Brain-synthesized estrogens regulate cortical migration in a sexually divergent manner

Estrogens play an important role in the sexual dimorphisms that occur during brain development, including the neural circuitry that underlies sex-typical and socio-aggressive behaviors. Aromatase, the enzyme responsible for the conversion of androgens to estrogens, is expressed at high levels during early development in both male and female cortices, suggesting a role for brain-synthesized estrogens during corticogenesis. This study investigated how the local synthesis of estrogens affects neurodevelopment of the cerebral cortex, and how this differs in males and females by knockdown expression of the Cyp19a1 gene, which encodes aromatase, between embryonic day 14.5 and postnatal day 0 (P0). The effects of Cyp19a1 knockdown on neural migration was then assessed. Aromatase was expressed in the developing cortex of both sexes, but at significantly higher levels in male than female mice. Under basal conditions, no obvious differences in cortical migration between male and female mice were observed. However, knockdown of Cyp19a1 increased the number GFP-positive cells in the cortical plate, with a concurrent decrease in the subventricular zone/ventricular zone in P0 male mice. The opposite effect was observed in females, with a significantly reduced number of GFP-positive cells migrating to the cortical plate. These findings have important implications for our understanding of the role of fetal steroids for neuronal migration during cerebral cortex development. Moreover, these data indicate that brain-synthesized estrogens regulate radial migration through distinct mechanisms in males and females.


Introduction 38
The unique organization and architecture of the cerebral cortex is established during 39 embryonic development. This organization is achieved in an "in-side-out" fashion, with 40 inner, or deep layers forming first, and outer, or superficial layers developing last 41 (Evsyukova et al., 2013). The development of the cortex and its laminar organization is 42 controlled in large part by the coordinated processes of neurogenesis and cell migration. 43 The process of radial neuronal migration is a multi-step process (Evsyukova et al., 2013). 44 Initially, newly born neurons, generated from neural stem cells, detach from the apical 45 surface of the germinal ventricular zone (VZ). These neurons adopt a multi-polar 46 morphology and move into the intermediate zone (IZ) (Noctor et al., 2004). Here, neurons 47 develop a bi-polar shape, and migrate along radial glia to their final position in the cortical 48 plate (CP) (Kawauchi, 2015). Once in this position, neurons can begin to form synaptic 49 connections, and thus contribute to circuit formation (Evsyukova et al., 2013). 50 51 Several areas of the brain undergo sexually dimorphic development. During 52 adolescent development, the brain undergoes differential development trajectories that lead 53 to sexual dimorphism in total cerebral volume and different local grey matter nuclei volumes 54 (Kaczkurkin et al., 2019). Notably, female total cerebral volume peaks earlier in 55 adolescence than males (Lenroot et al., 2007). In addition, analysis of neural circuitry and 56 behavior reveals that different systems and regions within the brain are engaged in a sex-57 dependent manner and specific nuclei within the brain are thought to be responsible for sex-58 specific behaviors (Gillies & McArthur, 2010;Choleris et al., 2018) The resulting amplicons were resolved on 1.5% agarose gels and visualized using ethidium 169 bromide staining with a GelDoc transilluminator (BioRad). 170 171

Immunohistochemistry (IHC) 172
Brains were mounted in OCT embedding media (Bright) and cut into 14 µm sections 173 across the coronal plane using a cryostat (Leica CM 1860 UV, Ag Protect) and collected on 174 SuperFrost Plus microscope slides (Thermo Scientific). Immunohistochemistry (IHC) was 175 carried out as previously described (Jones et al., 2019). In brief, sections were 176 simultaneously permeabilised and blocked in 0.1% Triton-X with 2% normal goat serum in 177 PBS for 1 h at room temperature, in a humidified chamber. They were then incubated 178 overnight at 4ºC in a humidified chamber with primary antibodies against chicken GFP 9 9 (1:1000; Abcam #ab13970) and rabbit aromatase (1:100 Abcam #ab18995). Sections were 180 then counterstained the appropriate secondary antibodies and counter stained with DAPI 181 (ThermoFischer D1306). Images were captured and analyzed as described below. 182 183

Image acquisition and data analysis 184
Confocal images of IHC stained P0 sections was carried out using a Nikon Spinning 185 Disk confocal equipped with either a 20X or 40X objective. Image z-stacks were acquired at 186 z-intervals optimised for the specific objective. Images to be used for subsequent intensity-187 based analysis were acquired using identical acquisition parameters. For migration 188 analysis, cortical sub-sections were identified by DAPI staining and regions of interest 189 (ROIs) were identified in the 488 nm channel using the epifluorescence microscope. The 190 effect of knockdown of aromatase on migration was analyzed by quantitative bin analysis 191 according to previously published methods (Kubo et al., 2010). In brief, the cortex was 192 divided into ten equal sections (bins) and percentage of GFP + cells within each bin was 193 determined. For each condition, a minimum of three images were collected over at least six 194

section. 195
Analysis of aromatase expression was carried out on sections immunostained for 196 aromatase and DAPI (endogenous aromatase expression) or immunostained for GFP,197 aromatase and DAPI (assessment of aromatase knockdown). ROIs were determined either 198 as described above or limited to GFP + cells. Images were background subtracted, and the 199 mean intensity of aromatase staining determined for five intendent ROIs per image; three 200 ROIs of background staining were also measured for each section. The mean intensity for 201 each section was normalised to background staining (average of 3x background ROIs + 2x 202 StDev). Between 3-4 sections per brain; 3 brains per condition was used for these analyses. aromatase expression revealed that aromatase expression was higher in the developing 221 cortex of male compared to female P0 mice ( Figure 1E). Taken together, these data 222 indicate that aromatase is expressed in the developing cortex of male and female mice, 223 suggesting a potential role for brain-synthesised estrogens during this developmental time 224

point. 225
As our data indicated that aromatase is more highly expressed in males compared to 226 females, we reasoned that this may be reflected in a difference in neuronal migration 227 11 11 between sex. Therefore, to analyze the effect of sex on neuronal migration, we in utero 228 electroporated embryos at E14.5 with a GFP expressing plasmid (pCAG-eGFP), and 229 quantified the positions of GFP-positive (GFP+) cells in P0 brains as previously described 230 (Kubo et al., 2010). Sex were again determined by assessing expression of sry. Analysis of 231 neuronal migration revealed that the differential expression of aromatase did not confer a 232 difference in the migration of GFP+ cells in either sex under these control conditions 233 (Figure 2A). This observation is confirmed by the relatively parity of GFP+ cells throughout 234 the developing P0 cortex ( Figure 2B). These data indicate that there are no differences in 235 neuronal migration between male and female mice at P0. 236 237

Validation of aromatase knockdown 238
Although our data indicates that there is no difference in neuronal migration between 239 male and females under control conditions, previous studies using ERβ knockout animals 240 have suggested a role for estradiol in cortical development (Wang et al., 2003). 241 Furthermore, estradiol has been shown to regulate proliferation and differentiation of neural 242 progenitor cells (Denley et al., 2018). Therefore, we were interested in understanding 243 whether brain-synthesised estrogens may contribute to neuronal migration in either male or 244 female mice. In order to do this, we employed a short hairpin RNA interference (shRNA) 245 approach to selectively knockdown expression of Cyp19a1, the gene encoding aromatase. 246 The efficiency of four individual shRNA to knockdown aromatase was first established in 247 hEK203 cells. Myc-tagged aromatase was exogenous expressed in hEK293 cells in the 248 presence of shRNA for aromatase or a control (scramble) shRNA ( Figure 3A). Of the four 249 shRNA tested shRNA_c (herein referred to as shRNA_arom) reduced myc-aromatase 250 expression by ~60% (Figure 3A and B) and was used in subsequent experiments. 251 12 12 Next, we assessed whether expression of shRNA_arom in the developing cortex by 252 in utero electroporation resulted in a significant knockdown of aromatase in males and 253 females. Embryos were electroporated with shRNA_arom expression construct or control 254 shRNA (shRNA_scram) at E14.5 (Figure 3 C and D). Corrected integrated intensity 255 measurements from confocal images taken of the P0 mouse cortex confirmed that 256 shRNA_arom effectively reduced aromatase expression by approximately 50% compared to 257 control condition in both sexes (Figure 3C-E). These data confirm the efficacy of aromatase 258 knockdown by shRNA in vivo. 259 260

Aromatase knockdown affects cortical migration divergently in male and females 261
To determine whether brain-synthesised estrogens play a role in the migration of 262 neocortical cells, we assessed distribution of GFP+ cells in control and aromatase 263 knockdown conditions (Figure 4). In P0 male mice, knockdown of aromatase resulted in an 264 increase of GFP+ cells within the upper most portion of the CP with a concurrent reduction 265 of GFP+ cells within the SVZ/VZ (Figure 4 A and B). Conversely, the opposite distribution 266 was observed in the shRNA_arom condition in females; a decreased number of GFP+ was 267 observed in the CP, where as an increase was detected in the SVZ/VZ (Figure 4C and D). 268 Taken together, these data indicate that that knockdown of aromatase may accelerate 269 radial neuronal migration in male, whereas migration is impaired in the female developing 270

Discussion 273
Aromatase is expressed in specific brain regions, where it controls the bioavailability 274 of brain-synthesized estradiol within female and male brains (Saldanha et al., 2011;275 Srivastava et al., 2013;Lu et al., 2019). Moreover, estradiol is present at significant levels in 276 the brain of both sexes, including the developing cortex (MacLusky et al., 1986;MacLusky 277 et al., 1994;Konkle & McCarthy, 2011). However, the functions of brain-synthesized 278 estradiol during early corticogenesis development are unclear (Denley et al., 2018). Here, 279 we demonstrated that aromatase is widely expressed within the developing cortex of The data presented in this study are consistent with a purported role for brain-296 have demonstrated that high levels of aromatase are expressed in multiple regions of the 298 brain, including the cortex (Beyer et al., 1994;Yague et al., 2008;Cisternas et al., 2015). 299 Importantly, the current results are consistent with this, but they further highlight that that 300 there is greater expression of aromatase in the male developing cortex compared with 301 females at the protein level. Although previous work implicated ERβ knockdown in the 302 development of the cortex (Wang et al., 2003), the current study provides evidence that 303 brain-synthesised estradiol regulates neuronal migration in the developing cortex of both 304 female and male mice. Whether systemic estradiol also impacts neuronal migration is 305 unclear from these studies and would need to be studied further in the future. 306 A striking finding of this study is that the effect of aromatase knockdown on neuronal 307 migration in the developing cortex is opposing in female and male mice. There are two 308 possible explanations for this observation. First, estradiol could be exerting multiple effects 309 on progenitor cells, such as controlling cell proliferation and/or apoptosis, as reported 310 previously in the hypothalamus (Denley et al., 2018;McCarthy et al., 2018). Second, 311 estradiol could be modulating the migration of newly born neurons in both sexes. No 312 differences in the number of GFP+ cells were found under different conditions and between 313 sex (data not shown), which indicates that the changes observed were dues to different 314 localization of the labelled cells rather than increases or decreases in the overall number of 315 cells. Thus, these effects are more likely an effect on the migration (or lack thereof) of newly 316 born neurons. The small GTPase Rap1 mediates the migration, polarity, and establishment 317 of neuronal morphology (Jossin & Cooper, 2011;Srivastava et al., 2012b). Since we 318 previously demonstrated that estradiol controls Rap1 activity in maturing cortical neurons 319 (Srivastava et al., 2008), it is possible that brain-synthesized estradiol mediates migration 320 via a Rap1-dependent mechanism. It is also important to note that although knockdown 321 15 15 approach using in utero electroporation allows us to examine the cell-autonomous effect of 322 aromatase in the specific time critical for radial neuronal migration. However, we should be 323 cautious in data interpretation, as off-target effect of shRNA may be confounded in the 324 results. Thus, it is important to investigate the effect of aromatase(Cyp19a1)-325 haploinsufficiency in mouse models to elucidate aromatase-mediated mechanisms 326 underpinning developmental phenotypes such as neuronal migration in the future. 327 The development of the cortex is fundamental for normal brain function. Interestingly, 328 multiple animal models for autism spectrum disorders aimed at understanding the 329 contribution of genetic and/or environmental risk factors to the underlying pathophysiology 330 of this disorder have revealed that disruptions in early brain development is prevalent in 331 these models. In particular, abnormalities in the development, migration, and organization of 332 the developing cortex have been reported (Fenlon et al., 2015;Varghese et al., 2017). 333 Moreover, there is accumulating evidence that elevated levels of fetal steroids, especially 334 testosterone ( Baron-Cohen et al., 2015;McCarthy & Wright, 2017), are linked with autism 335 spectrum disorders. Furthermore, rare mutations in the CYP191A gene have been reported 336 in autistic patients and reduced ERβ and aromatase expression has been measured in 337 autistic post-mortem tissue (Chakrabarti et al., 2009;Sarachana et al., 2011;Crider et al., 338 2014). These lines of evidence have led to suggestions that altered steroidogenic activity 339 and/or elevated levels of fetal testosterone could contribute to the pathophysiology of 340 autism. It should be noted that knocking down Cyp19a1 will both reduce estradiol levels and 341 likely increase the levels of testosterone and other androgens within the developing cortex 342 of these animals. Therefore, the current study may not only provide an insight into how 343 reduced brain-synthesised estradiol levels impact development of the cortex, but also the 344 impact of elevated levels of fetal testosterone on corticogenesisand therefore how 345 16 16 dysregulation of fetal steroids could contribute to the emergence of neurodevelopmental 346 disorders such as autism spectrum disorders. 347 In conclusion, the current study revealed that aromatase is expressed in the 348 developing cortex of both female and male mice, and at higher levels in males than 349 females. Knockdown of aromatase in cortical progenitor cells destined to migrate to layer 350 2/3, had marked sex-specific effects. Future studies focusing on understanding the 351 mechanism underlying these effects, including investigating the potential role of Rap1, and 352 also identifying the receptors that are responsible for the actions of the brain-synthesized 353 estrogens (i.e., do brain-synthesized estradiols function via the classical "genomic" mode of 354 action or do they act via a "membrane initiated" mode of action) are required. Together with 355 the current work, these studies will help reveal the potential role of fetal steroids in normal 356 development and how perturbations in this system may contribute to the emergence of 357 disease. 358 359

Conflict of Interest 360
The authors declare that the research was conducted in the absence of any commercial or 361 financial relationships that could be construed as a potential conflict of interest.