Involvement of Cxcl12a/Cxcr4b Chemokine System in Mediating the Stimulatory Effect of Embryonic Ethanol Exposure on Neuronal Density in Zebrafish Hypothalamus

Abstract

Background:

Embryonic exposure to ethanol (EtOH) produces marked disturbances in neuronal development and alcohol-related behaviors, with low-moderate EtOH doses stimulating neurogenesis without producing apoptosis and high doses having major cytotoxic effects while causing gross morphological abnormalities. With the pro-inflammatory chemokine system, Cxcl12 and its main receptor Cxcr4, known to promote processes of neurogenesis, we examined here this neuroimmune system in the embryonic hypothalamus to test directly if it mediates the stimulatory effects low-moderate EtOH doses have on neuronal development.

Methods:

We used the zebrafish (Danio rerio) model, which develops externally and allows one to investigate the developing brain in vivo with precise control of dose and timing of EtOH delivery in the absence of maternal influence. Zebrafish were exposed to low-moderate EtOH doses (0.1, 0.25, 0.5% v/v), specifically during a period of peak hypothalamic development from 22–24 hours post fertilization, and in some tests were pre-treated from 2–22 hpf with the Cxcr4 receptor antagonist, AMD3100. Measurements in the hypothalamus at 26 hpf were taken of cxcl12a and cxcr4b transcription, signaling, and neuronal density using qRT-PCR, RNAscope, and live-imaging of transgenic zebrafish.

Results:

Embryonic EtOH exposure, particularly at the 0.5% dose, significantly increased levels of cxcl12a and cxcr4b mRNA in whole-embryos, number of cxcl12a and cxcr4b transcripts in developing hypothalamus, and internalization of Cxcr4b receptors in hypothalamic cells. Embryonic EtOH also stimulated an increase in the number of hypothalamic neurons and coexpression of cxcl12a and cxcr4b transcripts within these neurons. Each of these stimulatory effects of EtOH in the embryo were blocked by pretreatment with Cxcr4 antagonist AMD3100.

Conclusions:

These results provide clear evidence that EtOH’s stimulatory effects at low-moderate doses on the number of hypothalamic neurons early in development are mediated, in part, by increased transcription and intracellular activation of this chemokine system, likely due to autocrine signaling of Cxcl12a at its Cxcr4b receptor within the neurons.

Introduction

In the United States, 11.5% of pregnant women report drinking alcohol during the prior 30 days, with 3.9% reporting instances of binge drinking (Denny et al., 2019). Exposure to alcohol during gestation produces a wide range of adverse effects on the developing offspring that fall under the umbrella term of fetal alcohol spectrum disorders (FASD) and commonly persist throughout life, with an estimated global prevalence of children and youth with FASD exceeding 1% in the general population (Lange et al., 2017). There is extensive evidence in humans and rodents, which shows maternal ethanol (EtOH) consumption during pregnancy to have major behavioral consequences in the offspring including an increase in alcohol drinking, anxiety and impulsivity (Caputo et al., 2016), and which link these alcohol-related behaviors to disturbances in neurochemical systems of the brain (Wille-Bille et al., 2018) in addition to neuroimmune systems (Crews et al., 2017). While lacking in the embryo, studies in adolescents and adults show EtOH exposure to have strong stimulatory effects on cytokine and chemokine systems in the periphery and brain (Crews and Vetreno, 2014, Crews et al., 2017) and these effects to be mimicked by peripheral administration of chemokines and inflammatory agents while blocked by their receptor antagonists and gene knockouts (Zhang et al., 2018, Roberto et al., 2017).

Investigations of FASD have generally examined relatively high doses of EtOH that have major cytotoxic effects, including increased apoptosis and gliogenesis, and produce facial abnormalities (Wilhelm and Guizzetti, 2016). While these high doses also disrupt the development of neurons and generally reduce neurogenesis (Granato and Dering, 2018), embryonic exposure to low-moderate EtOH doses, in addition to stimulating alcohol-related behaviors (Chang et al., 2015, Chang et al., 2018), is found in rodents to increase neurogenesis while having minimal cytotoxic effects (Santillano et al., 2005, Chang et al., 2012). Exposure to low-moderate EtOH during gestation also increases the neurogenesis of orexigenic peptide neurons known to promote alcohol consumption (Chang et al., 2018, Chang et al., 2015). These effects produced by lower EtOH doses may be more closely linked to alcohol-related neurodevelopmental disorder (ARND), which is more prevalent than FAS and characterized by neuronal and behavioral changes occurring without distinctive facial abnormalities (Coles et al., 2020).

To further explore this phenomenon of increased neurogenesis at lower EtOH doses as it relates to neuroimmune systems in the brain, we focused here on the pro-inflammatory chemokine Cxcl12 (also known as SDF-1) and its primary receptor Cxcr4, which are shown to have a major role in processes of neurogenesis, influencing the proliferation, differentiation and migration of neurons from neuroprogenitor cells shown predominantly in vitro (Hwang et al., 2019). In adult human subjects, EtOH and beer are found to stimulate Cxcr4 expression in peripheral tissue and increase circulating levels of Cxcl12 (Poulsen et al., 2019, Chiva-Blanch et al., 2014), while prolonged withdrawal or abstinence causes a decrease in Cxcl12 plasma levels (Garcia-Marchena et al., 2016). Whereas this EtOH-induced upregulation of Cxcl12 and Cxcr4 has been recapitulated in vitro (Karim et al., 2013) and in adult rodents (Gil-Bernabe et al., 2011), there is little evidence describing EtOH’s effects on this chemokine system in the embryo, with only our recent study in the rat providing evidence that prenatal EtOH exposure at a moderate dose stimulates the Cxcl12/Cxcr4 system in neurons and neuroprogenitor cells in the hypothalamus (Chang et al., 2020a).

To directly test if this neuroimmune system has a stimulatory effect on neuronal development in the hypothalamus, we turned to the zebrafish model which is found to be particularly useful for neurodevelopmental studies. Zebrafish have notable advantages, due to their transparency and small size that permit one to observe the embryonic brain in vivo and to their external development that allows one to precisely control the EtOH dose and exposure time without the influence of maternal physiology. In addition, there are a variety of useful transgenic lines that allow one to use live-imaging microscopy to study intracellular chemokine signaling and neuronal development, experiments that are difficult or impossible to perform using the rodent model. It is notable that the hypothalamus in zebrafish is well conserved (Machluf et al., 2011) and contains rich populations of neurons that express neuropeptides known promote EtOH consumption (Berman et al., 2009, Faraco et al., 2006), and there is a high degree of functional conservation between the Cxcl12/Cxcr4 system in mammalian species and the Cxcl12a ligand and Cxcr4b receptor in zebrafish (Wang and Knaut, 2014). Further, our recent studies have shown that low-moderate doses of embryonic EtOH exposure in zebrafish, as demonstrated in rats, stimulates the development of neurons in the hypothalamus and produces an increase in EtOH consumption and associated behaviors (Collier et al., 2019, Collier et al., 2020, Sterling et al., 2016). With the timing of embryonic exposure to environmental stimuli found to be crucial and their resulting effects determined by the specific developmental processes actively occurring (Hwang et al., 2019, Kleiber et al., 2013), it is notable that the stimulatory effect of EtOH on neuronal development in zebrafish is produced by embryonic exposure from 22–24 hours post-fertilization (hpf), precisely when the segmentation period is ending, the pharyngula phase of embryogenesis is beginning, and the hypothalamus is just starting to develop (Kimmel et al., 1995).

To study EtOH’s effects on neuronal development and the role of chemokines in this process, we examined in zebrafish the Cxcl12a and Cxcr4b system and hypothalamic neurons during their peak period of development and administered only low-moderate doses of EtOH (0.1%, 0.25% and 0.5%) known to increase hypothalamic neuronal density (Sterling et al., 2016, Collier et al., 2019), in contrast to higher doses (1.0% to 2.5%) that produce apoptosis and other neuronal deficits in zebrafish (Joya et al., 2014). Numerous reports indicate that these lower EtOH concentrations (i.e., 0.1%, 0.25%, 0.5%) presented for a short 2 hr exposure time, while not producing any gross morphological abnormalities or mortality (Fernandes and Gerlai, 2009), cause neuronal and behavioral changes that persist into adulthood, are comparable to changes in rodents (Chang et al., 2018), and are consistent with behavioral effects characteristic of milder cases of FASD that include ARND. Recent studies have shown the internal tissue ethanol concentration in zebrafish embryos to be about 1/3 of the external concentration in the ethanol bath solution (Lovely et al., 2014, Flentke et al., 2014, Zhang et al., 2013). Assuming that the tissue and blood EtOH levels are equal due to the lack of a complete closed cardiovascular system at 22–24 hpf (Ellertsdóttir et al., 2010), exposure to EtOH at the 0.5% dose results in a BAC equivalent of 0.12 g/dL, a level comparable to the BAC of human neonates at birth prenatally exposed to alcohol, shown to range between 0.005–0.212 g/dL (Burd et al., 2012).

We first tested in AB zebrafish whether embryonic exposure to EtOH at these lower doses, from 22–24 hpf affects the expression of cxcl12a and cxcr4b in whole embryo and the number of transcripts in the developing hypothalamus and their colocalization within hypothalamic cells where they may directly interact to affect the cell’s function. We next used live-imaging in transgenic zebrafish embryo to examine EtOH’s effects on the internalization of Cxcr4b receptors, reflecting a function of Cxcl12a activity and its intracellular signaling, and in further tests administered a Cxcr4 antagonist prior to EtOH exposure to test directly if these effects of EtOH on cxcl12a and cxcr4b transcripts, their colocalization, and intracellular signaling are mediated by activity at the Cxcr4b receptor. In our final experiments, we used live-imaging in transgenic zebrafish to examine the effects of EtOH on the number of hypothalamic neurons labeled with the early neuronal marker, HuC, and then directly tested whether EtOH affects the colocalization of both cxcl12a and cxcr4b transcripts within these HuC neurons where they may promote an increase in the number of differentiated HuC neurons, and whether the integrity of the Cxcr4b receptor is required for this effect of EtOH on neuroimmune signaling in embryonic neurons. The results of these experiments provide clear evidence indicating the involvement of the Cxcl12a/Cxcr4b system in mediating the stimulatory effects of low-moderate EtOH doses on neuronal development.

Materials and Methods

Animals and Housing

Three lines of zebrafish (Danio rerio) were used in this study: AB strain zebrafish bred in our facility from founders originally purchased from ZIRC (Eugene, Oregon), transgenic cxcr4b:cxcr4b-EGFP-IRES-kate2-CaaX (Venkiteswaran et al., 2013, Lau et al., 2020) zebrafish, and transgenic HuC:GFP (Park et al., 2000) zebrafish, which were generously gifted to us (see Acknowledgements). AB zebrafish were used as a wild-type strain and for their lack of transgenic expression. Cxcr4b:cxcr4b-Kate2-IRES-eGFP-CaaX transgenic line expresses Cxcr4b fused to the monomeric red fluorescent protein Kate2 from the cxcr4b promoter and membrane-tethered GFP from IRES. The HuC:GFP zebrafish were selected due to their cytoplasmic expression of GFP under the HuC promotor. Adult zebrafish were group-housed in 3-l tanks (Aquatic Habitat, Apopka, FL) with recirculating water flow at a temperature between 28 and 29°C and a pH between 6.9 and 7.4. All animals were maintained on a 12:12-hour light–dark cycle (8 am lights on and 8 pm lights off) within an Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited facility. Embryos were produced by natural spawning and raised in embryo media. They were maintained in 6-well cell culture plates in an incubator at 28 to 29°C and on a 12:12-hour light cycle. All protocols were approved by the Rockefeller University Institutional Animal Care and Use Committee and followed the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

Embryonic EtOH and AMD3100 treatment

The procedure used for embryonic EtOH exposure, described in detail in our previous papers (Collier et al., 2019, Sterling et al., 2016), is briefly summarized as follows. At 22 hpf, embryos were removed from the incubator, placed in a fresh solution of either 0.0%, 0.1%, 0.25% or 0.5% (v/v) EtOH diluted in embryo medium, and returned immediately to the incubator for 2 hours. After this 2-hour period, embryos were washed in fresh embryo medium and then returned to the incubator. For Cxcr4 antagonist experiments, 10 μM of AMD3100 was prepared using embryo medium, as previously described (Pillay et al., 2016), and zebrafish were immersed in this solution from 2–22 hpf followed by a wash in fresh embryo medium prior to exposure of 0.0% or 0.5% EtOH.

RNA isolation and cDNA synthesis

Total RNA from two groups of 100 26 hpf AB zebrafish and two groups of 100 48 hpf AB zebrafish were isolated with TRIzol reagent (Invitrogen, Carlsbad CA, USA). The homogenized tissue was mixed with chloroform (Sigma- Aldrich, USA) and incubated for 10 minutes in ice and then centrifuged at 12,000 × g for 15 minutes at 4° C. The top, clear aqueous phase was transferred into new tubes, and isopropyl alcohol (Sigma-Aldrich, USA) was added, mixed and incubated for 10 minutes at room temperature, and then centrifuged at 12,000 × g for 10 minutes at 4° C. The supernatant was removed without disturbing pellets, and the pellets were washed with 75% EtOH, mixed by gentle inversion and centrifuged at 7,500 × g for 5 minutes at 4° C. Then, 75% EtOH was discarded, and the pellets were left to air dry for 10 minutes in the fume hood. RNA pellet was resuspended in RNase-free water and incubated for 10 minutes at 55° C. The RNEasy Mini Kit (Qiagen, Germany) was used for removing contaminants and impurities from the RNA. For this procedure, β-mercapto-EtOH (Sigma-Aldrich, USA) and 75% EtOH were used in addition to the components in the kit. Finally, the concentration and purity of the RNA were checked using a NanoDrop Spectrophotometer ND-2000 (Thermo Fisher Scientific, USA), and the ratio value of A260:A280 over 2.0 was considered optimal in this condition. The RNA samples were stored at −80° C. The cDNA was synthesized from 2μg of total RNA that was pretreated with DNase I (Invitrogen, USA), using TaqMan Reverse Transcription reagents (Applied Biosystems, USA) with a final volume of 40.8 μL.

Reverse transcription-quantitative PCR (RT-qPCR).

RT-qPCR with SYBR Green (Applied biosystems, UK) was performed in MicroAmp Optical 96-well Reaction Plates with Barcode (Applied biosystems, USA) in QuantStudio 12K Flex (Applied Biosystems) with the following thermo-cycling conditions: 2 minutes at 50°C, 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. Housekeeping gene β-actin was used to normalize the expression of target genes, cxcl12a and cxcr4b. Their primers were: (1) β-actin: 5’-ATGAGTCTGGCCCATCCATC-3’ (forward) and 5’-CCTTTGCCAGTTTCCGCATC-3’ (reverse); (2) cxcl12a: 5’- CTGTCACAGTTGCTCCTGGAT-3’ (forward) and 5’-GGCTTGGCGTTGGAAATCG-3’ (reverse); (3) cxcr4b: 5’-GCGACCTCTCAGTCAGCAAT-3’ (forward) and 5’-TCACAAGCACCACAAGTCCA-3’ (reverse). The primer concentrations for β-actin, cxcl12a and cxcr4b were 100, 200 and 400 nM, respectively. Each reaction was performed in quadruplicate, and an average value was determined. The ratio of target gene expression to housekeeping gene expression (or mRNA fold change) was calculated using the standard delta-delta Ct method. The ANOVA was then run on this ratio calculated for each subject, with the effect of EtOH on target gene expression determined by comparing the average of the ratio in the experimental groups to the control groups. The mRNA gene expression data are presented as fold changes in the figures.

RNAscope FISH combined with immunofluorescence histochemistry:

We performed RNAscope in combination with immunofluorescence histochemistry (IF) as previously described (Gross-Thebing et al., 2014). Briefly, zebrafish embryos were fixed in 4% PFA in PBS at RT after hand dechorionation. A series of increasing methanol concentrations (25%, 50%, 75%, 100%) in 0.1% PBT (0.1% Tween-20 in PBS) was used to dehydrate the embryos stepwise in 5-minute washes. After the last methanol wash, embryos were stored at −20°C for at least one night. The embryos were then air-dried for 30 minutes at RT and subjected to the RNAscope-based signal amplification (Advanced Cell Diagnostics). Protease digestion of embryos using Pretreat 3 for 20 minutes at RT was followed by rinsing the embryos 3 × in 0.01% PBT (0.01% Tween-20 in PBS). Hybridization of cxcl12a and cxcr4b probes was then performed at 45°C overnight, followed by wash in 0.2x SSCT and post-fixation in 4% PFA for 10 minutes at RT. Cxcr4b was labeled with Atto 550 and cxcl12a was labeled with Atto 647. Pre-amplifier hybridization (30 minutes), signal enhancement (15 minutes), amplifier hybridization (30 minutes), and labeling (15 minutes) were all conducted at 40°C and separated by 3 × 15 minutes washes in 0.2x SSCT at RT. This was followed by incubation in blocking solution (0.3% Triton and 4% Normal Donkey Serum in PBS) overnight at 4°C, 3 × 15 minutes washes in 0.3% triton, and then incubation in primary antibody labeling the cellular membrane of HuC neurons (mouse anti-HuC; Invitrogen; 1:500) in blocking solution overnight at 4°C. The samples were then washed 3 × 15 minutes in 0.3% triton and incubated in secondary antibody (Alexa 488 anti-mouse 1:500) overnight in blocking solution containing 25 μg/mL of DAPI. The samples were washed 3 × PBS and stored in fresh PBS at 4°C until imaging.

Image Acquisition and Analysis

Live-imaging was conducted using an inverted Zeiss Axiovert 200 spinning disk confocal microscope with a 25x lens (z step = 0.2–0.4 μm). Zebrafish processed using RNAscope and IF were imaged using an inverted Zeiss LSM 780 laser scanning confocal microscope with a 40x objective (z step = 1 μm). Images were deconvoled using AutoQuant X3 software (Media Cybernetics, Rockville, USA), and analyzed using Imaris 9.5.1 software (Bitplane, Zurich, Switzerland). The “Crop 3D” feature of Imaris was used to visually delineate the hypothalamus from the entire forebrain images (Affaticati et al., 2015). These dimensions are presented in anatomical schematics illustrating the location of the developing hypothalamus from frontal, ventral and lateral views (Vaz et al., 2017) (Fig. 1A), as well as in photomicrographs of DAPI staining (Fig. 1B).

Figure 1:

Photomicrographs illustrating the location of the developing hypothalamus in 26 hpf AB embryos and the methodology used to quantify cxcl12a and cxcr4b colocalization within hypothalamic cells and HuC neurons. (A) Anatomical schematics illustrating the general location of the developing hypothalamus at 26 hpf from frontal, ventral and lateral views. (B) Confocal photomicrographs of representative DAPI staining of a 26 hpf embryo showing the location and dimensions of the developing hypothalamus (white boxes) in frontal, lateral and ventral views of the brain. Scale bar, 30 μm. (C) Representative confocal image of the left side of the embryo hypothalamus showing the staining used in this study of cxcl12a transcripts (white) and cxcr4b transcripts (red) labeled using RNAscope and of HuC neurons (green) labeled using immunofluorescence histochemistry. Scale bar, 8 μm. (D) Box 1 (from Fig. 1B) showing an example of cxcl12a and cxcr4b double-labeled transcript puncta within 1 μM of each other (left) and represented using Imaris spot labeling (right), suggesting their colocalization within hypothalamic cells. Scale bar, 3 μm. (E) Box 2 (from Fig. 1B) showing both cxcl12a and cxcr4b transcript puncta colocalized within 1 μM of an HuC-labeled neuron (left) and represented using Imaris rendering of HuC surface and chemokine transcript spot labeling with a white arrow illustrating triple-labeling. Scale bar, 5 μm. Abbreviation: Hyp, hypothalamus; Tel, telencephalon; v, ventricle; ORR, optic recess region.

For live-imaging, Cxcr4b internalized puncta and HuC neurons were quantified using the ‘Spot’ function in Imaris (1 and 10 μm XY diameter respectively, followed by manual correction). The number of membrane-bound Cxcr4b cells was quantified manually by multiple observers blind to the condition, while the overall number of cells in the RNAscope experiments could not be counted due to the high density of DAPI nuclear staining Cxcl12a and cxcr4b transcripts were quantified using the ‘Spot’ function in Imaris (2 μm estimated XY diameter, threshold = above 30). For cxcl12a + cxcr4b colocalization analysis, the “colocalize spots” tool in Imaris was used (threshold = 1 μm). Therefore, every instance of cxcl12a and cxcr4b transcripts within 1 μM from each other were defined as colocalized within cells which are generally > 10 μM (Fig. 1C, Box 1, enlarged in Fig. 1D). When HuC labeling using IF was used in conjunction with RNAscope, cxcl12a + HuC and cxcr4b + HuC colocalization analysis was done by creating a “Surface” rendering of HuC labeling followed by “find spots near surface” analysis in Imaris using 1 μm threshold to select the cxcl12a and cxcr4b transcripts located within a distance of 1 μm from the HuC surface indicating that they were neuronal (Fig. 1C, Box 2, enlarged in Fig. 1E). For triple labeling cxcl12a + cxcr4b + HuC analysis, first, cxcl12a transcripts that colocalized with cxcr4b within 1 μm were selected in the software, followed by “find spots near HuC surface” analysis with 1 μM threshold to quantify each instance of colocalized cxcl12a + cxcr4b transcripts that were within a distance of 1 μM from the HuC surface.

Statistical Analysis

All data are presented as mean ± SEM and were analyzed using Prism (version 8, GraphPad, San Diego, California, USA). Data from all experiments were evaluated using a one-way ANOVA followed by Dunnett’s post-hoc multiple comparisons test to evaluate differences between Control and 0.1%, 0.25% and 0.5% EtOH groups, or a Tukey’s multiple comparisons test to evaluate differences between Control, Control with AMD3100, 0.5% EtOH, and 0.5% EtOH with AMD3100 groups.

Results

Embryonic EtOH exposure increases mRNA expression of cxcl12a and cxcr4b in whole embryos

Our first goal was to evaluate whether 2-h exposure (22–24 hpf) to low-moderate doses of EtOH (0.1%, 0.25% and 0.5% v/v) affects mRNA expression of cxcl12a and cxcrb4 in whole AB strain zebrafish embryos at 26 hpf and to determine which doses are most effective. While analysis via one-way ANOVA failed to reveal a significant effect of EtOH on cxcl12a mRNA in whole embryos at 26 hpf (F(3, 12) = 2.14, p = 0.15), a Dunnett’s multiple comparisons analysis indicated a trend toward a stimulatory effect, as revealed by a near significant increase in cxcl12a expression compared to Control group (p = 0.068) specifically at the 0.5% dose but no difference at 0.1% and 0.25% EtOH (Fig. 2B). Analysis of cxcr4b mRNA levels via one-way ANOVA revealed a stronger stimulatory effect of EtOH at 26 hpf (F(3, 12) = 14.34, p < 0.001), with a Dunnett’s multiple comparisons test showing a significant increase in cxcrb4 mRNA levels after EtOH compared to Control at all three doses, 0.1% (p = 0.0004), 0.25% (p = 0.0008) and 0.5% (p = 0.0004) (Fig. 2C). In our next test to determine if the EtOH-induced increase in chemokine expression persists beyond 26 hpf, a one-way ANOVA revealed at 48 hpf a significant effect of EtOH on cxcl12a mRNA in whole embryos (F(3, 22) = 10.32, p = 0.0002), with a Dunnett’s multiple comparisons test showing a significant increase of cxcl12a expression after EtOH compared to Control at 0.25% (p < 0.0001) and 0.5% (p = 0.01) doses (Fig. 2E). Analysis of cxcr4b mRNA levels via one-way ANOVA also revealed at 48 hpf a stimulatory effect of EtOH (F(3, 19) = 6.44, p = 0.003), with Dunnett’s multiple comparisons test showing a significant increase of cxcr4b expression at 0.5% EtOH dose compared to Control (p = 0.03) but no effect at 0.1% (p = 0.15) and 0.25% (p = 0.98) EtOH. (Fig. 2F). These results in the whole zebrafish embryo indicate that embryonic EtOH exposure at low-moderate doses, particularly 0.5% EtOH, upregulates the expression of this chemokine and its receptor at 26 hpf immediately after exposure and that this effect persists until at least 48 hpf. There was no effect of EtOH on mRNA levels of β-actin at 26 hpf (F(3, 12) = 2.14, p = 0.15) (Fig. 2A), or at 48 hpf (F(3, 12) = 0.001, p > 0.99) (Fig. 2D).

Figure 2:

Effects of embryonic EtOH exposure on β-actin, cxcl12a and cxcr4b mRNA levels (fold change) in whole AB zebrafish embryos, measured using qRT-PCR. (A) EtOH has no effect on β-actin mRNA expression at 26 hpf. (B) EtOH produces a near significant increase in cxcl12a mRNA at 26 hpf at the 0.5% dose. (C) EtOH significantly increases cxcr4b mRNA at 26 hpf at all 3 doses tested. (D) EtOH has no effect on β-actin mRNA expression at 48 hpf. (E) EtOH significantly increases cxcl12a mRNA at 48 hpf at the 0.25% and 0.5% doses. (F) EtOH significantly increases cxcr4b mRNA at 48 hpf at the 0.5% dose. #p = 0.068, * p < 0.05 ***p < 0.001.

Embryonic EtOH stimulates cxcl12a and cxcr4b transcription in whole embryonic hypothalamus.

To examine EtOH’s effects on gene expression in the hypothalamus itself, an area too small to analyze in the embryo using RT-qPCR, this experiment used RNAscope FISH and Imaris software to examine the effect of EtOH exposure on the transcription of cxcl12a and cxcr4b in the whole hypothalamus of 26 hpf AB zebrafish. Analysis via one-way ANOVA of cxc12a and cxcrb4 transcripts revealed a significant effect of EtOH on the number of cxcl12a transcripts (F(3, 13) = 10.13, p = 0.001). Once again, the multiple comparisons analysis showed the 0.5% EtOH dose to produce a significant increase (p = 0.002) (Fig. 3A), as illustrated in the photomicrographs and enlarged in Box 1 (Fig. 3B). Similarly, EtOH had a significant effect on the number of cxcr4b transcripts (F(3, 13) = 9.74, p = 0.001), with this increase also produced only by the 0.5% dose (p = 0.0005) (Fig. 3C), as illustrated in the images (Fig. 3D). With our further analyses showing cxcl12a and cxcr4b transcripts to exist together within the same hypothalamic cells, we next tested whether embryonic EtOH exposure affects the number of cells that colocalize both of these transcripts. A one-way ANOVA showed an effect on the colocalization of cxcl12a with cxcr4b transcripts in the hypothalamic cells (F(3, 13) = 7.5, p = 0.004), with the 0.5% EtOH dose increasing the number of cxcl12a transcripts in cells containing cxcr4b transcripts (p = 0.002) (Fig. 3E), as shown in the images (Fig. 3F). These findings demonstrate that embryonic EtOH exposure at the 0.5% dose, in addition to stimulating cxcl12a and cxcr4b transcription, increases the hypothalamic cells that colocalize this chemokine with its main receptor.

Figure 3.

Effects of embryonic EtOH exposure compared to Control group on number and colocalization of cxcl12a and cxcr4b transcripts in cells of whole developing hypothalamus in 26 hpf AB zebrafish embryos. These are shown in the bar graphs (left) and representative RNAscope confocal images (right) of the entire hypothalamus visually dissected using Imaris, showing cxcl12a (white), cxcr4b (red), merged images (white + red), and colocalized cxcl12a and cxcr4b transcripts (white + red “spots”), with specific areas in white boxes enlarged (far right) to more clearly illustrate the individual transcripts. (A, B) EtOH at 0.5% dose increases the number of cxcl12a transcripts, as illustrated in Boxes 1 and 2. Scale bars, 20 μm and 5 μm. (C, D) EtOH at 0.5% dose increases the number of cxcr4b transcripts, as illustrated in Boxes 3 and 4. (E, F) EtOH at 0.5% dose also increases the colocalization of both cxcl12a and cxcr4b transcripts, as illustrated by merged images, enlarged in Boxes 5 and 6, and represented using Imaris spot labeling, enlarged in Boxes 7 and 8. Abbreviation: v, ventricle. n = 3–6, *p < 0.05, **p < 0.01.

Embryonic EtOH increases Cxcl12a signaling indicated by Cxcr4b receptor internalization in hypothalamic cells

In this experiment, we used live-imaging of transgenic cxcr4b:cxcr4b-Kate2-IRES-eGFP-CaaX zebrafish to determine whether embryonic EtOH exposure affects the intracellular signaling of Cxcl12a in the developing hypothalamus, as measured by internalization of the Cxcr4b receptor puncta within the cells at 26 hpf. A one-way ANOVA showed a significant effect of EtOH on the number of internalized Cxcr4b receptor puncta (F(3, 24) = 3.57, p = 0.028) and membrane-bound Cxcr4b cells (F(3, 24)=3.58, p = 0.029) at 26 hpf. Further, multiple comparisons analysis revealed a significant EtOH (0.5%)-induced increase in the number of internalized Cxcr4b receptor puncta (p = 0.01) and membrane-bound Cxcr4b cells (p = 0.01) (Fig. 4A and B), as illustrated in representative photomicrographs and enlarged in Boxes 1 and 2 (Fig. 4C). These findings suggest that embryonic exposure of the developing hypothalamus to 0.5% EtOH increases the activity of Cxcl12a at Cxcr4b, as reflected by an increased internalization of these receptors in hypothalamic cells.

Figure 4.

Effect of embryonic EtOH exposure compared to Control on the internalization of Cxcr4b receptors in cells of the whole developing hypothalamus in live 26 hpf cxcr4b:cxcr4b-Kate2-IRES-eGFP-CaaX embryos. (A) EtOH at the 0.5% dose increases the number of internalized Cxcr4b receptors. (B) EtOH at the 0.5% dose increases the number of membrane-bound Cxcr4b cells. (C) These effects are illustrated in representative confocal images of the entire hypothalamus visually dissected using Imaris, which show 0.5% EtOH compared to Control to have a greater number of internalized Cxcr4b receptor puncta (red) within hypothalamic cells expressing membrane-bound Cxcr4b (green), as clearly illustrated in the enlarged images (right) for Control (Box 1) and 0.5% EtOH (Box 2) Scale bars, 10 μm and 5 μm.. Abbreviation: v, ventricle. n = 5–10, *p < 0.05, **p < 0.01.

Cxcr4 antagonist AMD3100 blocks stimulatory effects of EtOH on Cxcl12a and Cxcr4b colocalization and signaling

To determine if Cxcl12a activity at the Cxcr4b receptor is responsible for the stimulatory effects of 0.5% EtOH on cxcl12a and cxcr4b transcription and signaling in the developing hypothalamus, we next tested if the Cxcr4b antagonist AMD3100 mitigates these effects of EtOH. Using RNAscope to examine AB zebrafish at 26 hpf, a one-way ANOVA revealed a significant effect of the drug treatment condition on the number of cxcl12a transcripts (F(3, 13) = 19.12, p < 0.0001). The multiple comparisons analysis showed significantly more cxcl12a transcripts in the 0.5% EtOH group compared to both Control group (p = 0.0001) and the Control+AMD3100 group (p < 0.0001). There was no change in transcript number in the Control+AMD3100 group compared to Control group (p = 0.67) and significantly reduced transcripts in the 0.5% EtOH+AMD3100 group compared to the 0.5% EtOH group (p = 0.0002) (Fig. 5A). This effect is illustrated in the photomicrographs and Boxes 1 and 2 to the right (Fig. 5B). Similarly, there was a significant effect of drug condition on the number of Cxcr4b transcripts (F(3, 13) = 5.62, p = 0.01). Multiple comparisons analysis showed a greater number of Cxcr4b transcripts in the 0.5% EtOH group compared to Control group (p = 0.007) and the Control+AMD3100 group (p =0.05). There was no difference in transcript number in the Control+AMD31000 group compared to Control group (p = 0.81), and reduced number of transcripts in the 0.5% EtOH+AMD3100 group compared to 0.5% EtOH group (p = 0.05) (Fig. 5C), as shown in the images (Fig. 5D). Further analyses of the effects of AMD3100 treatment in AB zebrafish before EtOH exposure revealed a significant effect on the colocalization of the cxcl12a and cxcr4b transcripts (F(3, 13) = 32.19, p < 0.0001). There was greater colocalization in the 0.5% EtOH group compared to Control group (p < 0.0001) and the Control+AMD31000 (p < 0.0001), no difference in the colocalization of the Control+AMD3100 group compared to Control group (p = 0.65), and significantly reduced colocalization in the 0.5% EtOH+AMD3100 group compared to the 0.5% EtOH group (p < 0.0001) (Fig. 5E), as illustrated in the images (Fig. 5F). These results show the dependence on the endogenous cxcr4b receptor of these stimulatory effects of 0.5% EtOH on cxcl12a and cxcr4b transcript expression and their colocalization within hypothalamic cells.

Figure 5.

Effects of Cxcr4 antagonist AMD3100 on the stimulatory effects of 0.5% EtOH on the number of cxcl12a and cxcr4b transcripts and their colocalization in cells of the developing whole hypothalamus of 26 hpf AB embryos. These are shown in the bar graphs (left) and representative RNAscope confocal images (right) of the entire hypothalamus visually dissected using Imaris, showing cxcl12a (white), cxcr4b (red), merged images (white + red) and colocalized cxcl12a and cxcr4b transcripts in hypothalamic cells (white + red spots), with specific areas in boxes enlarged to more clearly illustrate the individual transcripts. (A, B) 0.5% EtOH-induced increase in number of cxcl12a transcripts compared to Control group is prevented by AMD3100 administration prior to 0.5% EtOH exposure, as shown in Boxes 1–42. Scale bars, 20 μm and 5 μm. (C, D) 0.5% EtOH-induced increase in number of cxcr4b transcripts compared to Control is prevented by AMD3100, as shown in Boxes 5–8. (E, F) 0.5% EtOH-induced increase in colocalization of cxcl12a with cxcr4b transcripts compared to Control group is prevented by AMD3100, as illustrated by merged images enlarged in Boxes 9–12and represented using Imaris spot labeling enlarged in Boxes 13–16. Abbreviation: v, ventricle. n = 3–6, **p < 0.01, ***p < 0.001, **p < 0.0001.

Cxcr4 antagonist AMD3100 blocks stimulatory effect of EtOH on Cxcr4 internalization in hypothalamic cells

Using live-imaging of cxcr4b:cxcr4b-Kate2-IRES-eGFP-CaaX zebrafish, we also tested if AMD3100 pretreatment blocks the EtOH-induced increase in Cxcr4b internalization in hypothalamic cells. A one-way ANOVA revealed a significant drug treatment effect on Cxcr4b internalization (F(3, 21) = 6.35, p = 0.003) and membrane-bound Cxcr4b cells (F(3,24) = 11.79, p < 0.0001). The multiple comparisons analysis showed 0.5% EtOH to significantly increase the internalization of these receptors compared to Control group (p = 0.02) and increase the number of membrane-bound Cxcr4b cells (p = 0.002). While AMD3100 alone had no effect in the Control+AMD3100 compared to Control group for both Cxcr4b internalization (p = 0.93) and Cxcr4b cells (p = 0.71), this receptor antagonist before 0.5% EtOH exposure significantly reduced this stimulatory effect of EtOH on Cxcr4b internalization (p = 0.007) and Cxcr4b cells (p = 0.0003) (Fig. 6A and B), as illustrated in the photomicrographs and Boxes 1–4 to the right (Fig. 6C). These findings further demonstrate the ability of AMD3100 treatment prior to EtOH exposure to prevent the stimulatory effects of 0.5% EtOH on the activity of the Cxcl12a/Cxcr4b system in cells of the embryonic hypothalamus.

Figure 6.

Effects of Cxcr4 antagonist AMD3100 on the stimulatory effects of 0.5% EtOH on the internalization of Cxcr4b in cells of the developing whole hypothalamus in live 26 hpf cxcr4b:cxcr4b-Kate2-IRES-eGFP-CaaX embryos. (A) EtOH-induced increase in internalization of Cxcr4b receptors compared to Control group is blocked by administration of AMD3100 prior to EtOH exposure. (B) EtOH-induced increase in membrane-bound Cxcr4b cells compared to control is blocked by administration of AMD3100 prior to EtOH exposure (C). These effects are illustrated in representative confocal images of the entire developing hypothalamus visually dissected using Imaris, with AMD3100 alone in Control as compared to the Control group (top row) producing no change in the internalization of Cxcr4b receptors (red) or number of hypothalamic cells expressing membrane-bound Cxcr4b (green), enlarged in Boxes 1 and 2, but AMD3100 with 0.5% EtOH compared to 0.5% EtOH group (bottom row) totally blocking the EtOH-induced increase in internalized Cxcr4b receptors and Cxcr4b cells, enlarged in Boxes 3 and 4. Scale bars, 10 μm and 5 μm. Abbreviation: V, ventricle. n = 4–10, *p < 0.05,**p < 0.01, ***p < 0.001.

Embryonic EtOH increases the number of differentiated neurons

We next tested by performing live-imaging of HuC:GFP zebrafish embryos if embryonic EtOH exposure at the low-moderate doses (0.1%, 0.25% and 0.5%) produces a change in the number of HuC-positive neurons in the developing hypothalamus, an indicator of neuronal differentiation. A one-way ANOVA revealed a significant effect of EtOH on the number of HuC neurons (F(3, 10) = 3.72, p = 0.003). At the 0.5% EtOH dose, there was a significant increase in the number of HuC neurons compared to the Control group (p = 0.049) (Fig. 7A). This effect is illustrated in the photomicrographs (Fig. 7B).

Figure 7.

Effects of embryonic EtOH exposure on the number of HuC neurons in whole hypothalamus of live 26 hpf HuC:GFP embryos. (A) EtOH at 0.5% compared to Control group increases the number of HuC-positive neurons. (B) This effect is illustrated in representative confocal images of HuC neurons in the entire hypothalamus visually dissected using Imaris, comparing 0.5% EtOH-treated embryo to Control embryo. Scale bar, 15 μm. Abbreviation: v, ventricle. n = 4–6, *p < 0.05.

Embryonic EtOH increases the colocalization of cxcl12a and cxcr4b within HuC neurons

To determine the degree of colocalization of cxcl12a and cxcr4b within these hypothalamic neurons, we next performed RNAscope staining of cxcl12a and cxcr4b transcripts in conjunction with HuC labeling using IF. A one-way ANOVA revealed a significant effect of EtOH on the colocalization with HuC of cxcl12a (F(3, 13) = 17.41, p < 0.0001) and cxcr4b (F(3, 13) = 34.7, p < 0.0001). The 0.5% EtOH dose significantly increased the density of HuC-positive neurons that contain cxcl12a transcripts (p < 0.0001) (Fig. 8A), as illustrated in the photomicrographs (Fig. 8B), and also of neurons that contain cxcr4b transcripts (p < 0.0001) (Fig. 8C), as shown in the images (Fig. 8D) and Boxes 1 and 2. Analysis using triple labeling of the colocalization of both cxcl12a and cxcr4b transcripts in HuC neurons also revealed a significant effect of EtOH (F(3, 13) = 11.92, p = 0.0005). The 0.5% doses significantly increased the density of HuC-labeled neurons that contain both the ligand and its receptor (p = 0.0004) (Fig. 8E), as illustrated in the photomicrographs (Fig. 8F). This result indicates that EtOH, in addition to stimulating the differentiation of HuC-labeled neurons, increases their expression of both cxcl12a and cxcr4b.

Figure 8.

Effects of embryonic EtOH on the number of cxcl12a and cxcr4b transcript puncta colocalizing in HuC neurons of 26 hpf AB embryos. These are shown in the bar graphs (left) and the representative confocal RNAscope and HuC IF images (right) of cxcl12a transcript puncta (white) and HuC neurons (green), cxcr4b transcript puncta (red) and HuC, merged images (white + red + green), and colocalized cxcl12a (white spots) and cxcr4b (red spots) transcripts in HuC neurons, with specific areas in white boxes enlarged to more clearly illustrate the neuronal location of individual transcripts. (A, B) 0.5% EtOH compared to Control group increases the number of cxcl12a transcripts colocalized within HuC neurons, shown enlarged in Boxes 1 and 2. Scale bars, 15 μm and 5 μm. (C, D) 0.5% EtOH compared to Control group increases the number of cxcr4b transcripts colocalized within HuC neurons, shown enlarged in Boxes 3 and 4. (E, F) 0.5% EtOH compared to control increases the colocalization of both cxcl12a and cxcr4b transcripts within HuC neurons, as illustrated by merged images with enlargements in Boxes 5 and 6 and represented using Imaris spot labeling enlarged in Boxes 7 and 8, as indicated by white arrows. Abbreviation: v, ventricle. n = 3–6, ***p < 0.001, ****p < 0.0001.

Cxcr4 antagonist AMD3100 blocks EtOH’s stimulatory effect on the number of differentiated neurons

With EtOH found to increase the number of differentiated neurons, we used live-imaging in HuC:GFP zebrafish to test if treatment with AMD3100 prior to 0.5% EtOH exposure affects EtOH’s stimulatory effect on HuC neurons in the developing hypothalamus. There was a significant effect of drug treatment on the number of HuC neurons (F(3, 10) = 3.721, p = 0.0496). The 0.5% EtOH dose produced a significant increase in HuC number compared to the Control group (p = 0.032). AMD3100 produced no effect of its own in the Control+AMD3100 compared to Control group (p = 0.88) and compared to Control+AMD3100 group (p = 0.006). The administration of AMD3100 prior to EtOH blocked the stimulatory effect of 0.5% EtOH on HuC number (p = 0.17) (Fig. 9A). These results, as illustrated in the photomicrographs (Fig. 9B), suggest that the activity of the Cxcr4 receptor in hypothalamic neurons is involved in the EtOH-induced increase in their number.

Figure 9.

Effect of Cxcr4 antagonist AMD3100 on the stimulatory effects of 0.5% EtOH on the number of HuC neurons in the developing hypothalamus of live HuC:GFP 26 hpf embryos. (A) The 0.5% EtOH-induced increase in number of HuC neurons is prevented by AMD3100 administration prior to EtOH exposure. (B) These effects are illustrated in representative confocal images of the entire developing hypothalamus visually dissected using Imaris, showing AMD3100 alone compared to Control group (top row) to produce no change in the number of HuC neurons but to prevent the EtOH-induced increase in HuC neurons as shown in the 0.5% EtOH+AMD3100 group compared to 0.5% EtOH group (bottom row). Scale bar, 15 μm. Abbreviation: v, ventricle. n = 4–7, *p < 0.05, **p < 0.01.

Cxcr4 antagonist AMD3100 blocks EtOH’s effect on cxcl12a and cxcr4b colocalization within HuC neurons

Here we further tested if AMD3100 prevents the stimulatory effects of 0.5% EtOH on the colocalization of both cxcl12a and cxcr4b transcripts within the hypothalamic HuC neurons. We observed a significant effect of drug treatment on cxcl12a colocalization within these neurons (F(3, 11 = 22.91, p < 0.0001). There was a greater degree of colocalization of cxcl12a with HuC in the 0.5% EtOH group compared to both the Control group (p < 0.0001) and the Control+AMD3100 group (p = 0.0005). There was no difference between the Control group and Control+AMD3100 group (p = 0.99) and significantly reduced colocalization in the 0.5% EtOH+AMD3100 compared to 0.5% EtOH group (p = 0.0002) (Fig. 10A), as illustrated in the photomicrographs (Fig. 10B). Further, there was also a significant effect of drug treatment on cxcr4b colocalization with HuC neurons (F(3, 11) = 6.46, p = 0.009). This reflected an increased colocalization of cxcr4b with HuC in the EtOH group compared to the Control group (p = 0.007) and the Control+AMD3100 (p = 0.037), no effect in the Control+AMD3100 compared to Control group (p = 0.99), and a significant reduction in the 0.5% EtOH+AMD3100 compared to 0.5% EtOH group (p = 0.036) (Fig. 10C), as shown in the images (Fig. 10D). Finally, there was a significant effect of drug treatment on the colocalization of HuC neurons with the cxcl12a and cxcr4b transcripts (F(3, 11) = 15.2, p = 0.0003). There was increased colocalization of both transcripts with HuC neurons in the 0.5% EtOH group compared to the Control group (p = 0.0005) and the Control+AMD3100 group (p = 0.006) and significantly reduced colocalization in the EtOH+AMD3100 compared to the EtOH group (p = 0.0004) (Fig. 10E). AMD3100 treatment alone had no effect in the control group (p = 0.99). This effect is shown in the images (Fig. 10F). These results indicate that the endogenous Cxcr4b receptor is required for the stimulatory effect of embryonic EtOH exposure on the colocalization of both cxcl12a and cxcr4b within differentiated neurons in the whole developing hypothalamus.

Figure 10.

Effect of Cxcr4 antagonist AMD3100 on the stimulatory effects of 0.5% EtOH on the colocalization of cxcl12a and cxcr4b transcripts in HuC neurons of the whole developing hypothalamus in 26 hpf embryos. This is shown in the bar graphs (left) and the representative confocal RNAscope and HuC IF images (right) of cxcl12a transcript puncta (white) and HuC neurons (green), cxcr4b transcript puncta (red) and HuC, merged images (white + red + green), and colocalized cxcl12a (white spots) and cxcr4b (red spots) transcripts in HuC neurons, with specific areas in white boxes to more clearly illustrate the neuronal location of individual transcripts. (A, B) 0.5% EtOH-induced increase in colocalization of cxcl12a transcripts in HuC neurons is prevented by administration of AMD3100 prior to 0.5% EtOH exposure, shown enlarged in Boxes 1–4. Scale bars, 15 μm and 5 μm. (C, D) 0.5% EtOH-induced increase in colocalization of cxcr4b transcripts in HuC neurons is prevented by AMD3100 administration, shown enlarged in Boxes 5–8. (E, F) 0.5% EtOH-induced increase in colocalization of both cxcl12a and cxcr4b transcripts in HuC neurons is prevented by AMD3100, illustrated by merged images with an enlargement in Boxes 9–12 and represented using Imaris spot labeling enlarged in Boxes 13–16 as indicated by white arrows. Abbreviation: v, ventricle. n = 3–6, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Discussion

There is strong evidence in human and animal studies showing EtOH exposure during adolescence and adulthood to increase cytokines, chemokines and their receptors in the periphery and brain (Crews et al., 2017, Vetreno and Crews, 2014). While there are few investigations in the embryo, our recent studies in the developing hypothalamus of the rat, consistent with the present findings in zebrafish, have demonstrated a stimulatory effect of prenatal EtOH exposure at a moderate dose on gene expression and protein levels of both Cxcl12 and Cxcr4 (Chang et al., 2020a), as shown for another chemokine Ccl2 and its main receptor Ccr2 (Chang et al., 2020b). Whereas there is little evidence in the brain revealing the effects of high EtOH doses on chemokine systems, there is one study measuring plasma Cxcl12a in mice showing increased levels at low doses and decreased levels at high doses (Gil-Bernabe et al., 2011), suggesting that higher doses may suppress this chemokine system. The present study is the first to reveal a stimulatory effect of external embryonic EtOH exposure on Cxcl12a and Cxcr4b in zebrafish. We demonstrate an increase in levels of cxcl12a and cxcr4b mRNA in the whole-embryo immediately after EtOH exposure at 26 hpf and also at a later developmental time point of 48 hpf, similar to our reports in rats showing EtOH at low doses to stimulate chemokine expression in both embryos and adolescents (Chang et al., 2018, Chang et al., 2020a). We also show here in the zebrafish an increase in the number of both cxcl12a and cxcr4b transcripts in the developing embryonic hypothalamus.

The additional finding that cxcl12a and cxcr4b colocalize within the same hypothalamic cells, as previously described in neurons, neuroprogenitor cells, and other cell types (Stumm et al., 2002, Chang et al., 2020a, Peng et al., 2007), indicates that these cells are not only a source of cxcl12a but also a target of Cxcl12a signaling. Cxcl12a acts in part through autocrine signaling on Cxcr4b receptors in the same cell, possibly leading to functional changes. A potential molecular mechanism involved in mediating this stimulatory effect of embryonic EtOH exposure is the transcription factor, hypoxia-inducible factor-1 alpha (HIF1α) (Ostrowski and Zhang, 2020), which is found to be upregulated by EtOH in the rat cortex (Reddy et al., 2013). This transcription factor in ischemic tissue stimulates cxcl12a expression that acts to guide Cxcr4b-expressing progenitor cells to the site of injury (Ceradini et al., 2004, Petit et al., 2007), and siRNA for HIF1α is shown to reduce cxcr4b expression (Ishikawa et al., 2009).

Our results further demonstrate that embryonic exposure to 0.5% EtOH, in addition to stimulating cxcl12a mRNA and transcript number, increases the internalization of Cxcr4b in hypothalamic cells, an effect indicating an increased signaling of Cxcl12a at this receptor (Venkiteswaran et al., 2013). The possibility that this stimulatory effect of EtOH on Cxcl12a signaling promotes local cell proliferation is supported by evidence that Cxcl12a administration dose-dependently increases in vitro the proliferation of neural progenitor cells (Wu et al., 2009, Gong et al., 2006) and the number of nerve sheath tumor cells, an effect reversed by an anti-Cxcl12 antibody and blocked in Cxcr4 deficient cells (Mo et al., 2013). Our present results further substantiate this possibility in the brain, showing that 0.5% EtOH in addition to increasing the number of HuC neurons stimulates the number of membrane-bound Cxcr4b cells in the developing hypothalamus and that this effect is blocked by pretreatment with a Cxcr4 antagonist. With increased Cxcr4b receptor internalization in zebrafish known to decrease receptor availability at the membrane (Venkiteswaran et al., 2013), evidence that genetic knockdown of Cxcr4b influences the migration and axonal projections of peptide neurons originating in the olfactory placode (Palevitch et al., 2010) suggests that the EtOH-induced increase in Cxcr4b receptor internalization may affect the migration of cells in the hypothalamus and their functional connectivity with other systems.

The involvement of the endogenous Cxcr4b receptor in mediating some of the EtOH’s actions in the developing hypothalamus is demonstrated by our additional finding that pretreatment with the antagonist AMD3100, which has a high degree of specificity for the Cxcr4 receptor (De Clercq, 2010), blocks the stimulatory effects of EtOH on chemokines and neurons tested in this study. These include the increased expression of cxcl12a and cxcr4b transcripts, colocalization of cxcl12a with cxcr4b in the same cell, and internalization of Cxcr4b receptors as a result of activation of the Cxcl12a ligand at its Cxcr4b receptor. The convergence of these findings leads us to speculate on possible downstream events that follow these EtOH-induced changes. One potential pathway that deserves attention is the extracellular signaling-regulated kinase (ERK) 1/2 pathway, which has an established role in controlling cell proliferation and differentiation (Cruz and Cruz, 2007, Shioda et al., 2009). The binding of autocrine Cxcl12 to its own Cxcr4 receptor is shown to activate ERK 1/2 pathways in esophageal cancer stem cells (Wang et al., 2017), and the administration of Cxcl12 increases the phosphorylation of ERK 1/2 in breast cancer cells, an effect blocked by AMD3100 (Hattermann et al., 2014). Further, ERK signaling in the rat nucleus accumbens is increased following acute exposure to 1 g/kg EtOH, while having no effect at 0.5 or 2 g/kg (Rosas et al., 2014), and prenatal ethanol exposure at high concentrations suppresses ERK signaling in the rat thalamus and mouse hippocampus (Mooney and Miller, 2011, Samudio-Ruiz et al., 2009). Collectively, these results suggest that EtOH’s effects on ERK signaling in the brain, strongly dependent on dose, is a critical mechanism underlying the influence of EtOH on chemokine signaling and neuronal differentiation in the developing brain. Future studies are needed to elucidate such downstream signaling pathways of the Cxcl12/Cxcr4 axis that are essential in mediating these effects of low-dose EtOH, and also to determine the possible involvement of another Cxcl12 receptor, Cxcr7, which functions in maintaining a concentration gradient of Cxcl12 to provide directional cues for migrating cells (Tobia et al., 2019, Bussmann and Raz, 2015).

Along with these stimulatory effects of embryonic EtOH exposure on the Cxcl12a/Cxcr4b system, we demonstrate here that EtOH, at low doses (0.1%−0.5%) and for a short 2-hour exposure period (22–24 hpf) when the zebrafish hypothalamus is developing, significantly increases the number of differentiated neurons labeled with HuC, an effect opposite to the decreased proliferation of differentiated neurons (Joya et al., 2014) and neural progenitors (Yin et al., 2014) and the increased apoptosis induced by the higher EtOH doses (e.g., 0.75%−2.0%) and/or longer exposure periods (e.g., 0–24 hpf) (Ramlan et al., 2017). This stimulatory effect on the number of differentiated neurons in whole zebrafish hypothalamus at 26 hpf is in agreement with our previous findings, showing embryonic EtOH exposure at low-moderate doses for brief periods to stimulate hypothalamic neurogenesis, as indicated by an increased density of both HuC neurons co-labeling BrdU in 5 dpf zebrafish (Sterling et al., 2016) and mature neurons co-labeling BrdU in postnatal rats (Chang et al., 2012, Chang et al., 2015). While the effect of AMD3100 on neuronal proliferation stimulated by EtOH has yet to be tested, this receptor antagonist has been shown in rats to inhibit increased neurogenesis induced by other stimuli, an enriched environment (Zhou et al., 2017) and ischemia (Zhao et al., 2015). Together, this evidence provides strong support for the stimulatory effects of EtOH at lower doses, in contrast to higher doses, on the number of differentiated neurons.

Our further observation that EtOH increases the transcript expression of cxcl12a and cxcr4b in these HuC-labeled neurons provides evidence that increased autocrine chemokine signaling is involved in stimulating the number of differentiated neurons, with further studies needed to provide direct support for the role of this chemokine system in mediating this effect of EtOH. An example of autocrine Cxcl12/Cxcr4 signaling modulating neuronal function is provided by evidence that both Cxcl12 and Cxcr4 protein colocalize in peptide-expressing hypothalamic neurons and ventricular injection of Cxcl12 acts via Cxcr4 to alter their electrical activity (Callewaere et al., 2006). In adult rats, we have shown intracerebroventricular injection of Cxcl12 to increase the genesis and proliferation of hypothalamic neurons that express neuropeptides (Poon, 2016). Further, with prenatal exposure, we have also demonstrated in the rat a stimulatory effect of Cxcl12 on hypothalamic neurogenesis (Poon et al., 2017) and also of EtOH administration on the Ccl2 colocalization with its receptor Ccr2, an effect blocked by a Ccr2 antagonist (Chang et al., 2018, Chang et al., 2020a), and on the density of BLBP-labeled radial glia progenitor cells and their co-expression of Cxcr4 in the neuroepithelium, the primary source of hypothalamic neurons (Chang et al., 2020a). With EtOH shown to alter chemokine expression shortly after EtOH exposure (Neupane et al., 2016, Yang et al., 2000) and with chemokines known to stimulate neuroprogenitor cell proliferation (Wu et al., 2009) and both cxcr4b and cxcl12a shown to be expressed within zebrafish radial glia progenitor cells (Palevitch et al., 2010), this evidence provides a possible mechanistic link between ethanol stimulation of chemokine activity in radial glia progenitor cells and the number of differentiated neurons observed shortly after ethanol exposure. Our additional findings here in zebrafish, that AMD3100 blocks EtOH’s stimulatory effects in the embryo on the number of HuC neurons and their transcription and colocalization of cxcl12a with cxcr4b receptors, and the finding in rats, that AMD3100 blocks the stimulatory effect of CXCL12 treatment on neurogenesis (Mao et al., 2020), further support the idea that Cxcl12a activity at its Cxcr4b receptor, possibly acting through autocrine signaling and a positive-feedback loop, has an important role in mediating the increased number of differentiated neurons induced by embryonic EtOH exposure.

In conclusion, these results provide new information on how embryonic EtOH exposure, at low-moderate doses during a period of critical hypothalamic development, stimulates the Cxcl12/Cxcr4 axis in conjunction with increased neuronal number in the embryo and additionally how these effects of EtOH do, in fact, require the functional activity of Cxcl12 at its Cxcr4 receptor. With embryonic exposure to EtOH found to increase neuronal density at later ages in both zebrafish (Sterling et al., 2016) and rats (Chang et al., 2012), it is likely that these effects are long-lasting and that they persist into later ages when they may contribute to disturbances in alcohol-related behaviors also produced by early EtOH exposure.