Rosmus et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03110-x(2024) 21:168

Journal of Neuroinflammation

Open Access

RESEARCH

Redefining the ontogeny of hyalocytes
as yolk sac‑derived tissue‑resident macrophagesof the vitreous bodyDennis‑Dominik Rosmus1,2† , Jana Koch2,3,4† , Annika Hausmann3 , Aude Chiot5,6 , Franz Arnhold1,
Takahiro Masuda3,7, Katrin Kierdorf3,8,9, Stefanie Marie Hansen3, Heidrun Kuhrt1, Janine Fröba1, Julian Wolf4,10,11,
Stefaniya Boneva4 , Martin Gericke1, Bahareh Ajami5,6 , Marco Prinz12,3 , Clemens Lange4,13    andPeter Wieghofer1,2,3*   

Abstract
Background The eye is a highly specialized sensory organ which encompasses the retina as a part of the cen‑tral nervous system, but also non-neural compartments such as the transparent vitreous body ensuring stabilityof the eye globe and a clear optical axis. Hyalocytes are the tissue-resident macrophages of the vitreous body and areconsidered to play pivotal roles in health and diseases of the vitreoretinal interface, such as proliferative vitreoretin‑
opathy or diabetic retinopathy. However, in contrast to other ocular macrophages, their embryonic origin as wellas the extent to which these myeloid cells might be replenished by circulating monocytes remains elusive.
Results In this study, we combine transgenic reporter mice, embryonic and adult fate mapping approaches as wellas parabiosis experiments with multicolor immunofluorescence labeling and confocal laser-scanning microscopy
to comprehensively characterize the murine hyalocyte population throughout development and in adulthood. We
found that murine hyalocytes express numerous well-known myeloid cell markers, but concomitantly display a dis‑tinct immunophenotype that sets them apart from retinal microglia. Embryonic pulse labeling revealed a yolk sacderived origin of murine hyalocytes, whose precursors seed the developing eye prenatally. Finally, postnatal labelingand parabiosis established the longevity of hyalocytes which rely on Colony Stimulating Factor 1 Receptor (CSF1R)
signaling for their maintenance, independent of blood-derived monocytes.
Conclusion Our study identifies hyalocytes as long-living progeny of the yolk sac hematopoiesis and highlights theirrole as integral members of the innate immune system of the eye. As a consequence of their longevity, immunose‑
nescence processes may culminate in hyalocyte dysfunction, thereby contributing to the development of vitreoreti‑
nal diseases. Therefore, myeloid cell-targeted therapies that convey their effects through the modification of hyalocyte
properties may represent an interesting approach to alleviate the burden imposed by diseases of the vitreoretinal
interface.
Keywords Macrophages, Hyalocytes, Vitreous body, Cx3cr1, Fate mapping, Turnover, Development, CSF1R†

Dennis-Dominik Rosmus and Jana Koch contributed equally to this work.

*Correspondence:Peter Wieghofer
peter.wieghofer@med.uni-augsburg.de
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Rosmus et al. Journal of Neuroinflammation(2024) 21:168

Background
Macrophages are highly versatile immune cells residing
in different tissues and compartments of the body with
important roles in the host’s innate immunity. Originally
thought to be descendants of the “mononuclear phagocyte system” [1], the last decade of research uncovered a
remarkable heterogeneity of tissue-resident macrophages
in terms of their origin, fate and function in homeostasis and disease [2, 3]. Today, it is commonly accepted thatmyeloid cells arise from different hematopoietic waves
during pre- and postnatal development [4, 5], constituting a highly specialised network of myeloid cells adapted
to their respective local niches due to environmental
cues [6]. Studies in rodents revealed that the first tissueresident macrophages originate from early erythromyeloid precursors (EMPs) in the extra-embryonic yolksac (YS) at embryonic day 8.5 (E8.5) [7–10]. EMPs further differentiate into an A1 and a ­CX3CR1-expressing
A2 population which in turn seed prenatal tissueresident macrophage populations in nearly all tissues[11–13] including microglia, extraparenchymal CNSassociated macrophages (CAMs) [11, 14–16] and ocularmacrophages [17–19]. It is assumed that a later wave of
EMPs is generated in the YS. These EMPs subsequently
colonize the fetal liver (FL) between E8.5-E10.5 giving rise to EMP-derived FL monocytes that outcompete
YS-derived macrophages in the majority of organs [2,20]. Finally, hematopoietic stem cells (HSCs) develop in
the aorto-gonad-mesonephros region around E10.5 andestablish the intra-embryonic definitive hematopoiesis
which persists until adulthood [21, 22].
The murine eye is composed of different compartmentswith resident myeloid cell populations. Recent studies
using different fate mapping approaches demonstrated
that nearly all ocular compartments harbor prenatally
seeded macrophage populations [17, 18]. While retinal
microglia (rMG) derive from the YS and are maintainedby self-renewal, macrophages in the ciliary body, cornea
and the choroid have a dual origin with varying degrees
of replenishment by peripheral monocytes during adulthood [17, 18]. Hyalocytes, however, have long been overlooked with respect to their origin, heterogeneity, andfunction as tissue-resident macrophages of the vitreous[23]. Since their first description in the nineteenth century [24], hyalocytes have been implicated in a variety ofphysiological and pathological processes [23, 25–27]. In
the healthy eye, hyalocytes participate in the synthesis ofextracellular matrix components and are believed to contribute to the maintenance of the ocular immune privilege [25, 28]. Furthermore, hyalocytes are highly dynamic
cells and are regularly distributed above the inner limiting membrane (ILM) as shown by optical coherence
tomography in humans [29, 30]. The ILM represents

Page 2 of 20

a barrier consisting of distinct laminae that are built byboth the vitreous providing a dense meshwork of collagen fibrils and the retina abutting the vitreal side via theretinal glia limitans built by endfeet of Mueller glia cells
and astrocytes inhabiting the nerve fiber and ganglion
cell layer [31]. The glia limitans is connected to the abluminal side of the vitreal laminae via extracellular matrix
components. Hence, rMG reside in the CNS while hyalocytes are located close to the border of the CNS and thus
colonize distinct compartments of the vitreoretinal interface [32, 33].
Previous studies addressed the origin of hyalocytes
using bone marrow chimeras as a tool to investigate thecontribution of adult hematopoiesis to resident myeloid
cell populations and found a nearly complete replenishment of the resident hyalocyte population by ­GFP+
donor cells [23, 34]. However, the experimental paradigmof bone marrow chimerism often requires irradiationinduced depletion of the host’s bone marrow as a precondition, which can cause changes in vascular structuresand immunological barrier breakdowns. These may facilitate the engraftment of monocyte-derived macrophages
in the CNS [35–37] including the retina [38], which does
not occur under physiological conditions. Therefore, themain objective of our study was to scrutinize the previously established origin of murine hyalocytes and to
determine their postnatal turnover. To achieve this goal,
we utilized transgenic reporter mouse lines, modern
fate mapping tools as well as parabiosis techniques. We
demonstrate that, contrary to current conceptions, hyalocytes are mainly derived from precursors of the extraembryonic YS, represent a long-lived self-maintaining
cell population independent of adult hematopoiesis, andrely on functional Colony Stimulating Factor 1 Receptor(CSF1R) signaling as a prerequisite for their maintenance.
In summary, these data characterize murine hyalocytes
as YS-derived, tissue-resident macrophages that may
substantially contribute to physiological and pathologicalprocesses at the vitreoretinal interface.

Materials and methods
Mice

In this study, C57BL/6 J mice (RRID: IMSR_JAX:000664)
were used as wildtype (WT) mice. All transgenic lines
including ActbGFP/+ (RRID: IMSR_JAX:006567), UbcGFP/+(RRID: IMSR_JAX:004353), Cx3cr1GFP/+ (RRID: IMSR_
JAX:005582), Tmem119GFP/+ (RRID: IMSR_JAX:031823),
HexbtdT/tdT (kindly provided by Dr. Takahiro Masuda
and Prof. Dr. Marco Prinz), Cx3cr1CreER (RRID: IMSR_
JAX:020940), Csf1rEGFP/+ (RRID: IMSR_JAX:018549),
Csf1r∆FIRE/∆FIRE (RRID: IMSR_JAX:032783), Flt3Cre(RRID: IMSR_EM:11790), and Rosa26fl−stop−fl−EYFP(Rosa26-YFP, RRID: IMSR_JAX:006148) were bred on

Rosmus et al. Journal of Neuroinflammation(2024) 21:168

a C57BL/6 J background. All mice were housed in a
12-h light–dark cycle with ad libitum access to foodand water and were kept under specific pathogenfree conditions and devoid of Crb1 (RD8) mutations.
Cx3cr1CreER mice were crossed to Rosa26-YFP mice togenerate Cx3cr1CreER/+:Rosa26-YFPfl/fl animals that wereidentified by PCR screening. Flt3Cre mice were crossed to
Rosa26-YFP mice.
Tamoxifen (TAM) treatment

To induce nuclear CreER recombinase activity andexpression of YFP in adult Cx3cr1CreER:Rosa26-YFP mice,six-week-old mice were injected subcutaneously with
4 mg of TAM (T5648-1G, Sigma-Aldrich, Taufkirchen,
Germany) dissolved in corn oil (C8267, Sigma-Aldrich,
Taufkirchen, Germany) at 20 mg/ml. Overall, three
injections were performed, one every two days. To confirm recombination, blood was collected from the retroorbital sinus one week after the last injection, and YFP
expression of immune cells was assessed by flow cytometry. At 2 or 26 weeks post-injections, mice were deeply
anesthetized with ketamine (West-Ward Pharmaceuticals) and transcardially perfused with 0.1 M ice-cold
phosphate buffered saline (PBS). Blood and one cerebral
hemisphere per mouse were collected to assess YFP
percentage of different cell populations by flow cytometry. Both eyes were collected and post-fixed in 4% paraformaldehyde (PFA) for 1 h at room temperature (RT)for histological procedures. For embryonic pulse labeling experiments, pregnant females at day 9 post-coitum
underwent intraperitoneal (i.p.) injections with 200 µlof 20 mg/ml TAM (T5648-1G, Sigma-Aldrich, Taufenkirchen, Germany) and 10 mg/ml Progesterone (P0130,
Merck) dissolved in corn oil (C8267, Sigma-Aldrich,Taufenkirchen, Germany).
BM transplantation

Eight-week-old recipient wildtype mice (Actb+/+) were
irradiated and reconstituted by injecting 5 × ­106 bonemarrow cells derived from the femur and tibia of adultdonor (ActbGFP/+) mice into the tail vein of recipients.
Mice received whole-body irradiation (11 Gy) 24 h prior
to bone marrow reconstitution with an RS 2000 Biologica x-Ray Irradiator. After 4 weeks, reconstitution efficiency was assessed by flow cytometry and was found to
be > 90% in ­Ly6Chi monocytes.
Parabiosis

Six- to seven-week-old pairs of weight-matched
C57BL/6 J WT and UbcGFP/+ mice were surgically joined
as previously described [35]. Prior to surgery, mice werehoused together for two weeks and provided with soft
food. To confirm blood sharing, blood was collected

Page 3 of 20

from the tail vein, and GFP expression was assessed byflow cytometry two weeks post-surgery. At 4 or 28 weeks
post-surgery (i.e. 2 weeks and 26 weeks after establishment of blood sharing), mice were deeply anesthetizedwith ketamine (West-Ward Pharmaceuticals) and transcardially perfused with 0.1 M ice-cold PBS. Blood,
spleen and one cerebral hemisphere per mouse were collected to assess GFP percentage of cells by flow cytometry. Both eyes were collected and post-fixed in 4% PFA
for 1 h at RT for histological procedures.
Immunofluorescence

After transcardial perfusion with PBS followed by 4%
PFA, eyes were fixed in 4% PFA for 1 h at RT and processed either for flat mounts as previously described[39] or cryosectioning. Briefly, eye cups were incubated consecutively in 10%, 20%, 30% sucrose solution
after fixation and embedded in Tissue-Tek® O.C.T.TM
Compound (Sakura Finetek Germany GmbH). 12 µm
sections or retinal flat mounts were blocked with PBS
containing 2% (cryosections) or 1% (retinal flat mounts)
bovine serum albumin (BSA), 0.1% Triton-X 100 and
2% normal goat or donkey serum, respectively. Primary
antibodies were added overnight at a dilution of 1:500
for Iba-1 (234 013, Synaptic Systems, Göttingen, Germany, RRID: AB_2661873; 234 004, Synaptic Systems,
Göttingen, Germany, RRID: AB_2493179), 1:1000 for
anti-GFP (R1091P, Acris Antibodies Inc., San Diego,
USA, RRID: AB_1002036), 1:100 for F4/80 (MF48000,
Invitrogen, Waltham, USA, RRID: AB_10376289), 1:500
for TMEM119 (400 002, Synaptic Systems, Göttingen,Germany, RRID: AB_2721104), 1:100 for MHC class II(107 602, BioLegend, USA, RRID: AB_313317), 1:100
for CD206 (ab64693, abcam, UK, RRID: AB_1523910),
1:100 for CD163 (155 302, BioLegend, USA, RRID:
AB_2734239), 1:250 for LYVE1 (AF2125, R&D Systems,
USA, RRID: AB_2297188), 1:800 for CD11b (ab64347,
abcam, UK, RRID: AB_1140550), and 1:200 for Collagen IV (AB769, Millipore, USA, RRID: AB_92262).
For the visualization of myeloid cells and blood vessels, rhodamine-labeled Isolectin-B4 from Griffonia
simplicifolia (RL-1102, Vector Laboratories, RRID:
AB_2336492) was used. After washing three (cryosections) or five (retinal flat mounts) times with washing buffer containing 2% (cryosections) or 1% (retinal
flat mounts) BSA, 0.1% Triton X-100 and 0.2% normal goat or donkey serum, secondary antibodies wereadded at a dilution of 1:500 (Alexa Fluor® 488, Alexa
Fluor® 568 and Alexa Fluor® 647, Thermo Fisher Scientific, Waltham, USA) for 2 h at RT (cryosections)or overnight at 4 °C (retinal flat mounts). The following secondary antibodies were used in this study: Goat
anti-rabbit Alexa Fluor® 488 (A11034, Invitrogen, USA,

Rosmus et al. Journal of Neuroinflammation(2024) 21:168

RRID: AB_2576217), Goat anti-rat Alexa Fluor® 568(A11077, Invitrogen, USA, RRID: AB_2534121), Goat
anti-rabbit Alexa Fluor® 647 (A21245, Invitrogen, USA,
RRID: AB_2535813), Goat anti-guinea pig Alexa Fluor®
647 (A21450, Invitrogen, USA, RRID: AB_141882),
Donkey anti-rabbit Alexa Fluor® 568 (A10042, Invitrogen, USA, RRID: AB_2534017), and Donkey antigoat Alexa Fluor® 647 (A21447, Invitrogen, USA,
RRID: AB_2535864). Nuclei were counterstained with
4′,6-Diamidine-2-phenylindole (DAPI) at a dilution of
1:10,000 for 10 min followed by extensive washing with
PBS. Images were taken using a conventional fluorescence microscope (Olympus BX-61 with a color camera(Olympus DP71)) (Olympus, Tokyo, Japan) or a confocal microscope (Olympus Fluoview FV 1000 using a
20 × 0.95 NA XLUMPlanFL N, 20 × 0.75 NA U Plan S
Apo or a 40 × 0.95 NA U Plan S Apo objective or Leica
Stellaris 5 using a HC PL APO 20x/0.75 CS2). For confocal z-stacks, a z-step size of 1.5 µm was chosen andimages were taken from the retinal surface above theinner limiting membrane to the inner plexiform layer.
Confocal z-stacks were subsequently used to generate
projection images using maximum intensity projections
over the z-axis.
Flow cytometry

For isolation of microglial cells, brains were dissected andcut in half along the longitudinal fissure. One cerebral
hemisphere per animal was homogenized with a dounce
homogenizer in 0.1 M HBSS (Gibco), filtered througha 70 µm cell strainer (Falcon) and centrifuged (750 g,
7 min, 4 °C) (Thermo Scientific). To remove the myelin,cells were resuspended in 5 ml of 0.1 M HBSS + 2 ml of
RPMI (Gibco) + 3 ml of isotonic Percoll (ISP) (90% Percoll (GE Healthcare) + 10% 1 M HBSS). A 2 ml layer of
70% Percoll (70% ISP, 30% 0.1 M HBSS) was added at thebottom of the tube using spinal cord needles (BD Biosciences). Cells were centrifuged at 500 g for 15 min at
RT without break. Following centrifugation, myelin wasremoved from the top layer, and cells were recoveredfrom the interface between the 30% and 70% Percoll layers. Cells were washed twice using 0.1 M PBS (Gibco)
supplemented with 2% fetal bovine serum (FBS) (Atlas
Biologicals) followed by antibody staining. Blood wascollected in a heparin tube (BD Vacutainer) from cardiac
puncture when the mouse was sacrificed. Approximately
50 µl of blood was transferred to a 15-ml conical tube.
Erythrocytes from blood samples were lysed in 2 ml of
10X RBC lysis buffer diluted 1:10 in water (eBioscience)for 7 min at RT. Blood samples were then washed twotimes in 0.1 M PBS supplemented with 2% FBS, followed
by staining.

Page 4 of 20

Validation of recombination efficiency
in Cx3cr1CreER:Rosa26‑YFP mice

White blood cell and microglial cell Fc-receptors were
blocked using a purified anti-CD16/CD32 antibody(1:200, clone 93, BioLegend, USA, RRID: AB_312807) for
20 min at 4 °C. White blood cells were stained for 30 min
at 4 °C using a viability dye (1:2000, Ghost Dye Red 780,
TONBO) in addition to the following antibodies: CD11bBV421 (1:200, clone M1/70, BioLegend, USA, RRID:
AB_10897942), CD115-BV605 (1:200, clone AFS98,
BioLegend, USA, RRID: AB_2562760), CD45-BUV395(1:200, clone 30-F11, BD Biosciences, USA, RRID:
AB_2651134) and Ly6C-PE (1:16,000, clone HK1.4, BioLegend, USA, RRID: AB_1732082). Brain microglial
cells were stained for 30 min at 4 °C using a viability dye(1:2000, Ghost Dye Red 780, TONBO) in addition to thefollowing antibodies: CD11b-BV421 (1:200, clone M1/70,
BioLegend, USA, RRID: AB_10897942), CD45-BUV395(1:200, clone 30-F11, BD Biosciences, USA, RRID:
AB_2651134).
Validation of a successful post‑surgery shared circulationin parabiotic mice

White blood cell, splenocyte and microglial cell Fcreceptors were blocked using a purified anti-CD16/
CD32 antibody (1:200, clone 93, BioLegend, USA,
RRID: AB_312807) for 20 min at 4 °C. White blood
cells and splenocytes were then stained for 30 min at
4 °C, using a viability dye (1:2000, Ghost Dye Red 780,
TONBO) in addition to the following antibodies: CD11bBV421 (1:200, clone M1/70, BioLegend, USA, RRID:
AB_10897942), CD45-BUV395 (1:200, clone 30-F11,
BD Biosciences, USA, RRID: AB_2651134), CD19BUV737 (1:1600, clone 1D3, BD Biosciences, USA, RRID:
AB_2870111), CD3ε-APC (1:400, clone 145-2C11, BioLegend, USA, RRID: AB_312677) and Ly6G-PE (1:200,
clone 1A8, BioLegend, USA, RRID: AB_1186099). Microglial cells were stained as described above. Cells wereacquired on a BD FACSymphony Cell Analyzer (BDBiosciences, USA), and a multiparameter analysis was
performed using FlowJo Software Version 10.8.1 (BD
Biosciences, USA).
Statistical analysis

Using previous data as guidance [15, 18], we ensured acomparable group size for the quantitative analysis of theturnover in Cx3cr1CreER:Rosa26-YFP mice and parabiotic
mice. Statistical analysis was performed using GraphPad
Prism 7 (GraphPad Software, La Jolla, USA). Data were
tested for normality using the Kolmogorov–Smirnov
test. If normality was given, an unpaired t-test or oneway ANOVA was applied, if not indicated otherwise. If

Rosmus et al. Journal of Neuroinflammation(2024) 21:168

normality was not given, the Mann–Whitney or Kruskal–
Wallis test was applied. Differences were considered statistically significant for a p-value < 0.05.

Results
Immunophenotyping of murine hyalocytes
under homeostatic conditions

Previous studies have shown that hyalocytes are tissueresident macrophages that belong to the innate immune
system of the eye [40] and are widely regarded as themain cell population of the vitreous. Importantly, they
are distinct from other cell types at the vitreoretinal
interface such as fibroblasts [41]. However, a characterization of the marker profile of murine hyalocytes thatcombines modern genetic tools for the labeling of tissueresident macrophages with multi-color immunofluorescence to unveil the immunophenotype of hyalocytes, also
in relation to rMG would provide valuable insights into
the relationship between those two macrophage populations residing at the vitreoretinal interface. Therefore, wetook advantage of several transgenic reporter mouse linesand dissected eyes for retinal flat mounts, subsequent
immunofluorescence labeling and imaging using confocal laser-scanning microscopy (Fig. 1a). First of all, we
used the Cx3cr1GFP/+ reporter mouse line, in which thegreen fluorescent protein (GFP) is expressed under thecontrol of the fractalkine receptor ­(CX3CR1) promoter
and labels myeloid cells [42] (Fig. 1b). For the purpose ofthis study, we defined G­ FP+ cells located above the ILM,
which represents the border between the vitreous bodyand the underlying neuroretina, as preretinal hyalocytes(Fig. 1c). To visualize the anatomical localization of G
­ FP+
cells in relation to the ILM, we used a color-coded maximum intensity projection of distinct layers of the confocal z-stack to illustrate the depth of the tissue (Fig. 1c).
We found that hyalocytes are regularly distributed above

Page 5 of 20

the ILM and express common myeloid cell markers suchas the pan-macrophage marker IBA1 similar to microglia
in the retina, but also selectively express markers distinct from microglia such as F4/80, the mannose receptor CD206 and the scavenger receptor CD163 (Fig. 1d,
Additional file 1: Fig. S1). Hyalocytes were positive for
LYVE1 which is typically expressed in certain subpopulations of macrophages across organs [43] and was previously identified as a marker for vitreal macrophages[44]. Interestingly, neither rMG nor hyalocytes, show any
immunoreactivity for major histocompatibility complex
II (MHCII) (Fig. 1d).
To further exclude the possibility that these cells may
represent translocated microglia from the inner plexiform-(IPL) or ganglion cell layer (GCL), we utilized
TMEM119, which belongs to the core signature genesof microglia in the brain and the retina [18, 45–48], asa marker to discriminate between microglia and hyalocytes. We analyzed retinal flat mounts from Tmem119GFP mice expressing GFP under the control of theTmem119 promoter, without affecting the production of
the native protein through polycistronic gene expression(Fig. 1e), to specifically label microglia in the CNS including the retina [49]. Indeed, hyalocytes, which were positive for IBA1 and located above the ILM in the posterior
vitreous cortex, did not show any sign of GFP-labelingin comparison to rMG, which were consistently GFPpositive (Fig. 1f ). Finally, we used the recently generated
Hexb-tdTomato reporter mouse model, which enables
stable gene targeting of microglia in the CNS underhomeostatic and pathological conditions, to further characterize murine hyalocytes (Fig. 1g) [50]. Notably, hyalocytes did not express the Hexb-tdTomato transgenein contrast to rMG exhibiting a consistent tdTomatolabeling and expressing TMEM119, which further supports our findings from Tmem119-GFP mice (Fig. 1h). In(See figure on next page.)
Fig. 1 Immunophenotyping of murine hyalocytes underlines their myeloid cell Identity. a Graphical illustration of the experimental setup.
Retinal flat mounts were prepared of eyes of several transgenic reporter mouse models and analyzed using immunofluorescence labelingand confocal laser-scanning microscopy. b Graphical scheme of gene targeting in Cx3cr1-GFP mice where one allele of the Cx3cr1 locus is replacedby green fluorescent protein (GFP). c Representative images from retinal flat mount (left) and cryosection (right) from Cx3cr1-GFP mice depicting
the anatomical localization of hyalocytes and retinal microglia (rMG). Planes 1-4 (red) include hyalocytes (1) residing above the inner limiting
membrane (ILM, dashed line). Planes 7-14 (green) comprise rMG positioned in the inner plexiform layer (IPL) (2, 3) and outer plexifom layer (OPL) (4).
The z- step size is 1,5 µm. Vitr: vitreous, GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. d Representative images of retinal
flat mounts from Cx3cr1-GFP mice. In contrast to rMG (arrowheads), murine hyalocytes (asterisks) express F4/80, CD206 and LYVE1. Both cell types
did not show MHCII-immunoreactivity. Pictures are representative for three (CD206, LYVE1, MHCII) or five (F4/80) mice, respectively. e Graphicalscheme depicting gene targeting in Tmem119-GFP mice. GFP was introduced in the stop codon of the Tmem119 locus separated by a P2A-siteenabling functional expression of both proteins. f Representative images of retinal flat mounts from Tmem119-GFP mice. Hyalocytes were G­ FP−(asterisks) while rMG (arrowheads) are consistently ­GFP+. Images are representative for three mice. g Graphical illustration of gene targetingin Hexb-tdTomato reporter mice. A tdTomato cassette was introduced downstream of Exon 14 of the Hexb locus separated through a T2A-siteenabling the expression of functional HEXB protein and tdTomato. h Representative images of retinal flat mounts from Hexb-tdTomato mice.
Hyalocytes were identified as ­tdTomato−TMEM119− cells (asterisks) compared to ­tdTomato+TMEM119+ rMG (arrowheads). Images are representative
for two mice. All scale bars equal 50 µm

Rosmus et al. Journal of Neuroinflammation(2024) 21:168bamurine eye

Page 6 of 20c

Cx3cr1 gene locus
WT
allelemutantalleleretinal flat mounts confocal microscopy

flat mount
Z=1-4 Z=7-14

cryosection
Cx3cr1-GFP DAPI

3

1

2

Transcription+
Translation

3
1

CX3CR1

Cx3cr1-GFP IBA1 F4/80*

Cx3cr1-GFP**

Cx3cr1-GFP CD206 F4/80

*

IBA1

F4/80

Cx3cr1-GFP

CD206

F4/80

*

*

Tmem119 gene locus

F4/80

*

Cx3cr1-GFP

Cx3cr1-GFP

e

IBA1

4

OPL
ONL

GFP

Cx3cr1-GFP IBA1 LYVE1

*

IBA1

Cx3cr1-GFP

LYVE1

*
*

Cx3cr1-GFP IBA1MHCII

*

*
*

*

IBA1

LYVE1

Cx3cr1-GFP

IBA1

MHCII

*

F4/80

f

*

Cx3cr1-GFP

*

CD206

GCL
IPL
INL

2

4

d

Vitr
ILM

*

Cx3cr1-GFP

*

IBA1

MHCII

IBA1

Tmem119-GFP

IBA1

Tmem119-GFP

Tmem119-GFP
Tmem119-GFP

IBA1

IBA1 Tmem119-GFP

Transcription+
Translation*
TMEM119

g

*

*

*

GFP

Hexb gene locushHexb-tdTomato

IBA1

TMEM119

Hexb-tdTomato

Transcription+
Translation

*

HEXB

*

*

*

*

*

*

IBA1 Hexb-tdTomato TMEM119

*

*

*

tdTomato

Fig. 1 (See legend on previous page.)summary, hyalocytes in the vitreous body express macrophage cell markers, but concomitantly possess a distinct immunophenotype that clearly separates them frommicroglia in the retina.

Immunofluorescence labeling reveals spatial relationshipsof ocular macrophages during development

The embryonic development of the eye is a highlysophisticated process orchestrated by different

Rosmus et al. Journal of Neuroinflammation(2024) 21:168

regulatory mechanisms which collectively lead tothe establishment of this complex organ [51]. To get
deeper insights into the formation of the resident
hyalocyte population, our next step was to perform acomprehensive histological analysis of these cells at
different stages of pre- and postnatal development.
Using Cx3cr1-GFP reporter mice, we found that myeloid cells emerge as early as E9.5 in the mesenchyme(mes) surrounding the developing optic vesicle (OV)
consisting of neuroepithelium (n.ep.), which represents an evagination of the diencephalon in the forebrain [52, 53] (Fig. 2a). At E10.5, we observed G­ FP+
myeloid cells entering the optic cup, which develops
from the OV by invagination during the formation of
the lens placode (LP) [51]. The optic cup is part of the
diencephalon, connected to this part of the developing
CNS through the optic stalk (OS) in which ­GFP+ cells
can be found (Fig. 2b) [51]. At E11.5, E12.5, and E14.5,­GFP+ myeloid cells can be found in the developing
neuroretina in close proximity to the neuroblast (Nb)layer, most likely destined to mature to rMG. On theother side, G­ FP+ cells found in the vitreous body (Vitr)
near the neuroretina and the lens (L) vesicle likely represent early hyalocytes (Fig. 2c, 2d, 2e), which express
CD11b and F4/80 at the protein level, and can be identified via Isolectin-B4 labeling, which was previously
found to bind to microglial cells in the CNS [54] (Additional file 2: Fig. S2). At E16.5 and E18.5, the differentiation of the GCL and the IPL, where most of the rMG
can be found [55], characterizes neuroretinal development. At this stage, ­GFP+ hyalocytes are found in thevitreous and close to the posterior pole of the lens (L)(Fig. 2f, 2g). During postnatal development, microglia are regularly distributed across the IPL and outerplexiform layer (OPL) (Fig. 2h, 2i, 2j, 2k, 2l), whereas
hyalocytes reside in the vitreous body (Fig. 2h, 2i, 2k).
Taken together, these stainings reveal that hyalocytes
localize in the developing vitreous of the eye as early
as at E11.5 and are in close vicinity to the neuroretina
and the lens during prenatal development.

Page 7 of 20

Embryonic pulse labeling establishes a prenatal origin
of murine hyalocytes

The investigation of the origin and fate of murine hyalocytes in the past has largely been restricted to lethally
irradiated and bone marrow-reconstituted mice thatare consequently affected by an artificial engraftmentof peripheral monocytes into the tissue [18, 23, 35–38].
Indeed, we found that reconstitution of whole-body irradiated wildtype mice by intravenous injection of bonemarrow obtained from ActbGFP/+ animals (Fig. 3a) led
to an engraftment of ­GFP+ cells in the hyalocyte pool(Fig. 3b). These ­IBA1+GFP+ cells were located in theposterior vitreous cortex above the ILM and resembled
hyalocytes under homeostatic conditions (Fig. 3b). To
circumvent the use of artifact-causing irradiation, we
utilized the Cx3cr1CreER:Rosa26-YFP mouse line in anembryonic pulse labeling approach as described previously (Fig. 3c) [15, 18, 56, 57]. In this model, injection oftamoxifen (TAM) at E9.0 leads to nuclear translocationof the Cre-ER fusion protein in ­CX3CR1+ A2 progenitors
in the extra-embryonic YS, that give rise to tissue-resident macrophages in various prenatal organs including
the brain, eye, liver, and lung [11–13, 18, 58, 59], andsubsequent labeling of these cells and their progeny(Fig. 3d). Using this approach, we identified ­YFP+ hyalocytes in mice at postnatal day 0 (P0) (Fig. 3e), confirming the contribution of YS-derived progenitors to thehyalocyte population. Subsequent quantitative analysis
of hyalocytes, rMG and macrophages in the choroid at
P0 revealed that hyalocytes exhibited similar CreERinduced labeling or recombination rates (48.35 ± 7.01%)
to rMG (54.42 ± 10.06%) (Fig. 3f ), while the recombination rates in choroidal macrophages were significantly lower (12.13 ± 0.91%) (Fig. 3f ). In order to extend
our analysis over the perinatal period, we analyzed
Cx3cr1CreER:Rosa26-YFP mice that were exposed to TAM
treatment at E9.0 and sacrificed at P42. In support of ourdata at P0, we found a consistent YFP-labeling of rMG
and hyalocytes (Fig. 3g) with hyalocytes (68.91 ± 10.30%)
displaying similar recombination rates in comparison torMG (56.56 ± 11.33%) (Fig. 3h).

(See figure on next page.)
Fig. 2 Histological analysis of the spatiotemporal development of myeloid cell populations in the murine eye. a–g Cryosections from Cx3cr1-GFP
mice at different time points during prenatal development. ­GFP+ myeloid cells can be found in the periocular mesenchyme as early as embryonicday (E) E9.5 and enter the optic cup through the optic stalk at E10.5 (arrow), whereas hyalocytes (asterisks) can be distinguished from microglia(arrowheads) for the first time at E11.5 by localization. Scale bar = 200 µm (overview) or 50 µm (magnification). h–l Representative images
from cryosections of Cx3cr1-GFP mice during early postnatal development. Hyalocytes can be found in the vitreous (asterisk), retinal microglia(arrowheads) are evenly distributed across the emerging plexiform layers. Scale bar = 200 µm (overview) or 50 µm (magnification). telenc. vesicle
telencephalic vesicle, mes—mesenchyme, n.ep.—neuroepithelium, OV—optic vesicle, OS—optic stalk, LP—lens placode, L—lens, Nb—neuroblast
layer, Vitr—vitreous, GCL—ganglion cell layer, IPL—inner plexiform layer, INL—inner nuclear layer, OPL—outer plexiform layer, ONL—outer nuclear
layer, PRL—photoreceptor layer. Images are representative for n = 2 mice per time point

Rosmus et al. Journal of Neuroinflammationa(2024) 21:168

Page 8 of 20

b

E9.5

Cx3cr1-GFP
DAPI

E10.5
LP

mes
telenc.
vesiclemestelenc.
vesicle
n.ep.

OV

OS
n.ep.

c

E11.5

d

L

*

E12.5

L

Vitr

*

*

Nb

e

E14.5

Nb

f

L

Vitr

E16.5

L

*

* Vitr

Vitr
GCL
IPL

Nb

g

L

E18.5

Nb

h

P1

* Vitr

*

GCL
IPL

GCL
IPL
Nb

Nb

i

j

P7

*

P14

IPL

GCL

INL

IPL

OPL

INL

ONL

OPL

PRL

ONL

k

P21

l

*
GCL
IPL
INL

Fig. 2 (See legend on previous page.)

GCL

P60

GCL
IPL

OPL

INL
OPL

ONL

ONL

Rosmus et al. Journal of Neuroinflammation(2024) 21:168

In summary, our embryonic pulse labeling approach
establishes a prenatal origin of murine hyalocytes andpoints towards the extra-embryonic YS as the major
source.
Postnatal fate mapping identifies hyalocytes as long‑living
tissue‑resident macrophages

After having established a prenatal origin of murine
hyalocytes, our next step was to analyze the contribution of circulating monocytes to the resident hyalocyte
pool during postnatal development. Therefore, we took
advantage of a postnatal fate mapping approach in which
TAM is injected into six-week-old Cx3cr1CreER:Rosa26YFP animals as described before [18, 56, 57] to label tissue-resident macrophages. Retinal flat mounts of thesemice were analyzed 2 weeks and 26 weeks after injection(Fig. 4a, b). Hyalocytes (2 weeks: 95.93 ± 1.1, 26 weeks:
96.28 ± 1.1) showed, similar to rMG (2 weeks: 97.74 ± 0.5,
26 weeks: 98.01 ± 0.5), a high number of ­YFP+ cells(Fig. 4c, d), that remained stable over time resembling the
low turnover rates observed in brain microglia, CAMs,
and other ocular macrophages [15, 17–19]. Of note, theexpression of F4/80 was suitable to discriminate between
F4/80+ hyalocytes and rMG in Cx3cr1CreER:Rosa26-YFP
mice (Fig. 4e). However, for the reason of comparability
between both cell types and other ocular macrophages
as well as previous studies [18], we decided to use Iba1
for the quantitative analysis going forward. To complement our data, we used parabiosis by surgically connecting UbcGFP/+ mice, which express GFP under the controlof the Ubiquitin C promoter in all cell types including
monocytes in the blood stream, to Ubc+/+ mice and analyzed them 4 and 28 weeks after pairing (Fig. 5a). Indeed,­GFP+IBA1+ hyalocytes were not found in the vitreousof the wildtype parabiont indicating a negligible contribution of the adult hematopoiesis to the hyalocyte pool
under homeostatic conditions (Fig. 5b, c), whereas flow
cytometry demonstrated a consistent level of blood

Page 9 of 20

chimerism in wildtype parabionts (4 weeks: 46.93 ± 5.6%,
28 weeks: 52.98 ± 6.9%). These results were further supported by the use of Flt3Cre:Rosa26-YFP mice, in whichhematopoietic stem cells (HSCs) of the fetal and adulthematopoiesis and their progeny but not descendants of
the YS hematopoiesis are labeled (Fig. 5d) [12, 22, 60]. In
this model, ­YFP+ hyalocytes were not detected in the vitreous (Fig. 5e, f ). All in all, these data support the notionthat murine hyalocytes represent a long-living cell population, that seeds prenatally and is maintained throughlocal self-renewal and without contribution of adulthematopoiesis.
Hyalocyte maintenance depends on CSF1R signaling

Previous studies using pharmacological or genetic
approaches to interfere with the function of CSF1R provided compelling evidence that most tissue-resident macrophages rely on sufficient stimulation of CSF1R, whichis encoded by the Csf1r (historically known as c-fms)
gene [61], through its ligands CSF1 or Interleukin-34
for their maintenance [62, 63]. To investigate whether
murine hyalocytes also rely on CSF1R signaling for theirperpetuation, we used Csf1r-EGFP transgenic MacGreen mice [64] to confirm CSF1R expression in thesecells (Fig. 6a). Indeed, we detected consistent transgene
expression in murine hyalocytes and rMG, which, inthe latter case, were previously found to express CSF1R[65–67] (Fig. 6b). After having confirmed CSF1R expression in hyalocytes, we sought to investigate whether an
impairment of the CSF1R in macrophages would also
result in alterations of hyalocytes in the murine vitreous.
Therefore, we took advantage of a genetic approach using
the recently generated Csf1r∆FIRE/∆FIRE mouse line [68]. In
these mice, CRISPR/Cas9-based gene editing was used
to delete the fms-intronic regulatory element (FIRE), a
superenhancer of the Csf1r gene locus [69] positioned in
the second intron (Fig. 6c). Subsequent analysis revealed
that several tissue-resident macrophages including(See figure on next page.)
Fig. 3 Embryonic pulse labeling reveals a prenatal origin of murine hyalocytes. a Schematic of bone marrow chimera creation. Wildtype mice
(Actb+/+) were whole-body irradiated and reconstituted intravenously (i.v.). with bone marrow of ActbGFP/+ mice. b Images of retinal flat mountsof ActbGFP/+:Actb+/+ bone marrow chimeras. ­GFP+ donor-derived hyalocytes are present in the vitreous. Images are representative for three
mice. Scale bar = 100 µm. c Schematic of embryonic pulse labeling experiment. Cx3cr1CreER:Rosa26-YFP mice were injected with tamoxifen (TAM)
at embryonic day (E) 9 and analyzed at postnatal day (P) 0 or P42. d Illustration of embryonic pulse labeling approach. TAM administration
activates inducible Cre-recombinase which irreversibly removes a LoxP-site-flanked STOP-sequence in Cx3cr1-expressing cells, causing consistent
YFP-expression and labeling C­ X3CR1+ cells and their progeny. e Images of E9.0-labeled hyalocytes (asterisk), rMG (arrowhead) and choroidal
macrophages (chMacs) (arrow) in a cryosection from Cx3cr1CreER:Rosa26-YFP mice at P0. Images are representative for six mice from three
independent experiments. Scale bar = 50 µm. Vitr—vitreous, IPL—inner plexiform layer, NBL—neuroblast layer, Ch—choroid. f Quantificationof ­YFP+ cells among ­IBA1+ hyalocytes, rMG and chMacs in E9.0-labeled Cx3cr1CreER:Rosa26-YFP mice at P0. Graphs depict mean ± S.E.M for six micefrom three independent experiments. Statistics: one-way repeated measure ANOVA with post-hoc Tukey’s multiple-comparison test (* p < 0.05,** p < 0.01). g Images of retinal flat mounts depicting E9.0-labeled hyalocytes (asterisk) and rMG (arrowheads) from Cx3cr1CreER:Rosa26-YFP miceat P42. Images are representative for six mice. Scale bar = 50 µm. h Percentage of ­YFP+ cells among ­IBA1+ hyalocytes and rMG in E9.0-labeled
Cx3cr1CreER:Rosa26-YFP mice at P42. Graphs depict mean ± S.E.M for six mice. Statistics: paired t-test (n.s., p > 0.05)

Rosmus et al. Journal of Neuroinflammation(2024) 21:168

Page 10 of 20

b

a

IBA1
Actb+/+GFP

IBA1 GFP

Actb GFP/+
i.v.

c

d

Cx3cr1CreER:Rosa26-YFP

microscopic analysis

TAM-injection

P0

E9

P42

f

e
IBA1 YFPIBA1 YFP DAPI

*

100

Vitr
IPL

NBL

+

+

P0

*

E9

*

YFP cells of Iba1 cells [%]IBA1 DAPICh*IBA1 YFP

40
20

*

rMG

Hyalocytes chMacs
n.s.

100

*

+

+

YFP

**

60

0

YFP cells of Iba1 cells [%]

IBA1

*

80

h

E9

P42

g

n.s.

80
60
40
20
0

rMG

Hyalocytes

Fig. 3 (See legend on previous page.)microglia in the brain were depleted without causing thesevere phenotype observed in Csf1r-KO mice [68]. Consequently, we hypothesized that, given the tight ontogenetic relationship between hyalocytes and microglia,
ablation of FIRE would lead to reduced hyalocyte numbers in Csf1r∆FIRE/∆FIRE mice. Immunofluorescence labeling and subsequent quantitative analysis in retinal whole

mounts revealed a complete absence of hyalocytes andmicroglia in comparison to wildtype controls (Fig. 6d, e),thereby confirming our hypothesis.
Prenatally, the developing vitreous and lens rely on thehyaloid vasculature, which consists of the hyaloid artery,the vasa hyaloidea propria and the tunica vasculosa lentis, to meet their high metabolic demand [70]. Following

Rosmus et al. Journal of Neuroinflammation(2024) 21:168

the developmental growth phase, the metabolism of vitreous (and lens) decreases significantly which creates theneed of a regression of the hyaloid vessels, mediated bymurine hyalocytes as described in previous studies [71,72], to avoid light scattering. To address the question,
whether a depletion of murine hyalocytes is accompanied by a persistence of the hyaloid vasculature during
adulthood, we analyzed cryo-sections of murine eyesfrom 4-month-old Csf1r∆FIRE/∆FIRE mice and wildtype
controls. Interestingly, FIRE ablation was, aside from thecomplete absence of myeloid cells in the retina and vitreous, not accompanied by morphological alterations of thevitreoretinal compartment. In particular, we did not findany sign of persistent hyaloid vessels or alterations of thesuperficial and deep plexus in the IPL and OPL (Fig. 6f ).
All in all, our data demonstrate that CSF1R is expressedby murine hyalocytes and rMG and that an intact FIRE
sequence represents a prerequisite for the maintenanceof these cell populations but is not associated with thepersistence of the hyaloid vasculature during adulthood.

Discussion
Here, we provide evidence that murine hyalocytes, thetissue-resident macrophages of the vitreous body, display
a unique immunophenotype, originate from YS hematopoiesis and populate the developing eye as early as at
E11.5. Within the vitreous, they establish a self-maintaining cell population, which persists until adulthood andrelies on CSF1R for its maintenance, while being independent of blood monocytes (Fig. 7).
By performing immunofluorescence labeling on retinal
flat mounts from Cx3cr1-GFP mice, we identified preretinal hyalocytes as ­​CX​3C​R1​+I​BA1​+​F4/​80​+C​D16​3+​CD​206​+​
LYV​E1​+M​HCI​I−HEXB−TMEM119− cells confirming
their established myeloid cell identity, as previously suggested by immunocytochemical studies in tissue-cultured
hyalocytes [73] as well as in murine tissue sections [74].
Importantly, we show that murine hyalocytes exhibit

Page 11 of 20

a distinct immunophenotype in comparison to rMG,
which consistently express both HEXB and TMEM119,
as has already partially been described in previous studies[18, 47, 49, 50, 75, 76] including the work of Rajesh andcolleagues employing the Tmem119-GFP mouse model
to study tissue-resident macrophages at the vitreoretinal
interface [77]. Over and above that, this corroborates thefindings of recent studies investigating the transcriptional
profile of human hyalocytes demonstrating that humanhyalocytes express several myeloid signature genes [28],but concurrently display a unique gene expression profile
in comparison to microglia in the human retina [78, 79].
The expression of CD163 and CD206 in hyalocytes might
indicate similarities to CAMs in the brain, however, further research in necessary to support this hypothesis.
Interestingly, homeostatic hyalocytes in the mouse do
not express MHCII, which is in line with the findingsof Vagaja and colleagues [74], whereas MHCII-expression was upregulated in murine models of peripheral
lipopolysaccharide challenge and diabetic retinopathy
suggesting a role of hyalocytes in the induction of adaptive immunity [74]. The eye, including the vitreous body,
however, is regarded as an immune privileged site whichmeans that proinflammatory stimuli cause attenuatedimmune responses [80, 81]. Given their strategical position at the vitreoretinal interface, hyalocytes might contribute to this phenomenon [48, 82, 83], in parts by the
reduced expression of MHCII which is a known mechanism of immune privilege maintenance as shown for
microglia in the CNS [84]. Interestingly, human adulthyalocytes regularly express HLA-DR under homeostatic conditions [28, 78, 85]. Therefore, the mechanisms
by which the immune privilege of the eye is maintained
and, specifically, the role that hyalocytes might play indifferent species still warrants further investigation. In
addition, previous studies suggested ultrastructural differences between vitreoretinal hyalocytes and anterior
hyalocytes residing in close proximity to the ciliary body(See figure on next page.)
Fig. 4 Hyalocytes represent a long-living tissue-resident macrophage population. a Graphical scheme of the experimental setup. Six-week-old
Cx3cr1CreER:Rosa26-YFP mice were injected with tamoxifen (TAM) and retinal whole mounts subsequently analyzed by fluorescence microscopy
at 2 and 26 weeks post-injection (p.i.). b Graphical scheme illustrating the rationale of the adult turnover approach. TAM administration leads
to nuclear translocation of cytosolic Cre-ER fusion protein and subsequent Cre-mediated irreversible excision of a LoxP-site-flanked STOP-cassettein Cx3cr1-expressing cells. This causes a consistent level of YFP-expression under the control of the constitutively active Rosa26 promoter
and labeling of ­CX3CR1-positive cells and their progeny. c Confocal images of ­YFP+ and ­IBA1+ hyalocytes (asterisks) and microglia (arrowheads)
in Cx3cr1CreER:Rosa26-YFP at 2 weeks and 26 weeks after injection of TAM. Images are representative for eight animals (2 weeks) from three
independent experiments and ten animals (26 weeks) from two independent experiments, respectively. Scale bar = 50 µm. d Percentages of ­YFP+
cells among ­IBA1+ hyalocytes and rMG in Cx3cr1CreER:Rosa26-YFP mice 2 weeks (upper plot, N = 8, n.s., p > 0.05, paired t-test) and 26 weeks (lower
plot, N = 10, n.s., p > 0.05, Wilcoxon signed-rank test) post-injection. Recombination efficiency, as determined by the percentage of ­YFP+ brain
microglia using flow cytometry, was 90.38 ± 2.98% (2 weeks) and 97.17 ± 1.49% (26 weeks) among viable doublet-excluded ­CD45loCD11b+ cells.
Data are presented as mean ± S.E.M. e Representative confocal images of F4/80+YFP+ hyalocytes (asterisks) and F4/80−YFP+ rMG in retinal whole
mounts from Cx3cr1CreER:Rosa26-YFP at 2 weeks and 26 weeks after injection of TAM. Images are representative for four mice per timepoint. Scalebar = 50 µm

Rosmus et al. Journal of Neuroinflammationa

Cx3cr1CreER:Rosa26-YFP(2024) 21:168

Page 12 of 20b

microscopic analysis

TAM-injection

2 weeksp.i.

6 weeks

26 weeks
p.i.

c

d
YFP

IBA1 YFP

*

*

+

*

IBA1 YFP*

26 weeks p.i.

*
*

*

*
*

*
*

*

*

+

*

F4/80

YFP

2 weeks p.i.

*

*

*

*

*

*

*

*

20
0

rMG

Hyalocytes
n.s.

100
80
60
40
20
0

rMG

YFP

Hyalocytes

F4/80 YFP

*

*

F4/80

YFP

F4/80 YFP

F4/80

YFP

F4/80 YFP*

F4/80 YFP

*
*

40

*
*

YFP

*

*
*

*

*

F4/80

*

*

60

*

*

*

*
*

F4/80

F4/80 YFP*

26 weeks p.i.

80

+

YFP

YFP cells of Iba1 cells [%]

IBA1

e

n.s.

100

+

2 weeks p.i.

*

YFP cells of Iba1 cells [%]

IBA1

*

*

*

*
*

*

*
*

F4/80

*
YFP

*
F4/80 YFP*

Fig. 4 (See legend on previous page.)[40]. Although the scope of this study was to investigate
hyalocytes at the vitreoretinal interface, we acknowledge that single-cell profiling studies will be necessary tocapture the complete, molecular heterogeneity of murine
hyalocytes and to uncover differences between hyalocyte
subpopulations in different anatomical localizations.

aParabiosis
Ubc+/+ Ubc+/GFP(2024) 21:168b

microscopic analysis

IBA1Page 13 of 20

GFP

c

IBA1 GFP

* *

* *

**

4 weeks 28 weeksde

GFP+ cells of Iba1+ cells [%]Rosmus et al. Journal of Neuroinflammation

10

rMG

8

Hyalocytes

6
4
2
0

4 weeks

28 weeksrMG

Hyalocytes

*
*

*
*

*

*
*

*

*

*
*

*

*
*

*

+

IBA1 YFP+

YFP

YFP cells of Iba1 cells [%]

f
IBA1

10
8
6
4
2
0

Fig. 5 Circulating monocytes from adult hematopoiesis do not contribute to the resident hyalocyte pool under homeostasis. a Graphical schemeof parabiosis experiments. UbcGFP/+ donor mice were surgically connected to Ubc+/+ wildtype mice for parabiosis and retinal whole mounts
subsequently analyzed by fluorescence microscopy after 4 and 28 weeks, respectively. b Confocal images of retinal flat mounts from Ubc+/+
acceptor parabionts. Hyalocytes (asterisks) and rMG (arrowheads) in the inner plexiform layer can be identified. Images are representative for three
mice (4 weeks) and four mice (28 weeks), respectively. Scale bar = 50 µm. c Quantification of ­GFP+ cells among ­IBA1+ hyalocytes and rMG in Ubc+/+
parabiotic mice 4 (N = 3) and 28 (N = 4) weeks after surgery. Each symbol represents one animal. Blood chimerism of ­CD11b+ myeloid blood
cells in Ubc+/+ recipient parabionts, as assessed by flow cytometry, was 46.93 ± 5.6% for 4 weeks and 52.98 ± 6.9% for 28 weeks post surgery. dGraphical illustration describing the experimental setup. In Flt3Cre:Rosa26-YFP mice, constitutive activity of Cre-recombinase leads to YFP expression,under the control of the Rosa26-promoter, in all ­FLT3+ hematopoietic cells during fetal and postnatal hematopoiesis and their progeny. e
Fluorescent microscopic visualization of IBA1 (red) and YFP (green) in hyalocytes (asterisks) and rMG (arrowheads) in Flt3Cre:Rosa26-YFP mice. Images
are representative for four mice. Scale bar = 100 µm. f Quantification of ­YFP+ cells among ­IBA1+ hyalocytes and rMG in Flt3Cre:Rosa26-YFPmice (N = 4).
Each symbol represents one animal from one litter

By performing immunofluorescence labeling on eyes atdifferent stages of prenatal development, we found thatmyeloid cells were present in the periocular mesenchymeat E9.5 and hyalocyte precursors were identified in thedeveloping vitreous cavity as early as E11.5. This is in linewith studies examining the embryonic origin of microglia
in the brain where precursors of microglia first colonized
the adjacent mesenchyme at E9.0 and then entered theunderlying neuroepithelium shortly after [11]. Our data
indicate that myeloid cells may enter the developing eye
through the optic stalk, as part of the developing CNS,
which has been observed before in the context of rMG[86]. In this study, distinct waves of macrophages colonizing the murine retina were characterized, thereby
confirming previous reports from studies in quails inwhich rMG tangentially populated the retina from central to peripheral areas and through radial migrationfrom the vitreoretinal interface to the plexiform layers[87]. The presence of yet undefined myeloid cells at thevitreoretinal interface shortly after birth further suggests a common precursor of hyalocytes and rMG andfavors a model in which immature rMG radially infiltrate
the plexiform layers in the course of the ongoing stratification of the retina and undergo further maturation
processes whereas hyalocytes remain and mature at thevitreal side of the interface [75, 86]. However, it is also
imaginable that a bidirectional exchange between rMG
and hyalocyte precursors occurs until the ILM is established, thereby separating these different microanatomical niches. Such a colonization pattern would strongly
resemble the behavior of brain microglia and myeloid
cells destined to become intracerebroventricular macrophages in the CNS [88, 89], but requires further investigation in the eye. Finally, the anatomical peculiarities ofthe eye need to be considered as the ciliary zonules connecting the ciliary body and the lens were recently identified as a possible trafficking pathway for immune cells[90], given the fact that a recent study suggested that theRosmus et al. Journal of Neuroinflammationa(2024) 21:168

Page 14 of 20b

Csf1r-EGFP
IBA1

Transcription+
Translation

*

*

c

*

*

*

*

EGFP

d
IBA1

Csf1r ΔFIRE/ΔFIRE

FIRE

+/+

IBA1

FIRE

∆/∆

e
150

cell density in cells/mm 2

CSF1R

IBA1 Csf1r-EGFP

Csf1r-EGFP

+/+

FIRE
∆/∆
FIRE

100

*
*

50
0

n.d.
rMG

n.d.
Hyalocytesf
IBA1

Collagen IV

DAPI IBA1 Collagen IV

DAPI

IBA1

Collagen IV

DAPI IBA1 Collagen IV

FIREΔ/Δ

FIRE+/+DAPI

Fig. 6 CSF1R-dependence of murine hyalocytes and retinal microglia. a Graphical scheme depicting the experimental setup. In Csf1r-EGFP mice,enhanced green fluorescent protein (EGFP) is expressed under the control of the transgenic Csf1r promoter. Subsequent protein biosynthesis
leads to the simultaneous expression of CSF1R and EGFP in these mice. b Confocal images of IBA1 and anti-GFP immunofluorescenceco-staining on retinal flat mounts from Csf1r-EGFP mice. ­EGFP+ hyalocytes (asterisks) and rMG (arrowheads) can be regularly identified. Images
are representative for three mice. Scale bar = 50 µm. c Graphical illustration depicting the gene targeting approach in Csf1r∆FIRE/∆FIRE mice. CRISPR/
Cas9-based gene editing was applied to delete the fms-intronic regulatory element (FIRE) in the second intron of the Csf1r gene locus. d Confocal
images of IBA1 immunofluorescence labeling on Csf1r∆FIRE/∆FIRE mice and wildtype controls. Hyalocytes (asterisks) and rMG (arrowheads) can befound in wildtype mice, whereas ­IBA1+ myeloid cells are completely absent in Csf1r∆FIRE/∆FIRE mice. Images are representative for four mice per groupand two independent experiments. Scale bar = 50 µm. e Quantification of microglia and hyalocyte density in Csf1r∆FIRE/∆FIRE (N = 4) and wildtype
controls (N = 4). Data are presented as mean ± S.E.M. f Images from Collagen IV and IBA1 immunofluorescence co-staining on cryo-sections of eyesfrom wildtype controls (upper panel) and Csf1r∆FIRE/∆FIRE mice (lower panel). Images are representative for four mice per group. Scale bar = 50 µm

Rosmus et al. Journal of Neuroinflammation(2024) 21:168

Page 15 of 20

Fig. 7 Origin, turnover and phenotype of murine hyalocytes. Graphical summary of the findings in this study. Prenatally, the local hyalocyte
and microglial pool is recruited from yolk sac-derived C­ X3CR1+ A2 progenitors that are labeled with YFP (green) in Cx3cr1CreER:Rosa26-YFP
mice after tamoxifen (TAM) injection at E9.0 and enter the vitreous cavity through the blood stream. Postnatally, fate mapping in adultCx3cr1CreER:Rosa26-YFP mice has shown that hyalocytes, which exhibit a unique immunophenotype and rely on CSF1R-signaling for theirmaintenance, are long-living cells independent of replenishment from circulating peripheral myeloid cells from the definitive hematopoiesis.
Hyalocytes are largely maintained by local self-renewal and reside above the inner limiting membrane while retinal microglia are locatedbelow in the neuroretina. The inner limiting membrane is constituted of both vitreal and retinal laminae. The vitreal side has a dense collagen fibril
meshwork connected via extracellular matrices to the retinal glia limitans, built by the endfeet of the astrocytes (purple) residing in the nerve fiberand ganglion cell layer, separating hyalocytes and retinal microglia in two distinct compartments of the eye. Vitr—vitreous, ILM—inner limiting
membrane, GCL—ganglion cell layer, IPL—inner plexiform layer, INL—inner nuclear layer, OPL—outer plexiform layer, ONL—outer nuclear layer,
PRL—photoreceptor layer, RPE—retinal pigment epithelium

ciliary body may also serve as a source for rMG repopulation in a microglia cell depletion paradigm [91].
Previous studies investigating the origin and fate ofmurine hyalocytes have been restricted to bone marrowtransplantation techniques relying on whole-body irradiation that may cause artificial conditions under which
the engraftment of peripheral monocytes into tissues isfacilitated [36–38, 92, 93]. Indeed, we found an engraftment of ­GFP+ cells in ActbGFP/+→Actb+/+ bone marrow chimeras with a morphology strongly resembling
hyalocytes, which is in line with previous bone marrowtransplantation studies reporting a significant engraftment of peripheral myeloid cells into the retina [18, 34,
38, 92]. To challenge the previous conception, we used
an established inducible fate mapping model employing Cx3cr1CreER:Rosa26-YFP mice [15, 18, 56, 57], whichlabels ­CX3CR1+ A2 progenitors by TAM injection at
E9.0 and their descendants [11, 13] to trace the engraftment of YS-derived macrophages into different ocularcompartments in the absence of an artificial influx of
peripheral immune cells. We observed recombination
rates in murine hyalocytes that are comparable to thosefor other YS-derived macrophages [15, 18, 59]. These
data point towards a prenatal origin of murine hyalocytes [20]. Furthermore, as the injection of TAM into
the maternal body of Cx3cr1CreER:Rosa26-YFP mice at
E9.0, given its limited bioavailability, does not lead toa labeling of HSCs in the FL [59], our approach is specific for and clearly demonstrates the major contribution of YS hematopoiesis to the hyalocyte population.
Besides, the Flt3Cre:Rosa26-YFP mouse line used inthis study was shown to label HSC descendants of the
fetal and adult hematopoiesis [12, 15, 22, 60] and, given
the absence of Y
­ FP+ hyalocytes in this model, further
supports a YS-derived origin of murine hyalocytes
and argues against a significant contribution of HSCderived FL hematopoiesis.

Rosmus et al. Journal of Neuroinflammation(2024) 21:168

Concerning the turnover of macrophages, different
organs show remarkable differences. Whereas microglia
in the CNS are long-living cells [15, 18] with random andlimited self-expansion under homeostatic conditions[94], ocular macrophages in the choroid, cornea and ciliary body have at least in part been shown to be replenished by circulating monocytes [17, 18]. Using theCx3cr1CreER:Rosa26-YFP mice in an adult fate mapping
approach, we show that murine hyalocytes represent a
long-living cell population, which remains stable overtime. This is in line with a recent independent study by
Rajesh and colleagues demonstrating that vitreous macrophages retain YFP-labeling over a period of 4 weeks
after TAM-injection in adult Cx3cr1CreER:Rosa26-YFP
mice [77]. However, in a previous study, we were able todemonstrate that it may last up to 4 weeks after injection until peripheral monocytes lose their YFP-label [57].
Furthermore, given the fact that distinct compartmentsof the eye and brain, such as the cornea and the choroid
plexus, show a slow but steady turnover over months [15,18], the assumption can be made that a time window of
4 weeks might be too short to use the full potential of thisline [15, 18]. Therefore, our work provides a significant
and necessary extension of the findings in the study by
Rajesh and colleagues and allows to verify the hypothesis that hyalocytes are a self-maintaining cell population over an extended period of time. Moreover, our data
illustrate that the maintenance of murine hyalocytes is
independent of peripheral monocytes as shown by using
parabiotic mice with a shared blood circulation. Finally,
we further corroborated the independence of hyalocytes
from adult hematopoiesis using Flt3Cre:Rosa26-YFP micelabeling FLT3-expressing HSCs [95], in which we did notobserve a contribution of HSCs to the hyalocyte population. Taken together, these data clearly demonstrate thathyalocytes are a self-maintaining cell population underhomeostatic conditions. Of note, vitreous detachment
can often be observed at the human vitreoretinal interface which may impede local self-renewal in vitreoretinal hyalocytes and still requires further investigation.
Furthermore, previous studies linked hyalocytes to several pathological conditions of the vitreoretinal interface
like diabetic retinopathy [74, 96, 97] or proliferative vitreoretinopathy [27, 98–100]. These diseases may cause
severe alterations in the local microenvironment which
may ultimately lead to an additional influx of peripheral immune cells. In the retina, microglia represent the
predominant immune cell population in several retinal
disease models [17, 18, 101–106]. In the vitreous, however, the intravitreal injection of the proinflammatory
chemokine CCL2 was found to increase the numberof preretinal ­Ly6C+ monocytes [77] indicating that the
myeloid compartment may undergo significant changes

Page 16 of 20

in its composition during disease. Therefore, futurestudies using tools to differentiate monocytes from resident hyalocytes, as the Ccr2CreER mouse line [107], will
be necessary to dissect the contribution of distinct cell
populations to pathologies of the vitreoretinal interface.
The translation of these findings into the human setting
could open new therapeutic avenues as demonstrated for
Osteopontin in neovascular AMD [108, 109], which isalso expressed by hyalocytes on the transcriptional level[28, 78]. Moreover, aging is also associated with changes
in long-living tissue-resident macrophages due to immunosenescence that may affect their responses to pathological insults, as already demonstrated for microglia
in the brain [110, 111] and the retina [112]. In this context, targeting long-lived resident hyalocytes to reverse a
potential immunosenescence-associated hyalocyte dysfunction for the treatment of vitreoretinal diseases could
be achieved by local, intravitreal application of pharmacological agents, a practice performed in a daily mannerin the field of ophthalmology.
Finally, we confirmed that hyalocytes, but also rMG,
express CSF1R and, using Csf1r∆FIRE/∆FIRE mice, we
showed that CSF1R deletion in hyalocytes and rMG leads
to complete absence of these cells in the murine eye. To
our knowledge, this is the first report using this model toinvestigate macrophages in the eye and our results corroborate the findings of previous studies in which thedeletion of the FIRE sequence in the Csf1r gene led to thedepletion of several tissue-resident macrophage populations including brain microglia [68, 88, 113]. In the past,murine hyalocytes have been implied in the postnatal
regression of the hyaloid vasculature [71, 72]. Although
we found a complete absence of hyalocytes and rMG in
Csf1r∆FIRE/∆FIRE mice, we did not witness any morphological changes, such as persistent hyaloid vessels, inthe eyes of these animals during adulthood. Of note, we
examined eyes harvested from four-month-old animals.
Therefore, we cannot exclude that hyalocytes play a relevant homeostatic role earlier and that the regression of
the hyaloid vasculature is still delayed in Csf1r∆FIRE/∆FIRE
mice, which is compensated in their absence at later time
points through hyalocyte-independent mechanisms [71,72]. This warrants further investigation, as deeper knowledge of these mechanisms might be highly relevant for
new therapeutic interventions for diseases such as persistent hyperplastic primary vitreous, an idiopathic eye disease of childhood characterized by an absent regressionof hyaloid vessels [114, 115].

Conclusions
In conclusion, our study reveals hyalocytes as a newmember of the family of long-living, YS-derived tissueresident macrophages, which are independent of a steadyRosmus et al. Journal of Neuroinflammation(2024) 21:168

turnover by peripheral myeloid cells and rely on CSF1R
for their maintenance in vivo. These data shed new lighton the innate immune system of the eye and may pave
the way for the development of new myeloid cell-focused
therapies of vitreoretinal diseases adapted to the distinct
anatomical niches of the eye.
Abbreviations
AMD	Age-related macular degeneration
BSA	Bovine serum albumin
CAM	CNS-associated macrophage
CCL2	C–C chemokine receptor ligand 2
Ccr2	C–C chemokine receptor type 2
CSF1R	Colony Stimulating Factor 1 Receptor
CX3CR1	Fractalkine receptor
DAPI	4′,6-Diamidine-2-phenylindoleE	Embryonic day
EMP	Erythromyeloid precursor
FBS	Fetal bovine serum
FIRE	
fms-Intronic regulatory element
FL	Fetal liver
GCL	Ganglion cell layer
GFP	Green fluorescent protein
HSC	Hematopoietic stem cell
ILM	Inner limiting membrane
INL	Inner nuclear layer
i.p.	Intraperitoneal
IPL	Inner plexiform layer
ISP	Isotonic Percoll
L	Lens
LP	Lens placodeLYVE1	Lymphatic vessel endothelial hyaluronic acid receptor 1mes	Mesenchyme
MHCII	Major histocompatibility complex II
Nb	Neuroblast
n.ep.	Neuroepithelium
ONL	Outer nuclear layer
OPL	Outer plexiform layer
OS	Optic stalk
OV	Optic vesicle
P	Postnatal day
PBS	Phosphate buffered saline
PFA	Paraformaldehyde
PRL	Photoreceptor layerrMG	Retinal microglia
RT	Room temperature
RPE	Retinal pigment epithelium
TAM	Tamoxifen
Vitr	Vitreous body
WT	Wildtype
YFP	Yellow fluorescent protein
YS	Yolk sac

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03110-x.
Additional file 1: Figure S1. Expression of CD163 in tissue-resident
macrophages at the vitreoretinal interface. Representative images from
immunofluorescence labeling for CD163 with IBA1 in Cx3cr1-GFP mice.
Hyalocytes (asterisks) consistently show CD163 expression whereas rMG(arrowheads) are negative for this marker. Images are representative forthree mice. Scale Bar = 50 µm.
Additional file 2: Figure S2. Immunophenotype of murine hyalocytes
during prenatal development. Representative images from immunofluo‑
rescence labeling for CD11b, F4/80 and Isolectin B4 on Cx3cr1-GFP mice atPage 17 of 20

E14.5. Noteworthily, Isolectin B4 also stains hyaloid vessels (arrow). Scalebar = 50 µm.
Acknowledgements
The authors would like to thank Maria Oberle, Angela Ehrlich, Constanze
Hobusch, Alina Bogachuk, Hortenzia Imhof and Katherine H. Rott for excellent
technical assistance and Dr. Karsten Winter for his support with Imaging.
Csf1r∆FIRE/∆FIRE mice were a kind gift from Dr. Simon Johnson to B.A. Graphical
Schemes were created using BioRender.com.
Author contributions
Conceptualization: DDR, MP, CL, PW; Data curation: DDR, PW; Formal analysis:
DDR, JK, PW; Funding acquisition: DDR, AC, SB, BA, MP, CL, PW; Investigation:
DDR, JK, AH, AC, FA, TM, HK; Methodology: DDR, JK, AC, TM, HK, KK, PW; Project
administration: DDR, PW; Resources: AC, TM, JF, MG, KK, BA, MP; Supervision:
PW; Validation: DDR, JK, PW; Visualization: DDR, PW; Writing (original draft):
DDR, PW; Writing (review&editing): DDR, JK, AH, AC, FA, KK, SMH, HK, JF, JW,
SB, MG, BA, MP, CL, PW. SMH provided bone marrow chimera tissue. TM
performed embryonic pulse labeling experiments. JF and MG contributed toanalysis of Csf1r-EGFP mice. AC and BA performed parabiosis and provided
tissue from Cx3cr1CreER:Rosa26-YFP mice. All authors read and approved themanuscript. All authors confirm that they had full access to all the data in the
study and accept responsibility to submit for publication.
Funding
Open Access funding enabled and organized by Projekt DEAL. MP and CL
were supported by the German Research Foundation (DFG, SFB/TRR167). The
project was supported by the German Research Foundation (DFG, project
number 507881424). BA and AC were supported by OHSU institutional
support. SB was supported by the Berta-Ottenstein-Programme for Clinician
Scientists, Faculty of Medicine, University of Freiburg. DDR received scholar‑
ships from the German Academic Scholarship Foundation (“Studienstiftungdes deutschen Volkes”) and the German Ophthalmological Society (DOG).
DDR, JK and PW were supported for this study by the Medical Faculty of the
University of Augsburg.
Availability of data and materials
The datasets used and/or analysed during the current study are available fromthe corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
All animal experiments were approved by local administration and were per‑formed in accordance with the respective national, federal, and institutional
regulations.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Institute of Anatomy, Leipzig University, 04103 Leipzig, Germany. 2 Cel‑lular Neuroanatomy, Institute of Theoretical Medicine, Augsburg University,
Universitätsstrasse 2, 86159 Augsburg, Germany. 3 Institute of Neuropathology,
Medical Center, Faculty of Medicine, University of Freiburg, 79106 Freiburg,
Germany. 4 Eye Center, Medical Center, Faculty of Medicine, University
of Freiburg, 79106 Freiburg, Germany. 5 Department of Molecular Microbiology
and Immunology, Oregon Health & Science University, Portland, OR 97239,
USA. 6 Department of Behavioral Neuroscience, Oregon Health & Science
University, Portland, OR 97239, USA. 7 Division of Molecular Neuroimmunology,
Medical Institute of Bioregulation, Kyushu University, Fukuoka 812‑8582, Japan.
8Centre for Integrative Biological Signalling Studies, University of Freiburg,
79106 Freiburg, Germany. 9 Centre for Basics in NeuroModulation (NeuroMod‑
ulBasics), Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany.
10
Molecular Surgery Laboratory, Stanford University, Palo Alto, CA 94304, USA.

Rosmus et al. Journal of Neuroinflammation(2024) 21:168

11

Department of Ophthalmology, Byers Eye Institute, Stanford University, Palo
Alto, CA 94304, USA. 12 Signalling Research Centres BIOSS and CIBSS, University
of Freiburg, 79106 Freiburg, Germany. 13 Ophtha Lab, Department of Ophthal‑mology, St. Franziskus Hospital, 48145 Münster, Germany.
Received: 26 January 2024 Accepted: 22 April 2024

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