Depletion of embryonic microglia using the CSF1R inhibitor PLX5622 has ad- verse sex-specific effects on mice, including accelerated weight gain, hyperac- tivity and anxiolytic-like behaviour
Jessica M. Rosin, Siddharth R. Vora, Deborah M. Kurrasch PII: S0889-1591(18)30375-1
Reference: YBRBI 3458
To appear in: Brain, Behavior, and Immunity
Received Date: 12 April 2018
Revised Date: 5 July 2018
Accepted Date: 25 July 2018
Please cite this article as: Rosin, J.M., Vora, S.R., Kurrasch, D.M., Depletion of embryonic microglia using the CSF1R inhibitor PLX5622 has adverse sex-specific effects on mice, including accelerated weight gain, hyperactivity and anxiolytic-like behaviour, Brain, Behavior, and Immunity (2018), doi: https://doi.org/10.1016/j.bbi.2018.07.023
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1Depletion of embryonic microglia using the CSF1R inhibitor PLX5622 has
2adverse sex-specific effects on mice, including accelerated weight gain,
3hyperactivity and anxiolytic-like behaviour
Jessica M. Rosin1,2,3, Siddharth R. Vora4, and Deborah M. Kurrasch 5
61Department of Medical Genetics, Cumming School of Medicine, University of Calgary,
7Calgary, Alberta, CANADA
82Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta
103Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta CANADA
114Oral Health Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, British
17Deborah M Kurrasch
18Cummings School of Medicine
19University of Calgary
203330 Hospital Drive NW, Room HSC 2215
Calgary, AB T2N 4N1
Microglia, hypothalamus, embryonic development, POMC neurons, weight gain, hyperactivity
35Microglia are the resident immune cells in the central nervous system (CNS). Originally thought
36to be primarily responsible for disposing of cellular debris and responding to neural insults,
37emerging research now shows that microglia are highly dynamic cells involved in a variety of
38neurodevelopmental processes. The hypothalamus is a brain region critical for maintaining
39homeostatic processes such as energy balance, thirst, food intake, reproduction, and circadian
40rhythms. Given that microglia colonize the embryonic brain alongside key steps of hypothalamic
41development, here we tested whether microglia are required for the proper establishment of this
42brain region. The Colony-stimulating factor-1 receptor (Csf1r) is expressed by microglia,
43macrophages and osteoclasts, and is required for their proliferation, differentiation, and survival.
44Therefore, to eliminate microglia from the fetal brain, we treated pregnant dams with the CSF1R
45inhibitor PLX5622. We showed that approximately 99% of microglia were eliminated by
46embryonic day 15.5 (E15.5) after pregnant dams were placed on a PLX5622 diet starting at E3.5.
47Following microglia depletion, we observed elevated numbers of apoptotic cells accumulating
48throughout the developing hypothalamus. Once the PLX5622 diet was removed, microglia
49repopulated the postnatal brain within 7 days and did not appear to repopulate from Nestin+
50precursors. Embryonic microglia depletion also resulted in a decreased litter size, as well as an
51increase in the number of pups that died within the first two postnatal days of life. In pups that
52survived, the elimination of microglia in the fetal brain resulted in a decrease in the number of
53Pro-opiomelanocortin (POMC) neurons and a concomitant accelerated weight gain starting at
54postnatal day 5 (P5), suggesting that microglia could be important for the development of cell
55types key to hypothalamic satiety centers. Moreover, surviving PLX5622 exposed animals
56displayed craniofacial and dental abnormalities, perhaps due to non-CNS effects of PLX5622 on
57macrophages and/or osteoclasts. Finally, depletion of microglia during embryogenesis had long-
58term sex-specific effects on behaviour, including the development of hyperactivity and
59anxiolytic-like behaviour in juvenile and adult female mice, respectively. Together, these data
60demonstrate an important role for microglia during the development of the embryonic
hypothalamus, and perhaps the CNS more broadly.
63 1. Introduction
64 Microglia are the resident mononuclear phagocytic immune cells of the central nervous
65system (CNS). During embryogenesis, macrophages generated from erythro-myeloid progenitors
66(EMPs) in the yolk sac travel to the neuroectoderm and invade the developing CNS to generate
67microglia (Ginhoux et al., 2010; Gomez Perdiguero et al., 2015; Kierdorf et al., 2013). Microglia
68continue to develop and alter their morphology and expression signatures throughout the CNS
69during embryonic, postnatal, and adult stages (Butovsky et al., 2014; Kierdorf et al., 2013;
70Matcovitch-Natan et al., 2016; Thion et al., 2018), suggesting differing roles for microglia across
71defined developmental stages. Moreover, following the closure of the blood brain barrier,
72peripheral myeloid cells are rarely found to infiltrate the brain parenchyma during normal
73healthy CNS conditions (Ginhoux et al., 2010; Mildner et al., 2007), demonstrating that
74microglia truly represent a unique tissue-specific macrophage important for the CNS.
75 Originally thought to be primarily responsible for disposing of cellular debris and
76responding to neural insults, emerging research now shows that microglia are highly dynamic
77cells involved in a variety of developmental processes. Study of embryonic microglia shows that
78they can play a role in neural progenitor maintenance (Antony et al., 2011), in addition to neural
79progenitor engulfment and elimination at the termination of neurogenesis (Cunningham et al.,
802013). In the embryo, fetal microglia can direct cortical neuron migration and differentiation, as
81well as modulate the wiring and lamination of cortical neurons (Aarum et al., 2003; Squarzoni et
82al., 2014). Postnatally, microglia influence neuronal connectivity via their roles in dendritic spine
83formation (Miyamoto et al., 2016) and synaptic pruning (Paolicelli et al., 2011; Schafer et al.,
842012; Tremblay et al., 2010). This continuously growing body of literature supporting a role for
85microglia both as the tissue-specific immune cells of the brain, and as functional players
86embryonically and postnatally, highlight the importance of microglia for normal healthy CNS
87development. The functional role of microglia during embryonic and postnatal development is
88further supported by C-X3-C motif chemokine receptor 1 (Cx3cr1) knockout mice, whereby
89disrupting microglia signaling causes hyperactivity, anxiolytic-like behaviours, and phenotypes
90resembling depression in mutant female mice (Bolos et al., 2018).
91 The Colony-stimulating factor-1 receptor (Csf1r) is expressed by microglia, macrophages
92and osteoclasts, and is required for their proliferation, differentiation, and survival (Li et al.,
932006; Patel and Player, 2009). Csf1r knockout causes complete microglia ablation that leads to
94defects in the postnatal olfactory bulb, as well as an increase in cortical neurons and astrocytes
95with a concomitant decrease in oligodendrocyte cell number (Erblich et al., 2011). Moreover,
96these Csf1r knockout mice often do not survive to adulthood (Erblich et al., 2011; Ginhoux et al.,
972010). In contrast, microglia ablation in the adult using the CSF1R inhibitor PLX3397 results in
98~99% microglia depletion in the brain; however, no cognitive or behavioural impairments were
99identified (Elmore et al., 2014). Following removal of this CSF1R inhibitor, microglia repopulate
100the adult brain within one week, and were thought to repopulate from the proliferation of Nestin-
101expressing neural cells that differentiate into microglia (Elmore et al., 2014). However, a more
102recent study shows that microglia in the adult brain might actually repopulate from the self-
103renewal of the few microglia that remain following microglia depletion using the CSF1R
104inhibitor PLX5622 (Huang et al., 2018).
105 Microglia are also important for hypothalamic satiety signaling and circuitry (Gao et al.,
1062017a; Gao et al., 2017b; Urabe et al., 2013; Valdearcos et al., 2017). For example, mice
107deficient in Brain-derived neurotrophic factor (BDNF) specifically in haematopoietic cells
108develop hyperphagia, obesity and insulin resistance (Urabe et al., 2013). Moreover, microglia-
109specific lipoprotein lipase knockout mice, when challenged with a high-carbohydrate high-fat
110(HCHF) diet, display Pro-opiomelanocortin (POMC) neuronal loss and weight gain (Gao et al.,
1112017a). Furthermore, microglia express leptin receptor (Chang et al., 2017) and mice with
112myeloid-specific leptin receptor elimination show accelerated weight gain and hyperphagia (Gao
113et al., 2017b), demonstrating that microglia signaling plays a role in proper maintenance of
115 Here, we studied the timeline of PLX5622 mediated elimination of microglia in the
116embryonic hypothalamus, and examined the consequences of loss of fetal microglia on
117behavioural outcomes later in life. We showed that approximately 99% of microglia are
118eliminated by embryonic day 15.5 (E15.5) after treatment of pregnant dams with PLX5622 diet
119starting at E3.5. Following microglia depletion, we observed elevated numbers of dead cells
120accumulating throughout the developing hypothalamus. Moreover, elimination of microglia in
121the fetal brain decreased the number of POMC+ neurons and led to a concomitant accelerated
122weight gain starting at postnatal day 5 (P5). In addition, PLX5622 exposed animals displayed
123craniofacial and dental abnormalities. Finally, depletion of microglia during embryogenesis had
124long-term sex-specific effects on behaviour, including the development of hyperactivity and
125anxiolytic-like behaviour in juvenile and adult female mice, respectively. Together, these data
126demonstrate that microglia appear to play an important role during the development of the
embryonic hypothalamus, and perhaps across the entire CNS.
1292. Materials & Methods
131 CD1 mice (Charles River) were used for all experiments not involving transgenic lines.
Cx3cr1-CreERT2 mice (B6.129P2(Cg)-Cx3cr1
/WganJ Stock No. 021160, The
Jackson Laboratory) were crossed to Rosa26tdTomato
134tdTomato)Hze/J Stock no. 007914, The Jackson Laboratory) for embryonic flow cytometry
135experiments. Nestin-CreERT mice (B6.Cg-Tg(Nes-cre)1Kln/J Stock no. 003771, The Jackson
Laboratory) were crossed with Csf1r
/J Stock No. 021212, The
137Jackson Laboratory) and used with the Rosa26tdTomato reporter line for Nestin repopulation
138experiments. Microglia knockdown was achieved by administering the Plexxikon CSF1R
139inhibitor PLX5622 (1200 PPM added to chow AIN-76A, Research Diets) to pregnant dams
140starting at embryonic day 3.5 (E3.5). Control dams received control diet (AIN-76A, Research
141Diets). Both mice generated from Control diet and PLX5622 diet fed dams were put on a wet
142diet after weaning, in order to help mice experiencing feeding problems due to craniofacial
143and/or dental abnormalities. Cre recombinase was induced using 1mg 4-Hydroxytamoxifen
144(H7904, Sigma) dissolved in corn oil and administered intraperitoneally over two days as two 0.5
145mg doses. Animal protocols were approved by the University of Calgary Animal Care
Committee and followed the Guidelines for the Canadian Council of Animal Care.
148 2.2. Immunohistochemistry
149 Embryonic day 11.5 (E11.5), E13.5, E15.5, E17.5, and postnatal day 0 (P0), P2, P4
150brains were collected in ice-cold phosphate-buffered saline (PBS) and fixed in 4%
151paraformaldehyde (PFA) overnight at 4°C. The brains were then washed in PBS and equilibrated
152in 20% sucrose/PBS overnight at 4°C. Brains were embedded in Clear Frozen Section
153Compound (VWR, 95057-838) and cryosectioned (10–20 µm sections). For
154immunohistochemistry (IHC), cryosections were rehydrated in PBS, washed with PBT (PBS
155with 0.1% Triton-X), blocked using 5% normal donkey serum (NDS, Sigma) for 1 h at room
156temperature (RT), and exposed to rabbit anti-Fezf1 (1:100, Fitzgerald 70R-7693), rabbit anti-
157Iba1 (1:500, Wako 019-19741), goat anti-Iba1 (1:500, Abcam ab107159), mouse anti-Ki67
158(1:200, BD Pharmingen 550609), mouse anti-Nestin (1:4, DSHB AB2235915), rabbit anti-
159cleaved active Caspase 3 (1:500, BD Pharmingen 559565), rabbit anti-POMC (1:200, Phoenix
160Pharmaceuticals H-029-30), and/or goat anti-LepR (1:200, R&D Systems AF497) at 4°C
161overnight. Slides were then washed with PBT and exposed to secondary antibody (1:200, Alexa
162488 or 555 donkey anti-rabbit IgG, donkey anti-goat IgG, and/or donkey anti-mouse IgG, Life
163Technologies) for 2 h at RT. Sections were mounted using Aqua Poly/Mount (Polysciences Inc.).
164Fluorescent IHC images were captured on a Zeiss Axioplan 2 fluorescent microscope. Brightness
165and/or contrast of the entire image was adjusted using Adobe Photoshop CS5.1 if deemed
168 2.3. MicroCT imaging
169 Crania of euthanized P21 and P28 animals were scanned using the Scanco Medical
170µCT100 scanner at 55kVp, 200uA with an isometric resolution of 34.4µ. Following
171reconstruction, 3D volumes were rendered using Drishti software (V2.6.4). All scans were
visualized using similar thresholding and rendering parameters.
174 2.4. Flow cytometry
175 Whole E15.5 brains were extracted from Cx3cr1-CreERT2; Rosa26tdTomato embryos in
176HBSS (14025-092, Life Technologies). Whole brain tissue was dissociated into smaller pieces in
177EH media (DMEM (11965-092, Life Technologies), 10 mM HEPES (15630-080, Life
178Technologies), 2% FBS) using a scalpel. EH media (15 mL) was added to the dissociated tissues,
179and filtered through a 70 µm strainer (542070, Greiner Bio-One). The filtered cell suspension
180was then mixed with 5.4 mL Percoll (17-0891-02, GE Healthcare) stock solution (SIP: 9 parts
181(v/v) Percoll to 1 part 10 x PBS). 5 mL of Percoll d = 1,08 g/l was then placed under the cell
182suspension/SIP mixture. The sample was then centrifuged at 1200 g for 30 min at 20°C. Once
183the myelin debris was removed, the interface between the two liquid layers (cell suspension) was
184pipetted and placed into a clean tube, where EH media was added until the volume reached 45
185mL. The cell suspension was then centrifuged at 1350 rpm for 10 min at 4°C. The supernatant
186was removed and the cell pellet was reconstituted in 2 mL of Flow-PBS (1 x PBS with 3%
187BSA). The resulting cell suspension was analyzed by the University of Calgary’s Flow Core
Facility using a BD LSR II machine and analyzed using FlowJo software.
190 2.5. Mouse weight and size measurements
191 Control diet (n = 74-76) and PLX5622 diet (n = 45-52) pups (depending on postnatal
192deaths) generated from 6 dams for each experimental diet were weighed (g) on a scale every day
193from P0 to P21. 20 male mice (12 Control diet and 8 PLX5622 diet animals) and 36 female mice
194(28 Control and 8 PLX5622 animals) generated from 3 dams for each experimental diet were
195also weighed (g) at 6 weeks of age. Length (cm) and width (cm) measurements were taken from
196P0 (36 Control diet, 33 PLX5622 diet pups), P5 (34 Control diet, 30 PLX5622 diet pups) and
P21 mice (6 Control diet, 6 PLX5622 diet mice).
199 2.6. Developmental behavioural testing
200 Assessment of developmental behaviours began on P5 and continued through until P15.
201Behavioural analysis was performed on 40 Control diet and 16 PLX5622 diet pups generated
202from 3 dams for each experimental diet. Specific behaviours were assessed each day over the
indicated age range for each mouse pup.
205 2.6.1. Surface righting reflex (P5–P11)
206 Each pup was placed on its back on a flat surface. The time for the pup to return to its
four limbs was recorded. The cutoff time for this test was 30 sec.
209 2.6.2. Negative geotaxis (P5–P11)
210 Each pup was placed face down on a ramp with a 25-degree incline, covered with a paper
211 mesh to enable traction. The time for the pup to turn 180 degrees with its head oriented to the top
of the incline was recorded. The cutoff time for this test was 30 sec.
214 2.6.3. Locomotion (P5–P15)
215 Each pup was placed on a flat surface in the center of a circle (13 cm diameter). The time
216 for all four legs of the pup to exit the circle completely was recorded. The cutoff time for this test
was 30 sec.
219 2.7. Adolescent and adult behavioural testing
220 Behaviour was tested at 42–46 (adolescent) and 84–90 (adult) days of age using the same
22120 male mice (12 Control diet and 8 PLX5622 diet animals) and 36 female mice (28 Control and
2228 PLX5622 animals) generated from 3 dams for each experimental diet. Testing was conducted
223over 5 days (adolescent) or 7 days (adult), with at least 24 hours between each test. The tests
224were conducted in the order presented below. All adolescent and adult behavioural tests were
225performed in the University of Calgary’s HBI Advance Light and Optogenetics core facility
228 2.7.1. Open field (P42 adolescent, P84 adult)
229 Locomotion was assessed by observing the activity of mice in a square open field (60 cm
230x 60 cm) divided into a 5 x 5 grid (each of the squares comprising the grid were 12 cm x 12 cm).
231Open field lighting was set to 100 Lux. Mice were placed individually into the center of the field
232and allowed to explore for 10 min. The activity of the mice in the field was recorded using a
233movement tracking system (SMART 3 software) connected to a camera mounted above the field.
234The total distance travelled (cm), time spent resting (s), and percentage of time spent in each
235zone (open field was divided into 3 zones; zone 1 contained the outer squares, zone 2 contained
236the middle squares, and zone 3 contained the center square) was quantified. The mean scores +/-
S.E.M. were plotted.
239 2.7.2. Elevated plus maze (P43 adolescent, P85 adult)
240 Locomotion was assessed by observing the activity of mice in the elevated plus maze,
241which has two open and two closed arms (each arm is 21.6 cm in length and 7 cm wide).
242Elevated plus maze lighting was set to 60-70 Lux in the open arms, and 20-30 Lux in the closed
243arms. Mice were placed individually into the junction of the open and closed arms and allowed
244to explore for 10 min. The activity of the mice in the elevated plus maze was recorded using a
245movement tracking system (SMART 3 software) connected to a camera mounted above the
246elevated plus maze. The total distance travelled (cm), number of zone transitions, and percentage
247of time spent in each zone (open arms vs closed arms) was quantified. The mean scores +/-
S.E.M. were plotted.
250 2.7.3. Rotarod (P44-P46 adolescent, P86-P88 adult)
251 The rotarod is a test of motor ability in which the animal must continuously walk and
252maintain its balance on a rotating cylinder. The apparatus consists of a 10 cm wide lane with a
253cylinder, 3 cm in diameter, elevated to a height of 28 cm. Training was organized into 3 blocks
254of 3 trials conducted over 2 days. For each training day, a different rotational speed was used: 4
255rotations per minute (rpm) on day 1 and 10 rpm on day 2. During the day of testing, trials were
256organized into 3 blocks of 1 trial and the rotational speed was set to accelerate from 4 rpm to 40
257rpm over 5 min. For each trial, the mouse was placed on the cylinder facing against the direction
258of rotation and the latency to fall was recorded. The maximum length of training trials was
259180 sec, while the maximum length of the testing trials was 300 sec. For each animal, the score
260(latency before falling) for the best of three trials was plotted, which are depicted as mean scores
263 2.7.4. Three-chamber sociability test (P89-P90 adult)
264 Sociability between mice was evaluated using the three-chamber sociability test, whereby
265mice were placed in the center of a three-chamber field (see diagram in Supplementary Fig. 6E)
266and their activity in each of the three-chambers was evaluated. Three-chamber lighting was set to
26780 Lux. On day 1, mice were placed in the three-chamber for 10 min, where both chambers
268adjacent to the center chamber housed empty wire cages (used to determine if there is a side
269preference). During testing on day 2, mice were placed in the three-chamber for 5 min, where
270one chamber adjacent to the center chamber contained a cage with a stranger same-sex mouse
271and the other chamber adjacent to the center housed an empty wire cage. The activity of the mice
272in the three-chambers was recorded using a movement tracking system (SMART 3 software)
273connected to a camera mounted above the three-chamber field. The percentage of time spent in
each of the three-chambers was quantified. The mean scores +/- S.E.M. were plotted.
276 2.8. Quantification and statistical analysis
277 Every 10 µm Fezf1+ section (usually between 5-14 sections, depending on age) was used
278for microglia counts, cell death counts, and repopulation counts (n = 3 from 2-3 dams), while
279only the first three 10 µm Fezf1+ sections were used for POMC cell counts (n = 3, from 3 dams).
280Quantitative results for all cell counts, weight/size measurements, and behaviour are represented
281by mean scores +/- S.E.M. and were analyzed by two-tailed unpaired t-tests using Prism 6
2853.1. Maternal exposure to the CSF1R inhibitor PLX5622 dramatically reduced the number of
286microglia present in the embryonic brain
287 Considering that microglia colonize the embryonic brain alongside key steps of
288hypothalamic development (Marsters et al., 2016; Nesan and Kurrasch, 2016), we tested whether
289microglia are required for the proper establishment of the hypothalamus. To eliminate microglia
290from the fetal brain, we treated pregnant CD1 dams with the Colony-stimulating factor-1
291receptor (CSF1R) inhibitor PLX5622, since CSF1R activity is required for microglia survival (Li
292et al., 2006; Patel and Player, 2009). Pregnant dams were placed on either a control or PLX5622
293diet starting at embryonic day 3.5 (E3.5) in order to avoid disrupting the implantation process,
294given the importance of endometrial macrophages (Care et al., 2013; Wang et al., 2016).
295Samples were collected for immunohistochemistry (IHC) analysis and cell counts at E11.5,
296E13.5 and E15.5 following exposure to control or PLX5622 diet from E3.5 onwards (Fig. 1A).
297Iba1 antibody staining revealed that the number of microglia in the hypothalamus had
298dramatically decreased by E11.5 following exposure to the PLX5622 diet at E3.5 (Fig. 1B-C,
299arrowheads), and only approximately 12.5% of microglia remained (Fig. 1H; p = 0.0047). E13.5
300PLX5622 diet exposed embryos also showed a dramatic decrease in hypothalamic microglia
301numbers as compared to controls (Fig. 1D-E, arrowheads), with only approximately 7.2% of
302microglia remaining (Fig. 1H; p = 0.0001). By E15.5, the majority of microglia were found to be
303depleted from the embryonic hypothalamus of PLX5622 diet exposed animals (Fig. 1F-G,
304arrowheads), with approximately 99.9% of microglia eliminated from the E15.5 hypothalamus
305(Fig. 1H; p < 0.0001).
306 Considering that the few remaining microglia in the E15.5 hypothalamus following
307exposure to the PLX5622 diet appeared to be undergoing cell death (Supplementary Fig. 1L-M,
308arrowheads), we considered E15.5 to be our “depleted” time point for the hypothalamus moving
309forward. However, in order to ensure that this level of microglia depletion was not specific to the
310hypothalamus, and that it was indeed occurring throughout the E15.5 brain, we performed flow
311cytometry on E15.5 control and PLX5622 diet treated Cx3cr1-CreERT2; Rosa26tdTomato brains
312(Supplementary Fig. 1N). Whole brain analysis of all the cells in control diet fed animals
313demonstrated that the E15.5 embryonic brain is comprised of approximately 3.8% microglia
314(Supplementary Fig. 1O). Following exposure to the PLX5622 diet from E3.5 to E15.5, only
315approximately 1.7% of total microglia remain, demonstrating that approximately 98.3% are
316depleted throughout the entire E15.5 brain (Supplementary Fig. 1P). Together, these data
317indicate that exposure of pregnant dams to the CSF1R inhibitor PLX5622 from E3.5 to E15.5
318results in approximately 98.3-99.9% microglia depletion throughout the brain, and that at least
some of the ~1.7% remaining microglia appeared to be unhealthy and dying.
3213.2. Embryonic microglia depletion resulted in the accumulation of dead cells throughout the
322embryonic brain across development
323 Given that microglia are phagocytic immune cells that play an important role in cleaning
324up dead cells and cellular debris (Giulian et al., 1989), we asked if apoptotic cells were
325accumulating in the developing brain following the depletion of microglia. Similar to what was
326outlined above, E11.5, E13.5 and E15.5 brains were harvested from pregnant dams that were
327placed either on a control or PLX5622 diet starting at E3.5. Staining with an active cleaved
328Caspase 3 (CC3) antibody at both E11.5 and E13.5 revealed a significant increase in the number
329of CC3+ cells in the developing hypothalamus of PLX5622 exposed embryos as compared to
330controls (Fig. 1I-L, arrowheads), with an approximate 11-times increase in CC3+ cells in
331PLX5622 brains (Fig. 1O; E11.5 p < 0.0001, E13.5 p < 0.0001). Similarly, at E15.5 we observed
332a significant increase in cell death throughout the developing hypothalamus (Fig. 1M-N,
333arrowheads; Fig. 1O; p = 0.001). Together, Iba1 and CC3 double staining showed that as
334microglia numbers decreased there was a noticeable increase in cell death (Supplementary Fig.
3351B-G, arrowheads). However, when we examined the CC3+ cells more closely we only observed
336a few Iba1+/CC3+ dying microglia (Supplementary Fig. 1I, L-M, arrowhead) and NeuN+/CC3+
337dying neurons (Supplementary Fig. 1H, J-K, arrowhead), since the vast majority of CC3+ cells
338appeared to be apoptotic bodies. This indicates that during microglia depletion, dead and dying
339cells accumulate throughout the embryonic hypothalamus, and likely throughout the entire
342 3.3. Embryonic microglia do not appear to repopulate from Nestin+ neuronal cells
343 To determine whether embryonic microglia repopulated from Nestin+ neuronal cells, as
344has been proposed in the adult (Elmore et al., 2014), we removed pregnant dams from the
345PLX5622 diet at E15.5 and studied microglial repopulation. By E17.5, two days following the
346removal of the diet, we observed Ki67+ cycling microglia within the hypothalamus and
347throughout the brain (Fig. 2A-B, arrowhead); however, we were unable to find any obvious
348Iba1/Nestin double-positive repopulating microglia (Fig. 2C-E, arrowheads). To test whether our
349timing was off and in fact we were missing a transient Nestin-expressing Iba1+ microglia during
350the repopulation process, we generated transgenic E17.5 Nestin-CreERT; Rosa26tdTomato embryos
351that were exposed to the PLX5622 diet from E3.5 to E15.5 (Fig. 2F), thus, any repopulating
352microglia that was derived from a Nestin+ precursor would be detected by a tdTomato signal.
353We did not observe a strong overlap of Nestin-CreERT; Rosa26tdTomato signal in Iba1+
354repopulating microglia throughout the embryonic brain (Fig. 2G-H, arrowhead), and specifically
355when we analyzed the E17.5 embryonic hypothalamus we detected approximately 2.7% (2
356microglia/75 total microglia counted) that were double-positive (Fig. 2M); however, the
357tdTomato signal was also very weak (Fig. 2L-L’’’, arrowhead).
358 To test if Nestin+ neuronal precursors were required for microglia repopulation, we
;Rosa26tdTomato embryos. Since CSF1R is required for
360microglia proliferation, differentiation and survival (Li et al., 2006; Patel and Player, 2009), the
361elimination of this receptor in Nestin+ cells should shunt microglia repopulation if they are
362indeed arising from a Nestin+ precursor. Similar to what was previously outlined, pregnant dams
363were exposed to the PLX5622 diet from E3.5 to E15.5 and allowed to repopulate from E15.5 to
E17.5 (Fig. 2I). Staining for Iba1 in E17.5 Csf1r
as compared to Nestin-CreERT; Csf1r
animals showed that microglia still repopulate in Nestin-CreERT; Csf1r
366(Fig. 2J-K, arrowheads), and that there was no significant difference in the numbers of
367repopulating microglia (Fig. 2N; p = 0.1835). Together, these data suggest that although Nestin
368may be expressed in a limited number of repopulating microglia, Nestin is not required for their
371 3.4. Microglia repopulate the brain seven days after removal of the CSF1R inhibitor PLX5622
372 Given that microglia appeared to be 98.3-99.9% depleted by E15.5 (depending on the
373brain region examined), we next removed the pregnant dams from the PLX5622 diet at E15.5
374and studied microglia repopulation (Fig. 3A). Samples were collected for IHC analysis and
375counts at E17.5, postnatal day 0 (P0), P2 and P4 (Fig. 3A). Staining with Iba1 at E17.5 revealed
376~2.3% of total microglia numbers when compared to controls within 48 hours after removal of
377PLX5622 (Fig. 3B-C, arrowheads, Fig. 3J; p < 0.0001). By birth (E19/P0; approximately three
378days after removal of PLX5622), we observed a noticeable increase in the number of
379repopulating microglia in the hypothalamus of PLX5622 exposed animals as compared to
380controls (Fig. 3D-E), with microglia repopulation now approximately 43.4% of controls (Fig. 3J;
381p = 0.0402). At the same time, we noticed that embryonic microglia depletion caused a decreased
382litter size and an increase in the number of pups that died within the first two postnatal days of
383life (Table 1), which did not appear to preferentially effect either sex (data not shown). By two
384days after birth (P2; approximately five days after removal from PLX5622), we observed a
385striking increase in the number of repopulating microglia in the hypothalamus of PLX5622
386exposed animals as compared to controls (Fig. 3F-G, K-L), with microglia repopulation levels at
387approximately 80.1% of control (Fig. 3J; p = 0.0288). And finally, the number of microglia in
388the P4 (approximately seven days after PLX5622 removal) hypothalamus of PLX5622 diet
389exposed animals and controls appeared comparable (Fig. 3H-I, M-N), with some PLX5622
390animals showing up to 99.8% repopulation by this time-point (Fig. 3J; p = 0.2159). Combined,
391these data demonstrate that approximately seven days are required for microglia to repopulate
the brain following exposure to the CSF1R inhibitor PLX5622 during embryogenesis.
3943.5. Embryonic microglia depletion resulted in accelerated weight gain and a decrease in
395POMC neurons postnatally
396 Although embryonic microglia depletion resulted in a decreased litter size (Table 1), the
397majority of pups born to dams exposed to the PLX5622 diet do indeed survive. Therefore, we
398next assayed these surviving PLX5622 exposed pups for any obvious growth or developmental
399defects (Fig. 4A). At birth, P0 PLX5622 exposed pups were comparable in weight and length to
400controls (Fig. 4B, G; length p = 0.9825, weight p = 0.4121); however, P0 PLX5622 pups were
401slightly wider then controls (Fig. 4C; p = 0.0117). Moreover, the elimination of microglia in the
402fetal brain did not appear to influence their weight from P1 to P4 (Fig. 4G). By P5 PLX5622
403exposed pups were significantly shorter (Fig. 4D; p = 0.0005), wider (Fig. 4E; p < 0.0001), and
404heavier (Fig. 4G; p < 0.0001) than controls. Moreover, PLX5622 exposed pups continued to gain
405weight at an accelerated rate as compared to controls from P5 to P15 (Fig. 4G).
406 Given that microglia disruption in adult mice results in weight gain and loss of
407POMC neurons (Gao et al., 2017a; Gao et al., 2017b), we examined whether POMC neurons
408were affected in postnatal brains exposed to PLX5622 during embryonic development. IHC
409staining in P4 control and PLX5622 exposed animals showed a significant decrease in POMC+
410neurons (Fig. 4H-I, Supplementary Fig. 2A-F, arrowheads), with an approximately 45%
411reduction in the number of POMC neurons found in the tuberal hypothalamus of PLX5622
412brains as compared to controls (Fig. 4J; p = 0.0016). We also examined postnatal microglia for
413evidence of leptin signaling, similar to what is observed in the adult (Chang et al., 2017; Gao et
414al., 2017b), and did not detect leptin receptor expression at E15.5 (Fig. 4K, Supplementary Fig.
4152G-H, arrowheads) or P2 in hypothalamic microglia (Fig. 4L, Supplementary Fig. 2I-J,
416arrowheads). However, similar to previous findings in the adult (Chang et al., 2017), we detected
417leptin receptors on adult hypothalamic microglia (Fig. 4M, Supplementary Fig. 2K-L,
418arrowheads). Together, these data suggest that microglia might be important for the development
of specific neurons within hypothalamic satiety centers in a leptin receptor-independent manner.
4213.6. Embryonic exposure to the CSF1R inhibitor PLX5622 resulted in craniofacial and dental
423 Although embryonic microglia depletion resulted in accelerated weight gain from P5 to
424P15, we noticed that PLX5622 exposed pups began to lose weight starting at P16 (Fig. 4G; p =
4250.0488). PLX5622 exposed pups continued to lose weight up until weaning (P21), and were also
426significantly shorter compared to controls (Fig. 4F; p < 0.0001). Given that CSF1R is expressed
427on microglia, macrophages, and osteoclasts (Li et al., 2006; Patel and Player, 2009), and that P21
428PLX5622 exposed mice did not look as healthy as controls and appeared to be eating less of their
429food, we examined their craniofacial development. Gross morphological examination of the teeth
430of P21 control and PLX5622 exposed animals demonstrated that PLX5622 animals had altered
431morphology of upper incisors (Supplementary Fig. 3A-B, arrow) and noticeably smaller lower
432incisors (Supplementary Fig. 3A-B, arrowhead). We also observed eye defects in some PLX5622
433animals (50% of males in 3/3 litters; 50% of females in 1/3 litters), where they were unable to
434open one eye until later in adulthood (Supplementary Fig. 3C-D, dashed-circle).
435 Further examination of P21 and P28 control and PLX5622 exposed mice (Fig. 5A) using
436microCT (µCT) scanning revealed distinct doming at the posterior aspect of the cranial vault in
437PLX5622 exposed animals (Fig. 5B-E, #) and the absence of the expected flattening of the mid-
438cranial vault seen in control animals (Fig. 5B-E, green arrows). The upper and lower incisors in
439PLX5622 exposed animals lacked the typical tooth curvature seen in control animals and
440displayed distinct notches in enamel close to the incisal edges (Fig. 5B-G, asterisk) as well as
441ectopic enamel ridges further apically (Fig. 5B-G, arrowheads). Few animals also displayed
442malocclusions, with overlap of the incisor tips (data not shown). The continuous eruption of
443incisors did not appear to be inhibited, since most animals displayed contact of the upper and
444lower incisors (Fig. 5E). The first molars also displayed altered cuspal morphology, with a
445mesial enamel protuberance resembling an extra cusp visible in the PLX5622 diet group (Fig. F-
446G). The molar crowns were also mesio-distally elongated and narrow bucco-lingually compared
447to controls (Fig. 5F-G, dashed-arrow and unfilled arrowheads respectively). In addition, the
448molar roots appeared to be slightly taurodontic in the PLX5622 diet group compared to controls
449(data not shown). Notably, this phenotype was observed in all male and female animals exposed
450to the PLX5622 diet.
451 Closer examination of the dentition of PLX5622 animals prior to P21 demonstrated that
452these dental phenotypes were also apparent during this time (data not shown), which is consistent
453with the time in which we observed the PLX5622 exposed mice losing weight and doing poorly
454(Fig. 4G). While these dental defects continued to P28 (Fig. 5D-G), supplying PLX5622 exposed
455animals with a wet diet enabled both female and male mice to continue to grow to a healthy
456weight that was comparable to controls by 6 weeks of age (Fig. 4N-O; female p = 0.1342, male p
457= 0.8945). Together, these data demonstrate that exposure to the CSF1R inhibitor PLX5622 in
458utero resulted in craniofacial and dental defects postnatally, likely due to the effects of PLX5622
4613.7. Depletion of embryonic microglia resulted in hyperactivity and anxiolytic-like behaviour in
463 Considering that adult female Cx3cr1 knockout mice exhibit behavioural alterations
464(Bolos et al., 2018), we examined if microglia depletion embryonically causes neural defects that
465affect normal behaviours in juvenile, adolescent and/or adult PLX5622 exposed mice. First, we
466assayed whether embryonic microglia depletion affected juvenile behaviours between P5 and
467P15 (Fig. 6A). Although we did not observe significant changes in locomotion or negative
468geotaxis responses in PLX5622 exposed pups as compared to controls (Supplementary Fig. 4A,
469C), we detected a delay in PLX5622 pup surface righting as compared to controls
470(Supplementary Fig. 4B; P5 p < 0.0001), which recovered as the pups aged (Supplementary Fig.
472 Next, we examined whether embryonic microglia depletion affected adolescent behaviour
473at six weeks of age, specifically between P42 and P46 (Fig. 6A). Open field analysis on P42
474female control and PLX5622 exposed mice demonstrated that PLX5622 adolescent female mice
475were hyperactive (Fig. 6B-C), traveling a significantly greater distance (Fig. 6F; p = 0.0084),
476with a significantly lower resting time (Fig. 6G; p = 0.0028). In contrast, adolescent control and
477in utero PLX5622 exposed female mice spent a comparable amount of time in the inner and
478outer regions of the open field (Fig. 6H; outer p = 0.8788, inner p = 0.8784). Similarly, analysis
479of elevated plus maze results from P43 control and in utero PLX5622 exposed mice
480demonstrated that PLX5622 adolescent female mice were hyperactive (Fig. 6D-E), traveling a
481significantly greater distance (Fig. 6I; p = 0.0012) and showing a significant increase in the
482number of zone transitions (Fig. 6J; p = 0.0005). However, adolescent control and in utero
483PLX5622 exposed female mice spent a comparable amount of time in the open and closed arms
484of the elevated plus maze (Fig. 6K; closed arms p = 0.0594, open arms p = 0.3269). Adolescent
485P46 in utero PLX5622 exposed female mice also spent a significantly longer period of time on a
486rotarod accelerating from 4 to 40 rpm as compared to controls (Supplementary Fig. 7A; p =
488 Perhaps unexpectedly, adolescent in utero PLX5622 exposed male mice were not
489hyperactive (Supplementary Fig. 5A-D), since they did not show a change in distance traveled in
490the open field (Supplementary Fig. 5E; p = 0.4979) or the elevated plus maze (Supplementary
491Fig. 5H; p = 0.1390). Moreover, adolescent in utero PLX5622 exposed male mice showed no
492change in open field resting time (Supplementary Fig. 5F; p = 0.8344) or elevated plus maze
493zone transitions (Supplementary Fig. 5I; p = 0.2250). Adolescent male PLX5622 in utero
494exposed mice also showed no change in open field zone localization (Supplementary Fig. 5G;
495outer p = 0.3345, inner p = 0.3429) or elevated plus maze arm localization as compared to
496controls (Supplementary Fig. 5J; closed arms = 0.7930, open arms p = 0.4076). Adolescent P46
497in utero PLX5622 exposed male mice also showed no difference in latency to fall when placed
498on a rotarod (Supplementary Fig. 7B; p = 0.9832).
499 Similar to the adolescent male results, twelve-week old in utero PLX5622 exposed adult
500male mice likewise were not hyperactive (Supplementary Fig. 6A-D), given that they did not
501display a change in distance traveled in the open field (Supplementary Fig. 6E; p = 0.2985) or
502the elevated plus maze (Supplementary Fig. 6H; p = 0.2965). Moreover, adult male PLX5622 in
503utero exposed mice showed no change in open field resting time (Supplementary Fig. 6F; p =
5040.5017) or elevated plus maze zone transitions (Supplementary Fig. 6I; p = 0.8632). Adult male
505PLX5622 in utero exposed mice also showed no change in open field zone localization as
506compared to controls (Supplementary Fig. 6G; outer p = 0.6547, inner p = 0.6534) or elevated
507plus maze arm localization (Supplementary Fig. 6J; closed arms = 0.7250, open arms p =
5080.9810). Adult P88 in utero PLX5622 exposed male mice also showed no difference in latency
509to fall when placed on a rotarod (Supplementary Fig. 7D; p = 0.0700). Furthermore, three-
510chamber analysis (Supplementary Fig. 7E) of adult P90 control and in utero PLX5622 exposed
511male mice showed a comparable percent time spent in each of the three chambers
512(Supplementary Fig. 7F; empty cage p = 0.8512, center p = 0.8151, mouse p = 0.8106).
513 Finally, we examined whether embryonic microglia depletion affected adult female
514behaviour at twelve weeks of age, specifically between P84 and P90 (Fig. 6A). Open field
515analysis on P84 female control and in utero PLX5622 exposed mice showed no difference in
516distance traveled (Fig. 7A-B, E; p = 0.3289) and no difference in resting time (Fig. 7F; p =
5170.1111). Adult control and in utero PLX5622 exposed female mice also showed a comparable
518amount of time in the inner and outer regions of the open field (Fig. 7G; outer p = 0.6488, inner
519p = 0.6378). Similarly, analysis of elevated plus maze results from P85 control and in utero
520PLX5622 exposed female mice showed no difference in zone transitions (Fig. 7I; p = 0.2786),
521with PLX5622 exposed adult females actually traveling less than controls (Fig. 7C-D, H; p =
5220.0321). However, analysis of adult control and PLX5622 female mice showed that PLX5622 in
523utero exposed female mice spent significantly less time in the closed arms of the elevated plus
524maze (Fig. 7J; closed arms p = 0.0039, open arms p = 0.1440). Adult P88 PLX5622 in utero
525exposed female mice were found to show no difference in latency to fall when placed on a
526rotarod (Supplementary Fig. 7C; p = 0.2836). Furthermore, three-chamber testing
527(Supplementary Fig. 7E) of adult P90 control and in utero PLX5622 exposed female mice
528showed a comparable amount of time spent in each of the three chambers (Supplementary Fig.
5297F; empty cage p = 0.4685, center p = 0.2099, mouse p = 0.5361). Taken together, these data
530demonstrate that depletion of microglia during embryogenesis has long-term sex-specific effects
531on behaviour, including the development of hyperactivity and anxiolytic-like behaviour in
juvenile and adult female mice, respectively.
534 4. Discussion
535 To our knowledge, we are the first to show that approximately 99% of microglia are
536eliminated by E15.5 when pregnant dams are placed on the CSF1R inhibitor PLX5622 starting at
537E3.5. Following microglia depletion, we observed elevated numbers of apoptotic cells
538accumulating throughout the developing hypothalamus. Moreover, elimination of microglia in
539the fetal brain decreased the number of POMC neurons and led to a concomitant accelerated
540weight gain in postnatal animals. In addition, we observed craniofacial and tooth defects in the
541PLX5622 exposed animals. Finally, depletion of microglia during embryogenesis had long-term
542sex-specific effects on behaviour, including the development of hyperactivity and anxiolytic-like
543behaviour in juvenile and adult female mice, respectively. Together, these data demonstrate that
544microglia appear to play an important role during the development of the hypothalamus, and
likely other regions of the embryonic brain.
5474.1. In utero exposure to the CSF1R inhibitor PLX5622 resulted in ~99% microglia depletion
548and an accumulation of apoptotic cells in the embryonic brain
549 Other groups have used a diet to administer CSF1R inhibitors to adult mice and have
550demonstrated a range of success (~80% to 99% depletion) with varying timelines to total
551depletion (7 to 21 days) (Acharya et al., 2016; Chalmers et al., 2017; Elmore et al., 2014; Jin et
552al., 2017; Li et al., 2017; Rice et al., 2017; Spangenberg et al., 2016; Szalay et al., 2016).
553Specifically, the use of the PLX5622 diet on adults has been documented to result in ~90%
554depletion over two to six weeks in a model of radiation-induced cognitive deficits (Acharya et
555al., 2016), ~90% depletion over seven days in a neuronal lesion injury model (Rice et al., 2017),
556and ~80% depletion over 28 days in an Alzheimer model system (Spangenberg et al., 2016).
557Embryonically, there are no reports on the use of CSF1R inhibitors administered maternally
558through diet. At present, embryonic microglia have been disrupted using Cx3cr1-/-, CR3-/-, and
559DAP12-/- knockout animals (Bolos et al., 2018; Squarzoni et al., 2014). Moreover, microglia
560have been depleted using approaches such as Cx3cr1CreER to drive diphtheria toxin receptor
561expression in microglia (Parkhurst et al., 2013), liposomal clodronate injections (Cunningham et
562al., 2013), CSF1R-specific blocking antibodies (Hoeffel et al., 2015; Squarzoni et al., 2014), or
563Csf1r complete knockout animals (Erblich et al., 2011). In our system, in utero exposure of
564embryos to the CSF1R inhibitor PLX5622 from E3.5 to E15.5 via maternal diet resulted in
56598.3% to 99.9% microglia depletion, depending on the brain region analyzed. This allowed us to
566circumvent the early lethality of the Csf1r complete knockout models, as complete nulls often do
567not survive to adulthood (Erblich et al., 2011; Ginhoux et al., 2010), while still achieving a high
568level of microglia depletion.
569 Similar to other studies that have disrupted microglia signaling (Fourgeaud et al., 2016),
570we observed apoptotic cells accumulating throughout the hypothalamus as development
571proceeded in brains lacking microglia. Whether these apoptotic cells were caused by the absence
572of microglia, as if they required specific microglial signals for survival, or whether the presence
573of these apoptotic cells is a reflection of defects in normal phagocytosis, remains unknown.
574Moreover, while it is possible that the accumulation of these dead cells themselves could disrupt
575key brain developmental processes, such as neurogenesis/gliogenesis, neuronal/glial activity,
576and/or synaptic function, we propose it is the loss of microglia influence on specific neural
577developmental steps that is responsible for the phenotypic results we observe in PLX5622
578animals. Consistently, our results match well with groups that have used genetic knockout
579models to disrupt microglia signaling, such as Cx3cr1 knockout female mice that develop
580hyperactivity, anxiolytic-like behaviour and phenotypes resembling depression (Bolos et al.,
5812018). However, future analyses of the cellular environment and cytokine/chemokine changes
582following microglia depletion should be examined in order to better understand the signaling
deficits in the brain following microglia depletion.
585 4.2. Microglia do not appear to repopulate from Nestin+ neuronal cells during embryogenesis
586 Given that the use of CSF1R inhibitors does not affect the blood-brain barrier in adult
587animals (Elmore et al., 2014; Huang et al., 2018), we expected that microglia repopulation
588following depletion in the developing brain likewise occurs under a normal physiological state.
589Although others have shown that repopulating microglia express Nestin in the adult (Elmore et
590al., 2014), we observed very few repopulating Iba1/Nestin double-positive cells in the E17.5
591brain. Moreover, when we conditionally eliminated Csf1r specifically in Nestin+ cells, we did
592not observe a decrease in the number of repopulating microglia at E17.5, suggesting that
593embryonic microglia repopulate from a non-Nestin+ cell. However, given that we did detect a
594small proportion (~2.7%) of Nestin+/Iba1+ microglia, it is possible that Nestin becomes
595transiently expressed in repopulating microglia without being required. A recent study of
596microglia repopulation in the adult shows that newly born microglia arise from the few microglia
597that remain following depletion (Huang et al., 2018), and we propose this could also be the case
for embryonic microglia.
6004.3. Exposure to the CSF1R inhibitor PLX5622 during embryogenesis caused POMC neuronal
601loss, accelerated weight gain and craniofacial defects postnatally
602 Here, we uncovered accelerated weight gain in postnatal PLX5622 in utero exposed
603animals. Although the confounding dental phenotype prevented us from tracking this phenotype
604into adulthood, to the best of our knowledge this early postnatal weight gain is highly unusual,
605even among animal models with hypothalamic defects. Indeed, across various genetic
606backgrounds, usually the weight gain occurs later in adulthood and often time requires high-fat
607diet challenges (Gao et al., 2017a; Gao et al., 2017b; Urabe et al., 2013; Valdearcos et al., 2017;
608Yi et al., 2017; Zhan et al., 2013). Even Lepob/Lepob mice with a disruption in the leptin gene do
609not display increased body weight until >P15 (Bouyer and Simerly, 2013), thereby making this
610observed postnatal phenotype even more striking. Interestingly, the elimination of leptin receptor
611specifically in myeloid cells in adult animals causes accelerated weight gain (Gao et al., 2017b);
612however, we did not detect the expression of leptin receptors on embryonic or early postnatal
613hypothalamic microglia, thereby imply a different mechanism of action underlying the weight
614gain observed in these postnatal microglia depleted pups. Yet, at least in the adult, microglia are
615important for influencing hypothalamic energy balance signaling by other, non-leptin pathways
616(Gao et al., 2017a; Gao et al., 2017b; Urabe et al., 2013; Valdearcos et al., 2017). Specifically,
617BDNF deficiency (Urabe et al., 2013) or the disruption of lipoprotein lipase activity (Gao et al.,
6182017a) within microglia also causes a decrease in POMC neurons and/or weight gain. Therefore,
619additional studies are required to determine whether embryonic and/or early postnatal microglia
620target these same pathways or if they differentially influence the development of hypothalamic
621satiety centers. Furthermore, given that PLX5622 also causes depletion of macrophages and
622osteoclasts, it is possible that non-microglial effects mediate some or all of this weight gain
624 Surprisingly, a marked cranial and dental phenotype was observed in the animals exposed
625to the PLX5622 diet during gestation. Considering that CSF1R is expressed in microglia,
626macrophages, and osteoclasts (Li et al., 2006; Patel and Player, 2009), one or more of these cell
627types must also contribute to craniofacial morphogenesis. As teeth expand during growth,
628removal of bone at the tooth-bone interface (TBI) is carried out by Tartrate-resistant acid
629phosphatase (TRAP)-positive osteoclasts (Alfaqeeh et al., 2013). This removal of bone is
630presumably required for normal tooth morphogenesis. Moreover, it is known that CSF-1 and
631Receptor activator of nuclear factor kappa-B ligand (RANKL) are requisite for osteoclast-
632precursor cell proliferation, osteoclast differentiation, survival and migration (Arai et al., 1999;
633Biskobing et al., 1995; Fuller et al., 1993; Sakai et al., 2006). Hence, the dental phenotype found
634in in utero PLX5622 diet treated animals may be a consequence of disturbance in
635osteoclastogenesis at the TBI, and a resulting increase in constriction of the developing enamel
637 However, it is also possible that CSF1R expressed either in cells of the osteoclast,
638macrophage, microglial lineages and/or cells directly involved in tooth formation, play a role in
639various aspects of odontogenesis and tooth shape determination, independent of surrounding
640bone. The finding of ectopic enamel ridges towards the apical aspects of the incisors, and slightly
641taurodontic roots, supports such a direct role for CSF1R during tooth morphogenesis.
642Osteoclastic activity is certainly required for tooth eruption, with CSF-1 expression increased in
643the occlusal aspects of emerging murine teeth (Heinrich et al., 2005). However, in these pups
644exposed to PLX5622 during gestation, the eruption of teeth is not impeded. A possible
645explanation is that since the diet was discontinued at birth, osteoclast recruitment, expansion, and
646differentiation transpired normally at the time when teeth begin to erupt. The increased doming
647of the cranial vault may also reflect an altered morphology of the underlying brain during growth
648or a direct effect on bone formation and/or remodeling. Further investigations into the cranial
649and dental phenotypes resulting from PLX5622 exposure during gestation are being performed to
address these hypotheses.
6524.4. Microglia depletion in utero resulted in the development of hyperactivity and anxiolytic-like
653behaviours in female mice
654 Although previous studies using the CSF1R inhibitor PLX3397 showed that microglia
655depletion in the adult causes no behavioural deficits (Elmore et al., 2015; Elmore et al., 2014),
656here we demonstrated that the elimination of microglia embryonically resulted in behavioural
657phenotypes in both adolescent and adult animals. Specifically, PLX5622 gestationally exposed
658adolescent female mice were hyperactive using open field and elevated plus maze tests.
659Moreover, PLX5622 gestationally exposed adolescent female mice were found to perform better
660than controls on the rotarod. Interestingly, these phenotypes were not observed in PLX5622 in
661utero exposed adolescent male mice, demonstrating a sex-specific effect of microglia on the
662developing brain. Similarly, only in utero PLX5622 exposed adult female mice were shown to be
663less anxious than controls when analyzed using the elevated plus maze. However, this decrease
664in anxiety was only observed in the elevated plus maze and was not apparent in the open field,
665suggesting the phenotype is a mild decrease. Notably, our findings are consistent with those
666reported by Cx3cr1 complete knockout mice (Bolos et al., 2018), suggesting that disruption of
667microglia signaling through CX3CR1 to surrounding cells in the brain could be the cause of the
668behavioural phenotypes observed in our microglia depleted females. However, there are subtle
669differences in the timing of these behavioural alterations, and we did not test for depression-like
670behaviours in our animals. Therefore, additional behavioural tests should be performed on in
671utero exposed PLX5622 mice to directly compare the full spectrum of behavioural changes that
672result from microglial depletion. Combined, these studies suggest that microglia are important
673for development of key brain centers, which then leads to lasting effects on behaviour, since
674microglia have long repopulated in our model by the time the behavioural testing is conducted.
675 At the same time, we also acknowledge that our behavioural findings could be a result of
676a delay in microglia maturation and/or functional changes to the repopulating microglia during
677the postnatal period, which then influences behavioural measures later in life. Given that we did
678not observe complete microglia repopulation until P4 (seven days after removal of PLX5622),
679and that the repopulating microglia appeared to be more amoeboid and immature as compared to
680controls, these phenotypes could be indicative of changes to microglia signaling and/or function.
681Consistent with this notion, temporary depletion of microglia using liposomal clodronate
682specifically during the early postnatal period causes hyperactivity and anxiolytic-like behaviours
683in rats (VanRyzin et al., 2016), suggesting that microglia in the postnatal brain might also
684influence behavioural outcomes.
685 While our results demonstrate that fetal microglia are important for the development of
686normal mouse behaviours in females, we were surprised to find that microglia depletion
687embryonically did not result in any overt behavioural consequences in adolescent or adult male
688mice. These findings could reflect microglial sex differences, as microglia in the male brain
689mature on a different developmental trajectory as compared to microglia in the female brain
690(Bolton et al., 2017; Hanamsagar et al., 2018; Nelson et al., 2017; Schwarz et al., 2012; Thion et
691al., 2018). Especially considering that in P2 PLX5622 exposed brains the repopulating microglia
692were often more amoeboid, with thicker microglia projections, and it was not until around P4,
693the time at which repopulation was complete, that the repopulated microglia in PLX5622
694exposed animals were nearly indistinguishable from those found in control brains. These subtle
695differences in microglia morphology during the repopulation process postnatally could
696potentially contribute to the sex differences we observe, given the differences that exist in the
697normal developmental trajectory present in female and male mouse brains. Therefore, further
698behavioural testing of male mice exposed to PLX5622 gestationally will be required to more
699confidently conclude whether microglial loss during embryogenesis simply does not disrupt
700behaviour in males, or whether it differentially disrupts behaviours that have not been uncovered
701yet. Furthermore, it will be important to investigate early requirements for microglia in a sex-
702specific manner by analyzing changes in cell numbers (e.g., neuronal and glial), localization and
703connectivity, including changes in secreted factors and signaling molecules, separately in males
and females to try to determine what underlies these sex-specific changes in behaviour.
706 5. Conclusions
707 Here we explored the effect of embryonic microglia depletion on hypothalamic-mediated
708energy balance, as well as juvenile, adolescent and adult behaviours. Specifically, we
709demonstrated that the elimination of microglia in utero caused a decrease in POMC neurons and
710accelerated weight gain postnatally, suggesting that microglia could be important for the
711development of key neurons within hypothalamic satiety centers. Importantly, embryonic
712microglia depletion also showed long-term sex-specific effects on behaviour. Specifically,
713embryonic microglia depletion resulted in hyperactivity in juvenile female mice and the
714development of anxiolytic-like behaviour in adult female mice. Finally, we illustrate the use of
715CSF1R inhibitors through diet to pregnant dams as a means to efficiently eliminate microglia in
716the developing embryonic brain. Collectively, these findings demonstrate that microglia appear
717to play an important role during the development of the embryonic hypothalamus, and perhaps
across the entire CNS.
721J.M.R. is supported by a Canadian Institute of Health Research (CIHR) Postdoctoral Fellowship
(MEF-140891). D.M.K. is supported by CIHR (MOP-275053).
725JMR and DMK conceived and designed the experiments. SRV performed and analyzed all
726microCT results. JMR performed all other experiments. JMR prepared the manuscript. JMR,
SRV and DMK edited the manuscript. All authors read and approved the final manuscript.
730The authors thank Candi Grivel and Gaurav Kaushik for animal care, Taylor Chomiak for
731behavioural test training, Natasha Klenin for genotyping, and the entire Kurrasch lab for critical
732discussion. The authors also thank the University of Calgary’s Flow Core Facility and HBI
733Advance Light and Optogenetics core facility (HALO). We would also like to acknowledge
Plexxikon for providing the CSF1R inhibitor PLX5622 under the MTA.
736 Conflicts of interest
The authors declare no conflict of interest
739 Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version.
749Figure 1. Embryonic microglia are depleted by E15.5 and show an accumulation of dead
750cells within the tuberal hypothalamus when using the CSF1R inhibitor PLX5622. (A)
751Schematic of CD1 crossing, dam diet exposure period during pregnancy, and sample collection
752time-points. Expression of Iba1 in E11.5 (B, C), E13.5 (D, E), and E15.5 (F, G) control diet (B,
753D, F) and PLX5622 diet (C, E, G) exposed embryonic brains. White arrowheads mark Iba1+
754microglia. (H) Quantification of the number of Iba1+ microglia present in the tuberal
755hypothalamus of control or PLX5622 diet exposed embryos at E11.5 (p = 0.0047), E13.5 (p =
7560.0001), and E15.5 (p < 0.0001). Expression of active cleaved Caspase 3 (CC3) in E11.5 (I, J),
757E13.5 (K, L), and E15.5 (M, N) control diet (I, K, M) and PLX5622 diet (J, L, N) exposed
758embryonic brains. White arrowheads mark CC3+ cells. (O) Quantification of the number of
759CC3+ cells present in the tuberal hypothalamus of control or PLX5622 diet exposed embryos at
760E11.5 (p < 0.0001), E13.5 (p < 0.0001), and E15.5 (p = 0.001). Dashed-lines outline the ventricle
and pial surface. 3V, third ventricle.
763Figure 2. Microglia do not appear to repopulate from Nestin+ neural precursor cells
764following removal of the CSF1R inhibitor PLX5622. (A, B) Iba1 and Ki67 expression in the
765brains of E17.5 embryos allowed to recover from PLX5622 diet exposure (E3.5 to E15.5) for 2
766days. White arrowheads mark Iba1/Ki67 double-positive microglia. (C-E) Iba1 and Nestin
767expression in the brains of E17.5 embryos allowed to recover from PLX5622 diet exposure (E3.5
768to E15.5) for 2 days. White arrowheads mark Iba1+/Nestin- microglia. (F) Schematic of Nestin-
CreERT crossing to Rosa26
, dam diet exposure period during pregnancy (E3.5 to E15.5),
7704OHT injections (E15.5 and E16.5), and sample collection at E17.5. (G, H) Iba1 and Nestin-Cre
771expression in the brains of E17.5 embryos allowed to recover from PLX5622 diet exposure (E3.5
772to E15.5) for 2 days. White arrowheads mark Iba1+/Nestin- microglia. (I) Schematic of Nestin-
crossing to Rosa26tdTomato
, dam diet exposure period during
774 pregnancy (E3.5 to E15.5), 4OHT injections (E15.5 and E16.5), and sample collection at E17.5.
(J, K) Iba1 and Nestin-Cre expression in the brains of E17.5 Nestin-CreERT; Csf1r
776embryos allowed to recover from PLX5622 diet exposure (E3.5 to E15.5) for 2 days. White
777arrowheads mark Iba1+/Nestin- microglia. (L-L’’’) Iba1 and Nestin-Cre expression in the brains
of E17.5 Nestin-CreERT; Csf1r
knockout embryos allowed to recover from PLX5622 diet
779exposure (E3.5 to E15.5) for 2 days. White arrowheads mark Iba1+/Nestin+ double-positive
780microglia. (M) Quantification of the proportion of Iba1+ repopulating microglia that are
tdTomato+ in the tuberal hypothalamus of E17.5 Nestin-CreERT; Rosa26
782 to recover from PLX5622 diet exposure (E3.5 to E15.5) for 2 days. (N) Quantification of the
number of Iba1+ microglia in the tuberal hypothalamus of E17.5 Csf1r
control and Nestin-
knockout embryos allowed to recover from PLX5622 diet exposure (E3.5 to
E15.5) for 2 days (p = 0.1835).
787Figure 3. Microglia repopulate 7 days after removal of the CSF1R inhibitor PLX5622. (A)
788Schematic of CD1 crossing, dam diet exposure period during pregnancy (E3.5 to E15.5), and
789sample collection time-points. Expression of Iba1 in E17.5 (B, C), newborn (E19/P0; D, E), P2
790(F, G), and P4 (H, I) control diet (B, D, F, H) and PLX5622 diet (C, E, G, I) exposed brains.
791White arrowheads mark Iba1+ microglia. (J) Quantification of the number of Iba1+ microglia
792present in the tuberal hypothalamus of control or PLX5622 diet exposed embryos at E17.5 (p <
7930.0001), birth (E19/P0; p = 0.0402), P2 (p = 0.0288), and P4 (p = 0.2159). (K-N) Higher
794magnification images of Ibat+ microglia in P2 (K, L) and P4 (M, N) control diet (K, M) and
795PLX5622 diet (L, M) exposed brains. White arrowheads mark microglia projections. Dashed-
lines outline the ventricle and pial surface. 3V, third ventricle.
798Figure 4. Embryonic microglia depletion results in accelerated weight gain and a decrease
799in POMC neurons in postnatal animals. (A) Schematic of CD1 crossing, dam diet exposure
800period during pregnancy (E3.5 to P0), and sample collection or weight/measurement time-points.
801(B) Quantification of mouse pup length (mm) of P0 control and PLX5622 exposed animals (p =
8020.9825). (C) Quantification of mouse pup width (mm) of P0 control and PLX5622 exposed
803animals (p = 0.0117). (D) Quantification of mouse pup length (mm) of P5 control and PLX5622
804exposed animals (p = 0.0005). (E) Quantification of mouse pup width (mm) of P5 control and
805PLX5622 exposed animals (p < 0.0001). (F) Quantification of mouse length (cm) of P21 control
806and PLX5622 exposed animals (p < 0.0001). (G) Quantification of mouse pup weight (g) of
807control and PLX5622 exposed animals from P0 to P21. (H, I) Expression of
808proopiomelanocortin (POMC) in P4 control diet (H) and PLX5622 diet (I) exposed brains. White
809arrowheads mark POMC neurons. Dashed-lines outline the pial surface. (J) Quantification of the
810number of POMC neurons in the tuberal hypothalamus of P4 control and PLX5622 exposed
811animals (p = 0.0016). (K-M) Expression of Iba1 and leptin receptor (LepR) in E15.5 (K), P2 (L),
812and adult (M) wild-type brains. White arrowheads mark Iba1+ microglia that are either LepR- or
813LepR+. (N) Quantification of mouse weight (g) of 6-week old female control and PLX5622
814exposed animals (p 0.1342). (M) Quantification of mouse weight (g) of 6-week old male control
and PLX5622 exposed animals (p 0.8945).
817Figure 5. Embryonic exposure to the CSF1R inhibitor PLX5622 results in craniofacial
818defects. (A) Schematic of CD1 crossing, dam diet exposure period during pregnancy (E3.5 to
819P0), and sample collection for microCT (µCT) scanning. (B-G) µCT scans of crania of P21 (B,
820C) and P28 (D-G) animals exposed to control diet (B, D, F) or PLX5622 diet (C, E, G) during
821gestation. (B-E) Lateral views show the doming of the posterior aspect of the cranial vault in
822PLX5622 diet animals (C, E; #) and the absence of typical vault flattening observed in control
823animals (B, D; green arrows). Notching of the incisal edges of the enamel is observed in
824PLX5622 diet animals (C, E; *) with a lack of the expected curved morphology of incisors in
825controls (B, D; curved green arrow). Ectopic enamel ridges are observed on the facial surfaces of
826upper and lower incisors of PLX5622 animals (C, E, G; filled pink arrowheads). Altered first
827molar shape can also be observed with an elongation in the mesio-distal direction (G; dashed
828pink arrow) and constriction in the bucco-linugal direction (G; open pink arrowheads) in
829PLX5622 animals. Images are from representative scans of n = 4 animals at each time point (2M,
2F) per group. Scale bar = 10mm.
832Figure 6. Depletion of embryonic microglia results in hyperactivity in adolescent female
833mice. (A) Schematic of CD1 crossing, dam diet exposure period during pregnancy (E3.5 to P0),
834and juvenile, adolescent and adult testing periods. (B, C) Representation of open field results for
835a 6-week old female control diet (B) and PLX5622 diet (C) exposed animal. (D, E)
836Representation of elevated plus maze results for a 6-week old female control diet (D) and
837PLX5622 diet (E) exposed animal. (F) Quantification of the total distance (cm) traveled by 6-
838week old female control and PLX5622 mice in an open field over a 10-minute period (p 0.0084).
839(G) Quantification of the time (s) 6-week old female control and PLX5622 mice spent resting in
840an open field over a 10-minute period (p = 0.0028). (H) Quantification of the percent time 6-
841week old female control and PLX5622 mice spent in each zone of an open field over a 10-minute
842period (outer p = 0.8788, inner p = 0.8784). (I) Quantification of the total distance (cm) traveled
843by 6-week old female control and PLX5622 mice in an elevated plus maze over a 5-minute
844period (p 0.0012). (J) Quantification of the number of zone transitions made by 6-week old
845female control and PLX5622 mice in an elevated plus maze over a 5-minute period (p = 0.0005).
846(K) Quantification of the percent time 6-week old female control and PLX5622 mice spent in
847each region of an elevated plus maze over a 5-minute period (closed arms p = 0.0594, open arms
p = 0.3269).
850Figure 7. Depletion of embryonic microglia results in anxiolytic-like behaviour in adult
851female mice. (A, B) Representation of open field results for a 12-week old female control diet
852(A) and PLX5622 diet (B) exposed animal. (C, D) Representation of elevated plus maze results
853for a 12-week old female control diet (C) and PLX5622 diet (D) exposed animal. (E)
854Quantification of the total distance (cm) traveled by 12-week old female control and PLX5622
855mice in an open field over a 10-minute period (p 0.3289). (F) Quantification of the time (s) 12-
856week old female control and PLX5622 mice spent resting in an open field over a 10-minute
857period (p = 0.1111). (G) Quantification of the percent time 12-week old female control and
858PLX5622 mice spent in each zone of an open field over a 10-minute period (outer p = 0.6488,
859inner p = 0.6378). (H) Quantification of the total distance (cm) traveled by 12-week old female
860control and PLX5622 mice in an elevated plus maze over a 5-minute period (p 0.0321). (I)
861Quantification of the number of zone transitions made by 12-week old female control and
862PLX5622 mice in an elevated plus maze over a 5-minute period (p = 0.2786). (J) Quantification
863of the percent time 12-week old female control and PLX5622 mice spent in each region of an
elevated plus maze over a 5-minute period (closed arms p = 0.0039, open arms p = 0.1440).
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1066• The CSF1R inhibitor PLX5622 depleted ~99% of fetal microglia by E15.5
1067• Embryonic microglia depletion resulted in a decreased litter size
1068• Elimination of embryonic microglia resulted in a decrease in POMC neurons
1069• Accelerated weight gain is observed in microglia depleted animals
• Depletion of microglia during embryogenesis had sex-specific effects on behaviour