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RESEARCH ARTICLE

Molecular Reproduction & Development 82:365–376 (2015)

Changes in the Osmolarity of the EmbryonicMicroenvironment Induce Neural Tube Defects

YI-MEI JIN,1 GUANG WANG,1 NUAN ZHANG,1 YI-FAN WEI,1 SHUAI LI,1 YOU-PENG CHEN,2 MANLI CHUAI,3

HENRY SIU SUM LEE,5 BERTHOLD HOCHER2,4, AND XUESONG YANG1*

1 KeyLaboratory forRegenerativeMedicineof theMinistry of Education,Division ofHistology andEmbryology,MedicalCollege,Jinan University, Guangzhou, China

2 Department of Neonates, The First Affiliated Hospital of Jinan University, Guangzhou, China3 Division of Cell and Developmental Biology, University of Dundee, Dundee, United Kingdom4 Humboldt University of Berlin, University Hospital Charite, Center for Cardiovascular Research & Institute for Pharmacology,Berlin, Germany

5 Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom

SUMMARY

Many maternal disorders that modify the embryonic microenvironment, such as achange in osmolarity, can affect development, but how these changes influence theearly embryo remains obscure. Neural tube defects, for example, are commoncongenital disorders found in fetus and neonates. In this study, we investigatedthe impact of anisotonic osmolarity (unequal osmotic pressures) on neural tubedevelopment in the early chick embryo, finding that neuronal cell differentiation wasimpaired in the neural tube due to enhanced apoptosis and repressed cell prolifera-tion. Anisotonic osmolarity also affected normal development of the neural crest,which in turn influenced abnormal development of the neural tube. As neural tubedevelopment is highly dependent on the proper expression of bone morphogeneticprotein 4 (BMP4), paired box 7 (PAX7), and sonic hedgehog (SHH) genes in thedorsal and ventral regions along the tube, we investigated the impact of anisotonicosmolarity on their expression. Indeed, small changes in osmolarity could positivelyand negatively impact the expression of these regulatory genes, which profoundlyaffected neural tube development. Thus, both the central and peripheral nervoussystems were perturbed by anisotonic consitions as a consequence of the abnormalexpression of key genes within the developing neural tube.

Mol. Reprod. Dev. 82: 365�376, 2015. � 2015 Wiley Periodicals, Inc.

Received 3 October 2014; Accepted 24 March 2015

�Corresponding author:Department of Histology andEmbryologySchool of MedicineJinan UniversityNo.601 Huangpu Road WestGuangzhou 510632, China.E-mail: [email protected]

Yi-mei Jin and Guang Wang contributedequally to this work.

Grant sponsor: NSFC; Grant numbers:31401230, 31071054, 30971493;Grant sponsor: China PostdoctoralScience Foundation; Grant number:2014M560694; Grant sponsor:Guangdong Natural ScienceFoundation; Grant numbers:S2013010013392, S2011010001593;Grant sponsor: FundamentalResearch Funds for the CentralUniversities; Grant number: 21614319;Grant sponsor: Students ResearchTraining Program Fund;Grant numbers: CX14108,1210559035, 201310559063

Published online 14 April 2015 in Wiley Online Library(wileyonlinelibrary.com).DOI 10.1002/mrd.22482

Abbreviations: BMP [4], bone morphogenetic protein [4]; BrdU, bromodeox-yuridine; CCND1, cyclin D1; CDKN1, cyclin-dependent kinase inhibitor 1A/p21;

HNK1, human natural killer 1; PAX6/7, paired box 6 or 7; pHIS3, phosphorylatedhistone H3; SHH, sonic hedgehog; SNAI2, snail family zinc finger 2; TUNEL,terminal deoxynucleotidyl transferase dUTP nick-end labeling

� 2015 WILEY PERIODICALS, INC.

INTRODUCTION

Early-stage developing embryos are sensitive tochanges in their external environment, including the pres-ence of teratogens, chemical toxins, and even imbalancesin osmolality. Maternal disorders, such as diabetes insip-idus and dietary deficiencies during pregnancies, are alsocontributing factors that impact the embryonic environ-ment. Diabetes insipidus, for example, is a disorder char-acterized by the manifestation of polyuria and polydipsia. Itcan occur transiently during pregnancy, and is associatedwith acute fatty liver, pre-eclampsia, HELLP (hemolysis,elevated liver enzymes, low platelet count) syndrome, andmultiple pregnancies (Sherer et al., 2003). Profound hyper-natremia (electrolyte disturbance associated with in-creased sodium concentration in the blood) is a typicalmanifestation of diabetes insipidus, which can lead to thedevelopment of congenital holoprosencephaly (Vaqaret al., 2012), resulting in the failure of normal prosenceph-alon formation and thus the absence of two cerebral hemi-spheres. Chronic renal disease during pregnancy isanother source of anisotonic osmolarity. The progressiveloss of renal function illicits a number of compensatoryresponses that lead to hypernatremia and hyponatremia(Alcazar-Arroyo, 2008).

Anisotonic osmolarity can be transmitted from themoth-er to the fetus as its manifestation can depend on thevolume and hypotonicity of maternal plasma. Chronic hy-pertonicity, on the other hand,would induceadirected fetal-to-maternal flow of water that causes oligohydramnios,which in turn affects fetal development and survival (Shereret al., 2003). Indeed, post-term rats posses abnormal watertransfer, presumably due to anisotonic conditions in utero,while infants with hydranencephaly and chronic hyperna-tremia possess a disrupted thirst response and anti-diuretichormone (ADH) secretion (Endo et al., 1990), whichmarkedly elevates their plasma osmolarity. Additional stud-ies have found that holoprosencephaly and hydranence-phaly result from neural tube defects, but the mechanismunderlying this phenotype has not yet been established(Mitchell, 2005).

Neurulation is themorphogenetic process that forms theneural tube in vertebrate embryos, which ultimately devel-ops into the central nervous system, forming the rhomben-cephalon, mesencephalon, and prosencephalon in thecranial region and the spinal cord in the trunk. This processis initiated in the tri-laminar embryo by the proliferation anddifferentiation of neuroepithelial cells to form a neural plate.When cells along the edges of the neural plate proliferate,the sides of the plate begin to elevate and form neural foldsthat eventually bend and fuse together at the dorsal midlineto form a fluid-filled neural tube. Neural tube closure furtherrequires highly coordinated, complex, and dynamic mor-phological changes in the neuroepithelial cells that specifi-cally involve cell elongation and apical constriction(Karfunkel, 1974; Suzuki et al., 2012). Normal neuraltube development also requires the tight integration ofseveral important cellular processes, including cell prolifer-ation, apoptosis, and differentiation (Copp and Greene,

2010). Genetic mutations and factors that disrupt thesekey morphogenetic processes often result in neural tubedefects in neonates; indeed, the incidence of human neuraltube defects is reported at approximately 0.5�2 per 1,000pregnancies (Mitchell, 2005) and are mainly associatedwith abnormal development and failure to completely closethe neural tube (Copp and Greene, 2010).

Chick embryos can easily be exposed to salt solutions,making them goodmodels for analyzing the effects of hyper-osmolarity on development. The egg cannot excrete salt toregulate osmolarity, so hyper-osmolarity is maintainedthroughoutdevelopment; in contrast, placental animalsutilizethe renin�angiotensin system to regulate embryonic expo-sure to osmolarity via the blood circulation. In our previousstudy, we demonstrated that chick embryos exposed toexcess salt disrupts PAX6 expression, leading to impairedretina and lens development (Chenet al., 2014). In this study,we examined the effects of anisotonic osmolarity on neuraltube development and gene expression in chick embryos.

RESULTS

Anisotonic Osmolarity Induces Neural TubeDysplasia

Closure of the neural folds is fundamental to neural tubeformation. This process is dependent on precise gene-to-gene and gene-to-microenvironment interactions; any dis-turbance to these interactions has the potential to disruptneural tube closure, the most common congenital defectsreported in human fetuses (Copp and Greene, 2010). Herewe examined the influence of osmolarity on neurulationby exposing chick embryos to various osmolarities(240mosm/l as isotonic, 230mosm/l as hypotonic, and280mosm/l as hypertonic).

Embryos exposed to hypertonic and hypotonic osmolari-ties were grossly smaller than isotonic (control) embryos(Fig. 1A�C). The average weight of the anisotonic osmolari-ty-treated embryos was significantly reduced compared tocontrol embryos (control¼ 0.11� 0.02g, n¼ 5; 280mosm/l¼ 0.08� 0.01g, n¼ 7; 230mosm/l¼ 0.09� 0.01g, n¼ 6)(Fig. 1D). There was a higher incidence of mortality in thehypertonic than the hypotonic treatment groups (280mosm/l¼ 87%,n¼ 47;230mosm/l¼ 62%,n¼ 50) (Fig. 1E),where-as significantly more neural tube defects were observed inembryos exposed to hypotonic (230mosm/l¼ 75%, n¼ 40)(Figs. 1C,F) versus hypertonic (280mosm/l¼ 33%, n¼ 40)(Figs 1B,F) conditions. The neural folds in the hypertonic-treatedembryoswereable to elevate and fuse together at thedorsal midline, although the neural tubes that formed ap-peared morphologically abnormal (Figs. 1B) compared withcontrol embryos (Figs. 1A).

Anisotonic Osmolarity Disrupts F-ActinExpression in the Neural Tube

Wenext explored how osmolarity influenced neural tubeclosure by asking whether the cytoskeleton or cell-to-cell

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interactions in early-stage chick embryo were affected.Experimental embryos were fixed and stained for F-actinusing phalloidin-Alexa Fluor 488 (Fig. 2).We first examinedneural tube morphology in HH10 chick embryos, the Ham-burger and Hamilton stage at which embryos were firstexposed to the anisotonic osmolarity conditions. At somitelevel 1�6 of stage HH10 control embryos, the neural foldshave almost completely fused to form a closed neuraltube (Fig. 2A). Phalloidin staining showed F-actin highlyenriched in the apical surface of the neural tube(Fig. 2A2�A3). The effects of osmolality were then evalu-ated 6 hr after the initial treatment. In the isotonic controlgroup, the lumen of the neural tubes was smaller than inHH10 control embryos. F-Actin was also more-enriched atthe apical surface of the neural tube in these isotonicembryos compared to the HH10 control embryos(Fig. 2B2�B3). On the other hand, neural tube closure(white arrows) was disrupted in both hypertonic and hypo-tonic embryos (n¼ 5 or 6 for each group) (Figs. 2C,D) while

the intensity of F-actin staining was weaker in the neuraltubes of both hypertonic- and hypotonic-treated embryos(Fig. 2C2�C3 and D2�D3, respectively) than in isotonic-treated embryos (Fig. 2B2�B3).

Anisotonic Osmolarity Inhibits Cell Proliferationand Enhances Apoptosis

The neural tube relies on highly regulated cell prolifera-tion and apoptosis to develop normally. We therefore usedphosphorylated histone H3 (pHIS3) as a cell-proliferationmarker to investigate the extent of cell division in chickembryos exposed to isotonic, hypotonic, and hypertonicosmolarities for 24 hr (Fig. 3). Fewer pHIS3-positive cellswere present in the neural tube exposed to both hypertonic(Fig. 3B) and hypotonic conditions (Fig. 3C) compared tothe isotonic control group (Fig. 3A) (control¼ 15.40� 2.91;280mosm/l¼ 11.50� 2.00; 230mosm/l¼ 10.30� 2.42;n¼ 10 for each group) (Fig. 3G). To address the extent

Figure 1. Anisotonic osmolarity inducesneural tubedysplasia in developingchick embryos.Chick embryoswereexposed for 3 days to 240mosm/l(isotonic control), 230 mosm/l (hypotonic), and 280 mosm/l (hypertonic) osmolarities, produced by injection of NaCl solutions into the egg. A-C:Representative appearance of 4.5-day-old whole-mount chick embryos after isotonic (A), hypertonic (B), and hypotonic (C) treatments. A1-C1:High-magnification images showing the neural tube regions indicated by white arrows in A-C, respectively. A2/3-C2/3: Transverse sectionsshowing the morphology of the neural tube (levels indicated by white dashed lines in A-C) stained with hematoxylin & eosin. Black arrows indicatethe dorsal midline where the bilateral neural folds normally meet and fuse. D-F: Bar charts showing the embryos’ average body weight (D),percentage of embryo mortality (E), and frequency of neural tube defects (NTD) (F) after exposure to different osmolarities. *, P<0.05 and **,P<0.01 between experimental and control embryos. Scale bars, 2000mm (A-C), 500mm (A1-C1), and 400mm (A2-C2).

CHANGES IN THE OSMOLARITY INDUCE MALFORMATIONS

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of cell death in the neural tube following anisotonic osmo-larity treatment, we employed the TUNEL (terminal deox-ynucleotidyl transferase dUTP nick-end labeling) assay.Significantlymore apoptotic cellswere present in the neuraltubes of hypertonic- and hypotonic-treated embryos(Fig. 3E,F) than in isotonic control embryos (Fig 3D) (con-trol¼ 0.04� 0.02; 280mosm/l¼ 0.26� 0.05; 230mosm/l¼ 0.27� 0.06; n¼ 6 for each group) (Fig. 3H).

We next assessed the events that lead to the neural tubedefects in later-stage chick embryos. Bromodeoxyuridine(BrdU) incorporation was assessed to establish whether ornot cell proliferation was disrupted. We found that both hyper-tonic and hypotonic treatments significantly reduced the num-berofBrdU-positive cells present in thedevelopingneural tube(Fig. 4B,C) as compared to the control (Fig 4A) (control¼ 0.23� 0.02; 280mosm/l¼ 0.16� 0.02; 230mosm/l¼ 0.05� 0.01;

Figure 2. Anisotonic osmolarity disrupts neural tube closure and F-actin abundance. Chick embryos were exposed to isotonic, hypotonic,and hypertonic conditions for 6 hours, and then collected for phalloidin-Alexa Fluor 488 staining. A: Representative untreated HH10 embryoshowing that neural folds have almost closed at this stage.A1:Magnified view of the region indicated by dashed red box in ‘A’.A2-A3: Transversesection of the neural tube at the position indicatedby the dashed line in ‘A1’.B-D:Representativemorphology of neural tubeandF-Actin distributionin embryos after isotonic (B), hypertonic (C), and hypotonic (D) treatment. B1-D1: Areas indicated by dashed red boxes in ‘B’ through ‘D’,respectively.B2/3-D2/3:Transverse sections of the neural tubeat the position indicated by the dashed lines in ‘B1’ through ‘D1’, respectively. Scalebars, 500mm (A-D); 200mm (A1-D1); 50mm (A2/3-D2/3).


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n¼ 6 for each group) (Fig. 4G). Semi-quantitative reverse-transcriptase PCR analysis revealed that P21/cyclin-dependent kinase inhibitor 1A (CDKN1A) expressionwas upregulated, whereas cyclin D1 (CCND1) expressionwas down-regulated following anisotonic osmolaritytreatment (CDKN1A/GAPDH: control¼ 0.14� 0.005, n¼ 3;280mosm/l¼ 1.19� 0.06, n¼ 4; 230mosm/l¼ 1.21� 0.04,n¼ 4 | CCND1/GAPDH: control¼ 1.23� 0.02, n¼ 3;280mosm/l¼ 0.28� 0.01, n¼ 4; 230mosm/l¼ 0.28� 0.01,n¼ 4) (Fig. 4I). We also employed the TUNEL assay to deter-mine the extent of apoptosis in the neural tubes of these older,treated embryos. Significantly more apoptotic cells were pres-ent in the neural tubes of hypertonic- and hypotonic-treatedembryos (Fig. 4E,F) than in control embryos (Fig 4D) (control¼ 0.02� 0.01; 280mosm/l¼ 0.29� 0.04; 230mosm/l¼ 0.27� 0.04; n¼ 6 for each group) (Fig 4H).

Anisotonic Osmolarity Alters Gene Expression inthe Neural Tube

As normal neural tube development relies on pattern-determining genes to be correctly expressed in dorsal and

ventral region of the tube (Wilson and Maden, 2005), weused immunofluorescence staining and reverse-transcrip-tase PCR to investigate if regional expression of thesegenes was affected by anisotonic osmolarity. The spatio-temporal expression pattern of paired-box 7 (PAX7) wasexamined in 2.5- and 4.5-day-old embryos, specifically attransverse sections of the neural tube at the same level inthe embryos (white arrows) after treatment (Fig. 5). Immu-nofluorescence staining revealed that PAX7 was normallyexpressed in the dorsal neural tube, but this was signifi-cantly inhibited along all levels of the neural tube whenembryos were exposed to hypertonic and hypotonic osmo-larities (Fig. 5D�R). Bone morphogenic protein 4 (BMP4)signaling also plays an important role in specifying how thedorsal neural tube differentiates. Using semi-quantitativereverse-transcriptase PCR, we found that both hypertonicand hypotonic treatments inhibitedBMP4 expression in theneural tubes of 4.5-day-old embryos (control¼ 1.20� 0.01;280mosm/l¼ 0.44� 0.004; 230mosm/l¼ 0.36� 0.01;n¼ 3 for each group) (Fig. 5C). Furthermore, reverse-transcriptase PCR analysis of PAX7 confirmed the PAX7immunofluorescence results (control¼ 1.23� 0.01;

Figure 3. Cell proliferation and apoptosis in the developing neural tube are perturbed by anisotonic osmolarity in HH14 chick embryo.Phosphorylated histone H3 (pHIS3) immunofluorescence and TUNEL staining were used to determine the extent of cell proliferation and celldeath, respectively, in neural tubesexposed to isotonic, hypotonic, andhypertonicosmolarities inHH14chickembryos.A-C:DAPI stainingshowingthemorphologyof neural tubesafter isotonic (A), hypertonic (B), andhypotonic (C) treatment.A1-C1:pHIS3 immunofluorescence stainingshowingthe extent of cell proliferation in the neural tube after isotonic (A1), hypertonic (B1), and hypotonic (C1) treatments.A2-C2:merged images of DAPIand pHIS3 staining. D-F: TUNEL staining showing the extent of apoptosis in the neural tubes after isotonic (D), hypertonic (E), and hypotonic (F)treatment.G-H:Bar charts showing thenumber of pHIS3-positive cells (G) and ratio of TUNEL-positive cells present in the control andexperimentalneural tubes (H). **, P<0.01 and ***, P<0.001 between experimental and control embryos. Scale bars, 50mm (A-C), 20mm (D-F).


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280mosm/l¼ 1.05� 0.04; 230mosm/l¼ 0.22� 0.02; n¼ 3for each group) (Fig. 5C).

Sonic hedgehog (SHH) signaling is known to play a keyrole in specifying the differentiation of the ventral neuraltube.Using in situ hybridization, weobserved thatSHHwasexpressed in the notochord and the ventral neural tube inHH14 chick embryo (n¼ 6/6 embryos; Fig. 6A). Both hy-pertonic (n¼ 5/8 embryos; Fig. 6B) and hypotonic (n¼ 7/10embryos; Fig. 6C) treatment dramatically enhanced SHHtranscription in both the notochord and ventral neural tubecompared to isotonic treatment.

Anisotonic Osmolarity Disrupts Neural TubeDifferentiation

During neurogenesis, neural precursor cells migratefrom the luminal to the apical side of the neural tube, wherethey differentiate into neurons. These neurons thenextend processes out from the neural tube toward theirfated destination in the embryo via axonal guidance. We

used antibodies raised against neurofilament to visualizethe neuronal cells and evaluate their ability to differentiateunder hypertonic and hypotonic conditions. Based on neu-rofilament immunofluorescence staining, the trunk of neu-ral tubes do not close properly following either aniosotonicosmolarity treament (Fig. 7B,C) compared to control em-bryos (Fig. 7A). In addition, the intensity of neurofilamentstaining in these anisotonic osmolarity-treated embryoswas weaker on the ventral horns than corresponding siteson the control neural tubes, and their dorsal root gangliawere also smaller thannormal (Figs. 7A�C).Under isotonicconditions, dorsal root ganglia strongly expressed HNK1 (amarker for migrating neural crest cells) (Fig. 7D), whereasits abundance was significantly reduced in anisotonic os-molarity-treated embryos (Figs. 7E,F).

We also examined paired-box 6 (PAX6) expression intheneural tube since this gene is involved inmaintaining theproliferation and commitment of neural progenitor cells(Bel-Vialar et al., 2007). Semi-quantitative reverse-tran-scriptase PCR analysis determined that PAX6 was less

Figure 4. Cell proliferation and apoptosis in the developing neural tube are perturbed by anisotonic osmolarity in 4.5-day-old chick embryo. A-C:Representative transverse sections of the neural tube, showing the extent of BrdU incorporation (a marker of cell proliferation) after 3 days ofisotonic (A), hypertonic (B), and hypotonic (C) treatment.A1/2-C1/2:High-magnification images of regions indicated by dashed white boxes in ‘A’through ‘C’.D-F:TUNELstaining indicating theextent of cell death (brownnuclei) in the neural tubesof embryos.D1-F1:Highermagnification of theneural tube indicated by the dashedblack boxes in ‘D’ through ‘F’.G-H: Bar charts showing the ratios of BrdU-positive cells (G) andTUNEL-positivecells (H) present in control and experimental neural tubes. The results indicate that anisotonic osmolarity inhibits cell proliferation while enhancingcell death. I: Semi-quantitative reverse-transcriptase-PCR analyses showing the effects of anisotonic osmolarity on CDNK1A and CCND1expression. ***, P<0.001 between experimental and control embryos. Scale bars, 400mm (A-F), 20mm (A1/2-C1/2), and 20mm (D1-F1).


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abundant in both hypertonic- and hypotonic-treated embry-os than isotonic control embryos (control¼ 1.46� 0.05;280mosm/l¼ 0.17� 0.01; 230mosm/l¼ 0.15� 0.02;n¼ 3 for each group) (Fig. 7G). Conversely, Snail2(SNAI2) expression, amarker of pre-migratory neural crestcells, was not affected in either hypertonic or hypotonicosmolarity conditions (control¼ 1.18� 0.002; 280mosm/l¼ 1.19� 0.02; 230mosm/l¼ 1.04� 0.04; n¼ 3 for eachgroup) (Fig. 7G). By in situ RNA hybridization usingSNAI2-specific probes, we also found that despite expos-ing HH10 embryos to anisotonic osmolarity for 24 hr��wherein closure of the tubes was clearly affected inboth hypertonic and hypotonic conditions��SNAI2 expres-sion was the same in both hypertonic- (n¼ 4/5 embryos)

and hypotonic- (n¼ 4/6 embryos) treated embryos com-pared with isotonic control embryos (Fig. S1).

DISCUSSION

Using the early chick embryo, we demonstrated thataltering theosmolarity of theembryomicroenvironment caninduce neural tube defects. We specifically reported thatchanges in osmolarity inhibited neural tube malformationand neural crest cell migration. We have diagrammaticallyillustrated the possible mechanisms involved in Figure 8.

We selected HH10 chick embryos as the starting pointfor our anisotonic osmolarity treatment because the neural

Figure 5. Anisotonic osmolarity suppresses PAX7 and BMP4 expression. A-B: Drawings of 2.5- (A) and 4.5- (B) day-old chick embryos, withdashed lines indicating where the embryos have been sectioned. C: Semi-quantitative reverse-transcriptase-PCR results of 4.5-day-old embryosshowing the effects of anisotonic osmolarity on BMP4 and PAX7 expression. D-L: Transverse sections of 2.5-day-old neural tubes sectioned atlevels indicatedby the dashed lines in ‘A’ (A1-A3). Immunofluorescencestaining indicated that anisotonic osmolarity repressesPAX7abundance intheneural tube (outlinedbywhite dotted lines).M-R:Transversesectionsof 4.5-day-old embryossectionedat levels indicatedby thedashed lines in‘B’ (B1-B2). The immunofluorescence staining revealed the extent of PAX7 abundance in neural tubes after anisotonic and isotonic (control)treatments. Sectionswere counterstained with DAPI. *,P<0.05 and ***,P<0.001 between experimental and control embryos. Scale bar, 400mm(D-R).


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tube is almost completely closedat somite levels 1�6at thisstage of development. F-actin is highly enriched at theapical surface of the normal neural tube,which is consistentwith the observation that F-actin expression increases asthe apical region of cells became increasingly constrictedduring neural-fold closure (Suzuki et al., 2012). Neural tubedefects began to appear in both hypertonic and hypotonicgroups within 6 hr, and corresponded with irregular F-actindistribution in the neuroepithelium compared to the isotoniccontrol group. This implies that altered F-actin distributionmight be involved in the abnormal closure of the neuraltubes under anisotonic osmolarity conditions.

Cell proliferation and death play an important role in thedeveloping neural tube (Wei et al., 2012). Using pHIS3immunofluorescence, BrdU incorporation, and TUNEL as-says, we established that cell proliferation was inhibitedwhile apoptosis was enhanced after anisotonic osmolalitytreatement. Indeed, consistent with reports that P21/CDKN1A inhibits neural progenitor cell proliferation and

differentiation (Ribes and Briscoe, 2009) while cyclin D1promotes their growth (Bizen et al., 2013), we found thattheir respective genes were abnormally expressed underanisotonic osmolarity conditions. Therefore, mis-expres-sion of key cell-cycle-regulating genes may be an underly-ing cause of the abnormal development of the neural tubefollowing anisotronic osmolarity treatment.

SHH and PAX signaling play vital roles in dorsal-ventralpatterning of the neural tube. SHH is an important morpho-gen that has also been reported to inhibit local cell prolifer-ation (Wilson and Maden, 2005). It is secreted by thenotochord, and diffuses extracellularly toward the ventralneural tube to induce formation of the floor plate (Ribes andBriscoe, 2009). SHH establishes a dorsal�ventral gradientwithin the neural tube that then regulates the expression ofthePAXhomeoprotein family of transcription factors (Ribesand Briscoe, 2009). For example, low concentrations ofSHH induce cadherin-7 (CAD7) whereas high concentra-tions repress CAD7 expression. CAD7 controls axon

Figure 6. Anisotonic osmolarity upregulates SHH expression in the developing neural tube of HH14 chick embryos. Chick embryoswere exposedto isotonic, hypotonic, and hypertonic conditions for 1 day, and then collected for whole-mount in situ hybridization. A-C: Representative SHHexpression pattern in the neural tubes of embryos after isotonic (A), hypertonic (B), and hypotonic (C) treatment.A1-C1:Transverse sections of theneural tubes at positions indicated by the dotted lines in ‘A’ through ‘C’. Scale bars, 200mm (A-C) and 50mm (A1-C1).


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elongation and formation of the neural circuit, which couldbe inhibited by PAX7 (Luo et al., 2006). PAX6, on the otherhand, is important for regulating the differentiation andmigration of oligodendrocyte precursor cells (Di Lulloet al., 2011). In this study, we noted that anisotonic osmo-larity reduced PAX7 expression dorsally, while SHH ex-pression was upregulated in the ventral neural tube.Furthermore, PAX6 expression was reduced. Together,these changes in gene expression imply that anisotonicosmolarity-induced neural tube malformation was directlyor indirectly acting through altered SHH, PAX6, and PAX7-dependent regulation of neural differentiation and cellmigration..

SHH is not the sole factor regulating the developmentalfate of the neural tube; other molecules such as BMPsdynamically control the apical-basal polarity of neuroepi-thelial cells and help to bend, shape, and close the neuraltube (Eom et al., 2013). While SHH signaling governs theventral aspect of neural tube formation, BMP4 signaling ispredominant in the dorsal aspect (Wilson and Maden,2005). These signaling molecules together dictate thesubsequent differentiation and dorsal and ventral migrationof HNK1-positive neural crest cells (Lallier et al., 1992).We

therefore studied SNAI2 and HNK1 expression, whichrespectively identify pre-migratory and migrating cohortsof neural crest cells (Bannermanet al., 1998;Del Barrio andNieto, 2004). Following anisotonic osmolarity treatment,only HNK1 abundance was disrupted, which is consistentwith the failure of neural crest cells to migrate and establishthe dorsal root ganglia.

Normal neural tube development requires a tightlycoordinated balance between neuroepithelial cell prolifer-ation and differentiation. As reported here, osmoticchanges to the embryo’s microenvironment can readilyoffset this balance, causing the neural tube to developabnormally��although it is also worth noting that embryomortality also increased, especially following hypotonictreatments. Indeed, changes in osmolarity affected cellshape, proliferation, apoptosis, and differentiation in theneural tube. Interestingly, we also discovered thatthe cardiovascular system was correspondingly affected(unpublished). In human embryos, neural tube defect canresult in holoprosencephaly and hydranencephaly (Vaqaret al., 2012). The phenotypes generated from our chickmodel largely mimic these clinical congenital abnormali-ties, making this an ideal model to understand how

Figure 7. Anisotonic osmolarity impairs neural tube differentiation. Immunofluorescence and semi-quantitative reverse-transcriptase-PCRanalyses of chick embryos exposed to isotonic, hypotonic, and hypertonic microenvironments for 3 days. A-C: Transverse sections of neuraltubes stained for neurofilament expression in embryos after isotonic (A), hypertonic (B), and hypotonic (C) treatment. A1-C1: Neurofilament (NF)staining in sections (red) counterstained with DAPI (blue).D-F: Transverse sections of the neural tubes stained for HNK1 (red) and counterstainedwith DAPI (blue). G: Semi-quantitative reverse-transcriptase-PCR analysis showing the effects of various osmolarity treatments on SNAI2 andPAX6 expression. **, P<0.01 and ***, P<0.001 between experimental and control embryos. DRG, dorsal root ganglion. Scale bar, 400mm.


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microenvironmental osmolarity can impact vertebratedevelopment.

MATERIALS AND METHODS

Chick EmbryosFertilized chick eggswere obtained from theAvian Farm

of the South China Agriculture University. The eggs wereincubated in a humidified incubator (Yiheng Instrument,Shanghai, China) set at 38 8Cand70%humidity. After 36 hrof incubation, most of the chicks reached developmentalstage HH10. Two milliliters of albumen were then removedfrom the blunt end of the egg containing the embryos,and different concentrations of NaCl were introducedto modify the osmolarity within the egg (control, isotonicosmolarity, n¼ 50; 230mosm/l, hypotonic osmolarity,n¼ 50; 280mosm/l, hypertonic osmolarity, n¼ 50). Weincubated the eggs vertically, with the blunt end facingupwards, so that the embryos could be easily viewedthrough the hole made in the blunt end. A NaCl solutionwas injected into the hole above the embryo; due to thewaythe eggs were orientated, the NaCl solution was alwaysplaced at the same spot in the eggs.

We injected equal volumes of 0.7%NaCl (final osmolari-ty of the egg is 240mosm/l) as controls for each osmolaritytreatement. For the 230mosm/l hypotonic group, we in-jected2ml ofwater (final osmolarity of theegg is 230mosm/l), so we used 2ml of 0.7% NaCl for the controls. For the280mosm/l group, we injected 0.5ml of 11.23%NaCl (finalosmolarity of the egg is 280mosm/l) or 0.7% NaCl for thecontrols. The treated embryos were incubated for 6, 24, or72 hr, and weighed (dead embryos were excluded) beforethey were fixed in 4% paraformaldehyde for analysis.

Whole-Mount Embryo Immunofluorescent andF-Actin Staining

Chick embryos were harvested after treatment andfixed in 4% paraformaldehyde at 4 8C overnight. Forimmunofluorescence staining, whole-mount embryoswere incubated in the presence of the following antibod-ies: neurofilament (1:500; Invitrogen, Carlsbad, CA),PAX7 (1:100; Developmental Studies Hybridoma Bank,Iowa City, IA), p-Histone H3 (pHIS3) (1:400; Santa CruzBiotechnology, Shanghai, China), or HNK1 (1:500;Developmental Studies Hybridoma Bank). Briefly, theembryos were incubated with one of the primary anti-bodies at 4 8C overnight on a shaker. After extensiverinsing in phosphate-buffered saline (PBS), the embryoswere incubated with Alexa Fluor 555 anti-mouse-IgGor -IgM secondary antibodies (1:1,000; Invitrogen) at48C overnight on a shaker. For F-Actin detection,whole-mount embryos were stained using phalloidin-Alexa Fluor 488 (1:200; Invitrogen) at room temperaturefor 2 hr. All the embryos were counterstained with DAPI(1:1,000; Invitrogen) at room temperature for 1 hr.

In Situ HybridizationWhole-mount in situ hybridization was performed on

fixed chick embryos 24 hr after anisotonic osmolaritytreatment, as previously described (Henrique et al.,1995). Digoxigenin-labeled riboprobes were synthesizedto specifically detect the presence of SHH (Vaqar et al.,2012) and SNAI2 mRNAs (Nieto et al., 1994). Whole-mount-stained embryos were photographed, and then15-mm thick frozen sections were prepared from themon a cryostat microtome (Leica CM1900, Leica Micro-systems, Inc., Buffalo Grove, IL).

Figure 8. Model showing how anisotonic osmolarity affects neural differentiation, cell survival, neural tube closure, neural crest cell migration, andgeneexpression.Abnormal closureof theneural tube is proposed tobeaconsequenceof alteredcystoskeletal F-actindistribution in neuroepithelialcells. The cytoskeletal effects could be induced by altered expression of key morphogenic-related genes (BMP4, PAX7, and SHH), which in turnsaffects neuronal cell survival (via CDKN1A and CCDN1 expression) and differentiation (via PAX6 expression) in the developing neural tube.


374 Mol. Reprod. Dev. 82:365–376 (2015)

TUNEL StainingTUNEL stainingwas performed to establish the extent of

apoptosis in 4.5-day-old control and experimental chickembryos. The specimens were fixed in 4% paraformalde-hyde, dehydrated, and embedded in paraffin wax. Thespecimens were then sectioned at 4mm thickness, depar-affinized, and stained using a cell-death detection kit(Roche Diagnostics Corporation, Basel, Switzerland), ac-cording to the manufacturer’s instructions.

BrdU AtainingTo determine extent of cell proliferation, 100ml of BrdU

stock solution (10mM) was administered to control groupand anisotonic osmolarity-treated chick embryos for 5 hr.The embryos were then harvested, fixed in 4% paraformal-dehyde, and sectioned. Sections were stained with a BrdU-specific monoclonal antibody and developed for analysis,according to the manufacturer’s instruction (Roche Diag-nostics Corporation).

Semi-Quantitative Reverse-Transcriptase PCRAnalysis

Total RNA was isolated from 4.5-day-old control andexperimental chick neural tubes using a Trizol kit (Invi-trogen), according to manufacturer’s instructions. First-strand cDNA was synthesized in a final volume of 25mlusing a SuperScript III first-strand kit (Invitrogen). Follow-ing reverse transcription, amplification of the cDNA wasperformed as previously described (Amato et al., 2014).The primers used are listed in Table 1. PCR reactionswere performed in a Bio-Rad S1000TM Thermal cycler(Bio-Rad Corporation, Hercules, CA). The final reactionvolume was 50ml, and consisted of 1ml first-strand cDNA,25mM forward primer, 25mM reverse primer, 10ml PrimeSTARTM Buffer (Mg2þ plus), 4ml dNTPs Mixture (Ta-KaRa, Shiga, Japan), 0.5ml PrimeSTARTM HS DNAPolymerase (2.5U/ml TaKaRa), and RNase-free water.The cDNAs were amplified for 30 cycles. Each round ofamplification was performed at 94 8C for 30 sec, 58 8C for30 sec, and 72 8C for 30 sec. The PCR products (20ml)

were resolved in 1% agarose gels (Biowest, Hongkong,China) in 1� TAE buffer (0.04M Tris-acetate and 0.001MEDTA) and 10,000� GeneGreen Nucleic Acid Dye solu-tion (TIANGEN, Beijing, China). The resolved productswere visualized in a transilluminator (SYNGENE, Cam-bridge, UK), and photographs were captured using acomputer-assisted gel documentation system (SYN-GENE). The housekeeping gene GAPDH was run inparallel to confirm that equal amounts of RNA wereused in each reaction. The intensity of the fluorescentlystained bands were measured and normalized using animage-analysis system.

PhotographyImmunofluorescence-stained, whole-mount embryos

were first photographed using a stereo-fluorescent micro-scope (Olympus MVX10) run by Olympus Image-Pro Plus7.0 software. The embryoswere then sectioned into 15-mmthick slices using a cryostat microtome (Leica CM1900),and then photographed using an epi-fluorescent micro-scope (Olympus LX51, Leica DM 4000B) driven byCN4000 FISH Olympus software.

Data AnalysisData analyses were performed using Prism 5 software

(Graphpad Software, San Diego, CA). The results werepresented as the mean� standard deviation. All data wereanalyzed by ANOVA to test for differences among experi-mental and control groups. P<0.05 was considered to bestatistically significant.

ACKNOWLEDGMENTS

We would like to thank Zheng-lai Ma, Yun-lin Wu, andSheng-xin Li for technical assistance. This study wassupported by ‘‘973 Project’’ (2010CB529703); NSFC grant(31401230, 31071054, 30971493) and GuangdongNatural Science Foundation (S2013010013392,S2011010001593). It was also partly supported by

TABLE 1. List of Primers

Gene Primer Sequence Reference

PAX6 5’-CGACATCAAAGGCAAAGAG Ito and Walter, 20145’-GTCGATCCGGATAAATCTC

CDKN1A 5’-TCCTCCTCCTACCAGAGATG Scott-Drechsel et al., 20135’-TGTACCTGAGGCTCCTTGTC

CCND1 5’-TCGGTGTCCTACTTCAAGTG Scott-Drechsel et al., 20135’-GGAGTTGTCGGTGTAAATGC

SNAI2 5’-CCAATGACCTCTCTCCGCTTTCTG Endo et al., 20125’-ATCGCTAATGGGACTTTCTGAACCG

BMP4 5’-CAACTCCACCAACCACGCCATC Endo et al., 20125’-CAGCACCACCTTGTCATACTCATCC

PAX7 5’-GCTTACTGAAGAGGTCCGACTGTG Endo et al., 20125’-ACAAGTTGATGCGAGGTGGAAGG

GADPH 5’-GTCAACGGATTTGGCCGTAT Huber et al., 20085’-AATGCCAAAGTTGTCATGGATG


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Fundamental Research Funds for the Central Universities(21614319) and Students Research Training ProgramFund (201410559032, 1210559035, CX13181,CX14108, 201310559063).

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