D ). Next, to investigate whether TNFα inhibition is sufficient
   to protect bone matrix from osteoporotic insult, RANKL‐induced larvae
   were stained with calcein at 3 dphs to visualize mineralized matrix in
   the vertebral column. Upon RANKL induction, heterozygous tnfa ^+/−
   carriers showed similar bone defects as nonmutant siblings, which
   included bone loss in the neural arches and large lesions in the
   vertebral centra (Fig. [190]5E ). Homozygous tnfa ^−/− embryos, in
   contrast, had fewer and smaller lesions in the centra indicative of
   improved bone integrity. This suggests that TNFα is required for
   macrophage activation and osteoclast formation after RANKL induction in
   vivo.

Fig 5.

   Fig 5
   [191]Open in a new tab

   Macrophage recruitment and differentiation are impaired in tnfa
   mutants. (A) WT siblings, heterozygous and homozygous tnfa mutants with
   rankl:HSE:cfp/mpeg1:mCherry‐F/ctsk:GFP transgenic background were
   heat‐shocked at 9 days postfertilization (dpf) for 2 hours. Embryos
   without rankl:HSE:cfp transgene were used as controls (–RANKL). Blue
   box on bright field image depicts area imaged. White boxes encircle
   individual vertebrae. In –RANKL embryos, the distribution of
   macrophages in tnfa mutants is not different from that in WT siblings
   (tnfa ^+/+). Upon RANKL induction (+RANKL), there are less macrophages
   recruited in tnfa ^−/− embryos compared with the heterozygous or WT
   siblings. Also, numbers of total macrophages and ectopic osteoclasts in
   homozygous mutants are significantly less than in WT embryos. (B–D)
   Quantification of recruited macrophages, total macrophages, and ectopic
   osteoclasts, respectively. ns = Not significant. Error bars indicate
   mean numbers ± SD, **p < 0.01. Student's t test (unpaired, two‐tailed).
   (E) Calcein staining shows normal mineralization in tnfa ^+/− and tnfa
   ^−/− mutants and WT siblings in the absence of RANKL induction. After
   RANKL induction, tnfa ^−/− mutants exhibit less bone resorption with
   minor lesions (cyan arrowheads), compared with the severe defects in
   heterozygous and WT embryos (yellow arrowheads). dphs = days post
   heat‐shock. Scale bars: 100 μm (in A), 1 mm (in E, low magnification),
   200 μm (in E, high magnification).

   To confirm this, we finally treated nonmutant embryos with the chemical
   inhibitor PTX, which inhibits TNFα synthesis. Importantly, in the
   absence of ectopic RANKL, PTX treatment did not cause any obvious
   changes to the distribution and density of macrophages (Fig. [192]6A ).
   Moreover, upon RANKL induction, macrophages were recruited to the
   vertebral column to a similar extent in DMSO‐ and PTX‐treated embryos,
   as quantitated at 1 and 2 dphs (Fig. [193]6B,C ; white arrowheads). The
   total number of macrophages was slightly lower in PTX‐treated embryos
   at 1 dphs but increased to control levels at 2 dphs (Fig. [194]6D ).
   Thus, other than in tnfa ^−/− mutants, macrophage recruitment was not
   affected by PTX, which we attribute to a lower efficacy of the drug
   compared with the genetic deletion. Importantly, however, osteoclast
   differentiation was strongly impaired in PTX‐treated embryos when
   compared with DMSO controls at both 1 and 2 dphs (Fig. [195]6B,E ,
   green arrowheads). We then analyzed the resorptive activity of
   RANKL‐induced osteoclasts in control and PTX‐treated embryos using
   Alizarin Red staining. In the absence of ectopic RANKL, both PTX‐ and
   DMSO‐treated embryos showed normal mineralization with intact neural
   arches and vertebral bodies (Supplementary Fig. [196]S9). Upon RANKL
   induction, DMSO control embryos exhibited resorption of neural arches
   and severe lesions in the centra. In contrast, PTX treatment resulted
   in considerable bone protection (Supplementary Fig. [197]S9). Together,
   our data show that in medaka TNFα is required for macrophage activation
   and osteoclast formation.

Fig 6.

   Fig 6
   [198]Open in a new tab

   Chemical inhibition of tnfa impedes osteoclast formation. (A)
   Heat‐induced RANKL negative mpeg1:mCherry‐F/ctsk:GFP transgenic embryos
   treated with DMSO or pentoxifylline (PTX) showed normal macrophage
   density and distribution. (B) rankl:HSE:cfp/mpeg1:mCherry‐F/ctsk:GFP
   transgenic embryos were heat‐shocked for RANKL induction and
   immediately treated with PTX or DMSO. Upon RANKL induction, macrophage
   recruitment was not significantly different in DMSO‐ and PTX‐treated
   larvae (white arrowheads), but osteoclast formation was strongly
   impaired in PTX‐treated embryos at 1 and 2 days post heat‐shock (dphs;
   green arrowheads). (C,D) Quantification showed that the total number of
   macrophages at 1 dphs was slightly higher in DMSO control than in PTX
   treated embryos, but macrophage recruitment was not significantly
   different in both samples. (E) Quantification of ectopic osteoclasts
   shows significantly less osteoclast formation in PTX treated embryos at
   1 and 2 dphs. Quantifications in (C–E) show mean number of macrophages
   and osteoclasts ± SD, Student's t test (two‐tailed, unpaired),
   7≤N[DMSO_RANKL−]≤9, 22≤N[DMSO_RANKL+]≤24, 12≤N[PTX_RANKL−]≤16,
   27≤N[PTX_RANKL+]≤28, *p ˂ 0.05, ***p ˂ 0.001, data from three
   independent experiments. Mφ = macrophage; ns = nonsignificant; OC =
   osteoclast. Scale bar: 200 μm (in B), 1 mm (in A).

Discussion

   Small teleost fish, such as zebrafish and medaka, are widely used to
   model human bone diseases given their unique experimental advantages,
   as well as genetic and cellular similarities of medaka and human
   bone.^( [199]^42 ^) In the present study, we used live imaging to
   demonstrate that in medaka, similar to mammals,^( [200]^3 ^)
   osteoclasts are derived from the monocyte/macrophage lineage in
   RANKL‐induced conditions. Our macrophage depletion data suggest that in
   medaka no alternative cellular sources exist for osteoclasts, and that
   the formation of osteoclasts requires a high integrity of the
   macrophage population. Transcriptome profiling of medaka macrophages
   and osteoclasts identified sets of genes that are similarly regulated
   in human and mouse osteoclastogenesis. It also revealed the induction
   of genes in differentiated macrophages that are known to be involved in
   osteoblast formation and bone remodeling. We show that TNFα is
   essential for macrophage activation and osteoclast differentiation.
   Together, these results strengthen the medaka as a unique in vivo model
   accessible to live imaging and suitable for osteoporosis‐related
   studies.

Targeted migration and bone cell interaction of macrophages in medaka

   Macrophages express membrane‐bound receptors that are needed for their
   recruitment to target sites in response to pathogens or instructive
   signals coming from other cell types. After RANKL induction in medaka,
   macrophages are recruited towards bone matrix, where they interact with
   osteoblasts and osteoblast progenitors and differentiate into
   osteoclasts (Fig. [201]2). Importantly, although massively recruited to
   the vertebral column, macrophages only accumulate at mineralized matrix
   of vertebral bodies, but not at the nonmineralized intervertebral
   discs. This suggests that signals triggering macrophage recruitment
   could be released either by resident cells lining the bone surface, ie,
   osteoblasts and osteoblast progenitors, or the matrix itself. Such
   signals appear to be absent from intervertebral discs or alternatively
   are distinct from those that induce chronic macrophage recruitment seen
   in intervertebral disc‐degeneration diseases.^( [202]^43 ^) Previous
   studies indicated that osteoclasts can form from mouse spleen or bone
   marrow cells in the absence of bone matrix when cocultured with
   osteoblasts and supplemented with calcitriol (1 α,25(OH)2 vitamin
   D3).^( [203]^44 ^) This suggests that ossified matrix is dispensable
   for osteoclast formation and that osteoblasts instead produce the
   necessary differentiation signals. However, whether osteoblasts also
   release signals to recruit macrophages is still unknown. In mammals,
   osteoblasts express RANKL, which is important for osteoclast
   differentiation. In the medaka model, however, heat‐shock induced RANKL
   is ubiquitous, yet only mineralized tissue attracts macrophages. This
   suggests that ectopic RANKL potentially acts directly on osteoblasts to
   trigger a release of attractants needed for macrophage recruitment.
   Consistent with this idea, we recently reported that medaka osteoblasts
   express the chemokine ligand Cxcl9l, which controls migration of
   macrophages towards bone matrix.^( [204]^19 ^) Future characterization
   of osteoblasts is likely to identify additional factors controlling
   macrophage recruitment.

Macrophages are the exclusive source for osteoclasts in medaka

   Macrophages are highly dynamic and have a variety of functions ranging
   from the clearance of foreign effectors to mediating communication
   across different cell types under developmental and pathological
   conditions.^( [205]^45 , [206]^46 ^) By time‐lapse analysis, we found
   that all ectopically induced ctsk‐expressing osteoclasts in the
   vertebral column originate from recruited mpeg1 macrophages, suggesting
   that these macrophages are the exclusive cellular source for
   osteoclasts. Consistently, the transient depletion of mpeg1 cells by
   Lipo‐Clo significantly reduced osteoclast numbers upon RANKL induction
   (Fig. [207]3). The few remaining osteoclasts that escaped Lipo‐Clo
   inhibition also originated from macrophages, but not any other cell
   type. These results appear in contrast to earlier studies that reported
   persistent osteoclast formation in macrophage‐depleted mice.^( [208]^47
   ^) This difference may imply that mice potentially have osteoclast
   precursors other than macrophages, and that these precursors are absent
   in medaka. The other possibility, however, is that the macrophage
   ablation in mice obtained by a tamoxifen‐inducible Cre‐lox apoptosis
   system was incomplete, allowing remaining macrophages to differentiate
   into osteoclasts, similar to our findings in medaka. Consistent with
   our findings, Csf1r mouse mutants exhibit a significant reduction of
   macrophage numbers in different tissues and also a decrease in
   osteoclast formation.^( [209]^48 , [210]^49 ^) Thus, other more
   efficient ablation techniques are needed to confirm that remaining
   osteoclasts in mice are differentiated from nondepleted macrophages or
   other cellular sources. In medaka, however, our live‐imaging approach
   confirmed that macrophages are the exclusive source for RANKL‐induced
   osteoclasts.

TNFα is critical for macrophage recruitment and differentiation

   Earlier cell culture studies showed that TNFα induces osteoclast
   formation from mouse bone marrow‐derived macrophages.^( [211]^15 ,
   [212]^17 ^) Serum levels of TNFa are also increased in humans with low
   thyroid‐stimulating hormone (TSH) levels and in mice carrying mutations
   in TSH receptor (Tshr ^−/− ).^( [213]^50 , [214]^51 ^) Both experience
   low BMD and an increased risk of bone fractures. Interestingly, in Tshr
   ^−/− mouse mutants, deletion of Tnfa partially rescued the low bone
   mass phenotype.^( [215]^52 ^) Also, blocking TNFα by injecting a
   binding protein or deletion of Tnfa inhibited bone loss induced by
   ovariectomy.^( [216]^53 , [217]^54 ^) Conversely, TNFα addition
   promoted osteoclast survival and prevented apoptosis.^( [218]^55 ^)
   Thus, these findings in mice strongly suggest an osteoclast‐promoting
   effect of TNFα. Consistent with this, we showed that in the medaka
   osteoporosis model, genetic or chemical inhibition of tnfa expression
   reduced osteoclast formation upon RANKL induction (Figs. [219]5 and
   [220]6). In vivo imaging further revealed that in the absence of
   ectopic RANKL, TNFα deficiency did not cause any obvious effects on
   macrophage populations. It was only after RANKL induction that tnfa
   mutants exhibited significantly reduced macrophage numbers overall and
   in the vertebral column. This strongly suggests that TNFα selectively
   contributes to osteoclast differentiation under osteoporotic
   conditions, but not in normal development. Consistently, our qPCR
   analysis revealed that tnfa transcription reached a maximum at 1 day
   after RANKL induction and was then reduced at 2 and 3 dphs
   (Supplementary Fig. [221]S8). This implies that TNFα may participate in
   the initial phase of macrophage recruitment. Its high levels at the
   beginning appear critical for macrophage differentiation into
   osteoclasts. Blocking TNFα has been an efficient treatment for
   rheumatoid arthritis, which is characterized by strong inflammation
   mediated by macrophages.^( [222]^13 ^) Our results suggest that
   targeting TNFα signaling might be a promising therapeutic strategy for
   osteoporosis.

Dynamic transcriptome changes in activated macrophages suggest multiple
functions in bone remodeling

   A dynamic regulation of cytokines and their receptors is critical for
   immune cells to function accurately in response to foreign stimuli or
   microenvironmental changes.^( [223]^56 ^) Uncontrolled overproduction
   of cytokines can lead to destructive chronic inflammation, which
   results in severe tissue defects.^( [224]^57 ^) Certain cytokines
   receptors, such as IL22Ra and IL20R, have been shown to function as
   drivers for the migration and recruitment of different immune cell
   types including T and B cells, dendritic cells, and neutrophils.^(
   [225]^58 , [226]^59 ^) The expression of these genes is also found
   upregulated in patients suffering from bone loss caused by rheumatoid
   arthritis and osteoporosis.^( [227]^60 , [228]^61 , [229]^62 ^)
   Interestingly, our transcriptome profiling of FAC‐sorted cells showed
   that these genes were downregulated in macrophages undergoing
   osteoclast differentiation (Supplementary Fig. [230]S5). The reduced
   cytokine receptor expression coincided with a reduction of macrophage
   dynamics after they reached the bone matrix and started
   differentiating. We speculate that it is critical for macrophages to
   reduce motility before they attach tightly to bone matrix and form
   actin rings to facilitate bone resorption. Although cytokine receptor
   expression was downregulated under osteoporotic conditions, pathways
   involved in chondrocyte and osteoblast differentiation surprisingly
   were upregulated in RANKL‐induced macrophages (Table [231]2 and
   Supplementary Fig. [232]S5). This could reflect a critical role for
   activated macrophages in bone cell‐coupling: by promoting formation of
   chondrocytes and osteoblasts to achieve bone homeostasis. Our findings
   of upregulated col6a1, col6a2, col6a3, and col12a1b expression in
   RANKL‐induced macrophages are suggestive for a role in osteogenesis.
   Such a role was earlier proposed by Izu and colleagues, who found that
   collagens VI and XII form a complex to mediate osteoblast interaction
   during osteogenesis.^( [233]^63 ^) In the medaka model, we also found
   upregulation of different integrin subunits and other adhesion
   molecules (Supplementary Fig. [234]S5). This is in line with our
   observation that RANKL‐induced macrophages interact dynamically with
   osteoblasts at the bone surface when maturing into osteoclasts (see
   [235]Supplementary Movie S7). We speculate that these matrix and
   adhesion proteins are critical for the tight interaction of
   preosteoclasts with osteoblasts and facilitate osteoclast
   differentiation. In an alternative scenario, our observation of
   upregulated osteogenic transcripts in osteoclasts could reflect the
   uptake of osteoblast‐released exosomes, a process that was recently
   shown to occur in regenerating zebrafish scales.^( [236]^64 ^) These
   osteoblast‐derived exosomes could not only contain factors driving
   osteoclast differentiation, as reported by Kobayashi‐Sun and
   colleagues,^( [237]^64 ^) but also osteogenic mRNAs. However, we
   observed that the majority of upregulated osteogenic genes (i) is
   already expressed at basal levels in noninduced macrophages, and (ii)
   encode secreted or transmembrane proteins. This observation favors the
   idea of a non‐cell autonomous role for activated macrophages in
   chondro‐ and osteogenesis. Clearly, further studies are needed to
   investigate the complex roles of macrophages in bone remodeling.
   Together, our study highlights the dynamic nature of macrophages and
   the complex intercellular interactions implicated in bone homeostasis.

Disclosure

   The authors declare that they do not have any conflict of interest. All
   data presented in this article will be shared upon request.

AUTHOR CONTRIBUTIONS

   Quang Tien Phan: Conceptualized study; performed experiments; analyzed
   data; prepared figures, wrote and edited manuscript. Ranran Liu:
   Designed and performed experiments; analyzed data; prepared figures,
   wrote and edited manuscript. Tan Wen Hui: Performed experiments;
   analyzed data; prepared figures and edited manuscript. Nurgul Imangali:
   Planned and performed apoptosis experiments; analyzed data and prepared
   figures. Benedict Cheong: Performed expression analysis; analyzed data.
   Manfred Schartl: Performed phylogenetic and synteny data analysis;
   wrote and edited manuscript. Christoph Winkler: Conceptualized study;
   analyzed data; prepared figures; wrote and edited manuscript.

PEER REVIEW

   The peer review history for this article is available at
   [238]https://publons.com/publon/10.1002/jbm4.10409.

Supporting information

   Supplementary Figure S1 Macrophages become less motile after
   differentiation into osteoclasts. (A) Cell tracking showed that most
   macrophages remained in the AGM of ‐RANKL embryos. On the contrary,
   many macrophages were directionally recruited towards bone matrix of
   the vertebral column upon RANKL induction. The recruited cells
   clustered mainly at neural arches and vertebral bodies but not in the
   intervertebral discs. Macrophage dynamics was determined at 4 hphs.
   (B). At 1–2 dphs, macrophages that differentiated into osteoclasts
   (mpeg+/ctsk+) have significantly reduced motility and are confined to
   bone matrix. Macrophages that were recruited later stayed
   undifferentiated and remained highly motile. As the cells were more
   dynamic, they had angular shapes with lower sphericity. In contrast,
   mature osteoclasts became more static with rounded shapes (high
   sphericity) after fusion. Macrophages dynamics was determined at 34
   hphs. Student's t test was used to determine the significance of
   difference between groups, error bars show standard deviation. AGM:
   aorta gonad mesonephros; V: vertebral column; iv: intervertebral disc;
   vb: vertebral body; na: neural arch; Mφ: macrophage; OC: osteoclast.
   Scale bar: 100 μm.
   [239]Click here for additional data file.^ (2.6MB, tif)

   Supplementary Figure S2 Macrophages clear apoptotic osteoclasts. (A)
   Still images from a time‐lapse movie recorded at 30 hphs. Circles show
   that apoptotic osteoclasts (green cells) at the neural arch are
   engulfed and digested by conventional phagocytosing macrophages.
   Time:hh:mm. (B) Immunostaining of cleaved‐Caspase‐3 on cryosections
   revealed apoptosis (white arrowheads) of osteoclasts in RANKL‐induced
   embryo. Scale bar: 20 μm (in A), 40 μm (in B).
   [240]Click here for additional data file.^ (4.1MB, tif)

   Supplementary Figure S3 Quantification of macrophage and osteoclast
   numbers. (A) rankl:HSE:cfp/mpeg1:mCherry‐F/ctsk:GFP and
   mpeg1:mCherry‐F/ctsk:GFP transgenic embryos were heat‐shocked and
   imaged repeatedly at 1, 2 and 3 dphs. In the absence of ectopic RANKL
   expression, macrophages were distributed at different positions along
   the body, predominantly in the AGM (white arrowheads). There were no
   ectopic osteoclasts in the trunk (magenta arrowheads). Upon RANKL
   induction, macrophages were directionally recruited towards the
   vertebral column, particularly to the neural arches and the vertebral
   bodies (cyan arrowheads). Osteoclasts were later formed by
   differentiation of recruited macrophages along the trunk (yellow
   arrowheads). The ectopic osteoclasts started to vanish at 3 dphs while
   new macrophages continued to be recruited. (B) Quantification showed
   mean numbers of recruited macrophages, total macrophages and ectopic
   osteoclasts ± SD; Student's t test (two‐tailed, unpaired), **p˂0.01,
   ****p˂0.0001, ns: non‐significant, 10 ≤ N[Larvae] ≤ 19, data from three
   independent experiments. Mφ: macrophage; OC: osteoclast. Scale bar:
   1 mm.
   [241]Click here for additional data file.^ (3.5MB, tif)

   Supplementary Figure S4 Gene Ontology analysis of up‐ and downregulated
   genes in osteoclast compared with macrophage. (A,B) Functional groups
   in down‐ and upregulated genes, respectively. Listed genes were used as
   input for GO terms analysis using ShinyGO. Upregulated genes involved
   in osteoblast differentiation and bone remodeling are labeled in blue,
   and genes related to adhesion were underlined (B).
   [242]Click here for additional data file.^ (2.4MB, tif)

   Supplementary Figure S5 qPCR validation of gene expression in
   FAC‐sorted macrophages and osteoclasts. Gene expression was determined
   at 1 dphs. (A) Among downregulated genes, il22ra2a, il22ra2b and
   pip5k1ba showed trends of downregulation in osteoclasts compared with
   macrophages, consistent with RNAseq data. In contrast, cx3cr1 and fes
   show upregulation, while socs1b, sh2dp1 and sbno2 were not regulated.
   (B) Among upregulated genes, genes encoding collagen VI, collagen XII
   and integrin subunit beta 3 and their binding subunits were
   significantly higher expressed in osteoclasts, consistent with RNAseq
   data. Similarly, expression of osteoclast makers nfatc1, trap and ctsk
   were also upregulated. Error bars indicate mean fold change ± SD,
   *p < 0.05, **p < 0.01, ***p < 0.001, ns: non‐significant, Student's t
   test, data were obtained from three biological replicates with three
   technical repeats each and β‐actin was used as loading control.
   [243]Click here for additional data file.^ (505.2KB, tif)

   Supplementary Figure S6 Phylogeny analysis of CC chemokine receptors. A
   molecular phylogeny was computed for all so far identified medaka Ccr
   receptors as well as human CCRs 1–9, human CX3R and known orthologs
   from the clawed frog Xenopus tropicalis, spotted gar Lepisosteus
   oculatus and zebrafish Danio rerio. In the resulting tree, the medaka
   receptor ENSORLP00000035998 (red arrow) identified in this study is
   nested together with the human CX3C chemokine receptor (blue arrow).
   This orthology assignment is confirmed by conserved synteny with ccr8
   and entpd3 in medaka and human, and also in spotted gar and zebrafish.
   We therefore refer to the encoding gene as medaka cx3cr‐like.
   Interestingly, this gene underwent independent lineage specific local
   gene duplications in zebrafish and medaka, while the other chemokine
   receptors have no such paralogs.
   [244]Click here for additional data file.^ (1.7MB, tif)

   Supplementary Figure S7 Phylogeny analysis of interleukin receptors. A
   molecular phylogeny was computed for proteins annotated as Il22ra2 and
   Il20ra in ENSEMBL (ENSORLP00000023762, ENSORLP00000033651; red arrows),
   respectively. This showed that both proteins cluster together with
   human IL22RA2 (blue arrow) and thus likely represent the product of a
   fish‐specific gene duplication. We therefore refer to the encoding
   genes as il22ra2a and il22ra2b. Conserved synteny with lfngr1 was shown
   for il22ra2a that is present in human, zebrafish, spotted gar and
   medaka genomes. No synteny relation was detected for il22ra2b outside
   Perciformes, thus this gene represents a specific local gene
   duplication in this lineage.
   [245]Click here for additional data file.^ (1.9MB, tif)

   Supplementary Figure S8 tnfa expression and mutant establishment in
   medaka. (A) tnfa expression was analyzed by qPCR at 1, 2 and 3 dphs.
   Non‐heat shocked embryos were used as control. In embryos without RANKL
   transgene (‐RANKL), tnfa was downregulated after heat shock. In
   contrast, tnfa was upregulated in RANKL‐induced embryos (+RANKL) at 1
   dphs, and significantly reduced at 2 and 3 dphs. Error bars indicate
   mean fold change ± SD, *0.01 < p < 0.05, **p < 0.01, ***p < 0.001,
   Student's t test, data from three sets of biological samples. (B)
   Design of guide RNAs for CRISPR/Cas9 to generate tnfa mutants. Three
   primers were used for genotyping. FP2 was used to differentiate between
   homozygous and heterozygous mutants. (C) Representative gel image
   showing PCR‐based genotyping. (D) 1010 bp deletion in tnfa leads to a
   predicted truncated protein.
   [246]Click here for additional data file.^ (641.9KB, tif)

   Supplementary Figure S9 Bone protection in PTX‐treated embryos during
   RANKL induction. Embryos were stained with Alizarin Red at 3 dphs. Both
   DMSO control and PTX‐treated larvae showed normal bone development in
   the absence of ectopic RANKL (blue arrowheads). Upon RANKL induction,
   control embryos exhibited severe bone loss in arches and vertebral
   bodies (black arrowheads). PTX‐treated embryos, on the other hand,
   showed only few mild lesions in arches (small black arrowheads) in
   otherwise normally mineralized centra (blue arrowheads). Scale bar:
   500 μm.
   [247]Click here for additional data file.^ (2.6MB, tif)

   Movie S1 Macrophage behavior without RANKL overexpression. The 9 dpf
   mpeg1:mCherry‐F/ctsk:GFP transgenic embryo was incubated at 39°C for
   2 hours and subjected to confocal imaging at 2 hphs. Tracks showed
   mpeg1‐positive macrophages actively patrolled around the body. Most
   macrophages were found in the AGM while only a few cells located
   proximal to the arches and vertebral bodies. AGM: aorta gonad
   mesonephros; VC: vertebral column; time: hh:mm:ss. Scale bar: 50 μm.
   [248]Click here for additional data file.^ (17.1MB, avi)

   Movie S2 Macrophage recruitment upon RANKL induction. Transgenic embryo
   rankl:HSE:cfp/mpeg1:mCherry‐F/ctsk:GFP was induced for RANKL
   overexpression by heat‐shock at 9 dpf and subjected to confocal imaging
   at 2 hphs. Mpeg1‐positive macrophages were massively recruited towards
   the vertebral column. The cells dynamically localized themselves
   particularly at the ossified matrix, neural arches, and vertebral
   bodies, but not at the intervertebral disc region. Boxes outline the
   vertebral bodies. AGM: aorta gonad mesonephros; V: vertebral body; na:
   neural arch; time: hh:mm:ss.
   [249]Click here for additional data file.^ (17.8MB, avi)

   Movie S3 Macrophage differentiation and clearance of apoptotic
   osteoclasts. RANKL‐induced rankl:HSE:cfp/mpeg1:mCherry‐F/ctsk:GFP
   embryo was subjected for confocal time‐lapse at 30 hphs. Arrowheads
   showed recruited macrophages along the trunk gradually differentiated
   into ctsk‐positive cells. Box highlighted the phagocytosis of apoptotic
   osteoclasts by macrophages. AGM: aorta gonad mesonephros; na: neural
   arch; time: hh:mm. Scale bar: 50 μm.
   [250]Click here for additional data file.^ (3.7MB, avi)

   Movie S4 Fusion of differentiating osteoclasts. A RANKL‐induced
   rankl:HSE:cfp/mpeg1:mCherry‐F/ctsk:GFP embryo was subjected to confocal
   time‐lapse at 54 hphs. During terminal differentiation, osteoclasts
   fused together into larger cells, both at the arches and the centra
   (blue boxes). White box showed an apoptosis event. AGM: aorta gonad
   mesonephros; time: hh:mm:ss. Scale bar: 50 μm.
   [251]Click here for additional data file.^ (9.1MB, avi)

   Movie S5 Normal osteoclast formation in Lipo‐PBS injected embryo. A
   transgenic rankl:HSE:cfp/mpeg1:mCherry‐F/ctsk:GFP embryo was injected
   with Lipo‐PBS, induced for RANKL and then subjected to confocal imaging
   at 1 dphs. Osteoclasts formed normally from the recruited macrophages
   (white arrowheads). AGM: aorta gonad mesonephros; VC: vertebral column;
   time: hh:mm. Scale bar: 50 μm.
   [252]Click here for additional data file.^ (10.4MB, avi)

   Movie S6 Absence of osteoclast formation in macrophage‐depleted embryo.
   A macrophage‐ablated transgenic rankl:HSE:cfp/mpeg1:mCherry‐F/ctsk:GFP
   embryo was induced for RANKL and subjected to confocal imaging at 1
   dphs. Osteoclast formation was impaired after macrophages were ablated.
   AGM: aorta gonad mesonephros; VC: vertebral column; time: hh:mm. Scale
   bar: 50 μm.
   [253]Click here for additional data file.^ (10.4MB, avi)

   Movie S7 Multiplication of recruited macrophages and dynamic macrophage
   interaction with bone cells. A transgenic
   rankl:HSE:cfp/col10a1:GFP/mpeg1:mCherry‐F embryo at 9 dpf was
   RANKL‐induced at 39°C for 2 hours. Time‐lapse movie was started at 6
   hphs. Arrows show macrophage multiplication upon recruitment to the
   vertebral column. White box highlights the dynamic interaction of
   macrophages with osteoblast precursors (col10a1 cells). Time: hh:mm:ss.
   Scale bar: 50 μm.
   [254]Click here for additional data file.^ (3.1MB, avi)

   Supplementary Table S1.
   [255]Click here for additional data file.^ (276.6KB, docx)

Acknowledgments