Abnormal Paraventricular Nucleus of Hypothalamus and Growth Retardation Associated with Loss of Nuclear Receptor Gene COUP-TFII

The paraventricular nucleus of hypothalamus plays important roles in the regulation of energy balance and fetal growth. However, the molecular mechanisms underlying its formation and function have not been clearly elucidated. Various mutations in the human COUP-TFII gene, which encodes a nuclear receptor, result in growth retardation, congenital diaphragmatic hernia and congenital heart defects. Here, we show that COUP-TFII gene is expressed in the developing hypothalamus in mouse. The ventral forebrain-specific RXCre/+; COUP-TFIIF/F mutant mice display growth retardation. The development of the paraventricular nucleus of hypothalamus is compromised in the COUP-TFII mutant mainly because of increased apoptosis and mis-migration of the Brn2+ neurons. Moreover, hypoplastic anterior pituitary with blood cell clusters and shrunken posterior pituitary lacking AVP/OT neuron innervations are observed in the mutant, indicating the failure of formation of the hypothalamic-pituitary axis. Mechanistic studies show that the expression of Bdnf and Nrp1 genes is reduced in the mutant embryo, and that Bdnf is a direct downstream target of the COUP-TFII protein. Thus, our findings provide a novel functional validation that COUP-TFII gene promotes the expression of Bdnf and Nrp1 genes to ensure the appropriate morphogenesis of the hypothalamic-pituitary axis, especially the paraventricular nucleus of hypothalamus, and to prevent growth retardation.


COUP-TFII gene is expressed in the developing hypothalamus of mouse.
Since patients with various 15q26 deletions including the COUP-TFII gene display growth retardation and COUP-TFII gene heterozygous mutant mice generate developmental delay and poor postnatal viability 36 , we asked whether COUP-TFII in the hypothalamus contributes to the regulation of growth deficit. To answer this question, we first assessed the expression of COUP-TFII in mouse embryonic forebrain by immunofluorescence assays. At E10.5, the expression of COUP-TFII was detected in the ventricular zone of the hypothalamus, where reside neuronal progenitor cells (NPCs) (Fig. 1A,B). The expression of COUP-TFII was confined to the hypothalamic region at E12.5 (Fig. 1C,D) and E14.5 (Fig. 1F,G). Thus, COUP-TFII is expressed in the NPCs and early differentiating neurons of the mouse hypothalamus.
It has been shown that the activity of RXCre recombinase is detected in the mouse embryonic ventral forebrain including the eye and the hypothalamus 38 , and LacZ expression can be used as an indicator for the deletion of COUP-TFII gene 39 . We performed double immunofluorescence staining assays with antibodies against COUP-TFII and LacZ on coronal sections of a RXCre/+; COUP-TFII F/+ heterozygous mutant embryo at E14.5. COUP-TFII was expressed at the hypothalamus (Fig. 1I,M). The expression of LacZ was also detected at the hypothalamus and the caudal ganglionic eminence (Fig. 1J,N). Merged images revealed that the green COUP-TFII signals and the red LacZ signals were highly colocalized at the hypothalamus (Fig. 1K,O), suggesting that RXCre recombinase can efficiently excise the COUP-TFII gene in the embryonic hypothalamus.

RXCre/+; COUP-TFII F/F mutant mice display growth retardation and compromised PVH nucleus.
RXCre mouse was used to generate the RXCre/+; COUP-TFII F/F conditional homozygous mutant mouse, referred to as COUP-TFII mutant or mutant hereafter. At birth, all the pups in the same litter were similar. Nevertheless, the mutant pups were smaller than their control littermates at postnatal day 3 (P3) and day 4 (P4) (data not shown). Some mutant mice did not survive between P18 and P26, and some survived to adulthood. No obvious differences were observed among COUP-TFII F/+ , COUP-TFII F/F and RXCre/+; COUP-TFII F/+ mice; therefore, they were used as the control in the study. The body weight of the mutant mice was only approximately half that of the control mice at wean ( Fig. 2A). Clearly, RXCre/+; COUP-TFII F/F homozygous mutant mice displayed growth retardation.
To investigate the cause of growth restriction, we performed H&E staining on coronal sections from the control and the mutant mouse at 3 weeks (3 W) and 3 months (3 M) after birth. The same phenotypes were observed at both stages, and images generated from mice at 3 M were shown. Compared with the control (Fig. 2B,D), the PVH nucleus was barely observed in the mutant (Fig. 2C,E). Brn2 gene is specifically expressed in the PVH neurons 17,18 . Immunofluorescence staining showed that compared with the control (Fig. 2F and insert), there were much fewer Brn2 + PVH neurons in the mutant ( Fig. 2G and insert). Quantitative analysis from three pairs of mice showed that the reduction of Brn2 + PVH neurons in the mutant was significant (Fig. 2H). Overall, therefore, the development of the PVH nucleus is morphologically and molecularly compromised in the COUP-TFII mutant mouse.
MCH neurons and Orexin B neurons in the LHA promote food intake, and MCH mutant or Orexin B neuron-ablating mouse displays hypophagia [11][12][13] . Compared with the control (Fig. 2I), the number of MCH neurons was significantly reduced in the mutant LHA (Fig. 2J,K), as was the number of Orexin B neurons in the mutant (Fig. 2L-N). POMC and NPY neurons in the arcuate nucleus also participate in the regulation of energy balance [4][5][6] . The expression of POMC and NPY in the arcuate nucleus was comparable between the control and the mutant mouse (data not shown). GHRH neurons in the arcuate nucleus control body growth through regulating growth hormone pathway [40][41][42] . Compared with the control (Fig. S1A,C), there were much few GHRH neurons in the arcuate nucleus in the mutant at 1 M (Fig. S1B,D), and the reduction was significant (Fig. S1E). Similar as the RXCre/+; COUP-TFII F/+ control mouse (Fig. S1F,H), the expression of LacZ was barely detected in the arcuate nucleus in the RXCre/+; COUP-TFII F/F mutant mouse (Fig. S1G,I), indicating COUP-TFII gene is not deleted in the mutant arcuate nucleus. The data above suggest that the reduced MCH, Orexin B and GHRH neurons in the hypothalamic regions may contribute to growth defect in the COUP-TFII mutant.
The expression of COUP-TFII in the ventromedial nucleus of hypothalamus (VMH) of mouse is related to hypoglycemia-associated autonomic failure 43 . Consistent with a previous report 43 , the expression of COUP-TFII was readily detected in the majority of SF1 + VMH neurons at 1 M (Fig. 3A-D). Double immunostaining with antibodies against COUP-TFII and LacZ was conducted in the RXCre/+; COUP-TFII F/+ heterozygous and RXCre/+; COUP-TFII F/F homozygous mice at 3 M. The expression of both COUP-TFII and LacZ in the VMH nucleus was similar between the control and the mutant mouse ( Fig. 3E-L). It seems that RXCre recombinase does not target the VMH neurons, and the development of the VMH nucleus is normal in the COUP-TFII mutant mouse. Thus, in the mutant hypothalamus, the differentiation of Brn2 + neurons in the PVH nucleus, MCH and Orexin B neurons in the LHA was affected, but not POMC and NPY neurons in the arcuate nucleus and COUP-TFII + neurons in the VMH nucleus.
COUP-TFII is expressed in Brn2 + early differentiating PVH neurons. Hypocellular PVH nucleus is the most obvious defect observed in the COUP-TFII mutant (Fig. 2B-H). It has been reported that Brn2 gene plays a pivotal role in the differentiation of the PVH neuron 17,18 . We performed double immunofluorescence staining with antibodies against COUP-TFII and Brn2 on coronal sections containing the PVH nucleus at E14.5, E17.5, P1 and 6 M. The expression of Brn2 was detected at the prospective PVH nucleus at E14.5 (Fig. 4A), in the late differentiating PVH neuron at E17.5 (Fig. 4E) and at P1 (Fig. 4I), and in the mature PVH neurons at 6 M (Fig. 4M). The expression of COUP-TFII was broadly detected in the hypothalamic regions, and was colocalized with Brn2 in the early differentiating PVH neurons at E14.5 ( Fig. 4B-D). At E17.5, the expression of COUP-TFII was sharply decreased in the Brn2 + differentiating PVH neurons, but remained high in the neurons localized dorsally to the PVH nucleus ( Fig. 4F-H). The expression of COUP-TFII was barely detectable in the Brn2 + late differentiating PVH neurons at P1 (Fig. 4J-L) and the Brn2 + mature PVH neurons at 6 M ( Fig. 4N-P). These data show that COUP-TFII gene is expressed in the early differentiating PVH neurons, but not in the late differentiating and the mature PVH neurons.
Reduced Brn2 + early differentiating PVH neurons, hypocellular SON nucleus, and increased apoptosis in the COUP-TFII mutant embryo. Next, we asked whether the development of the PVH nucleus is normal at embryonic stages. As shown in Fig. 5Aa,c,e, there were many Brn2 + neurons at the PVH region along the rostro-caudal axis in the control at E15.5. In contrast, there were very few Brn2 + neurons at the prospected mutant PVH nucleus (Fig. 5Ab,d,f). Quantitative data from three pairs of animals at the same stage revealed that compared with the control, the reduction of the Brn2 + early differentiating PVH neurons was significant in the mutant (Fig. 5Ag).
Interestingly, many Brn2 + neurons were localized laterally to the prospected caudal PVH nucleus in the mutant (Fig. 5Ad,f). Calbindin is another marker of the SON and PVH neurons 17,44 . Similar to the Brn2 + neurons, compared with the control (Fig. S2A,C,E,G), there were more mis-migrating Calbindin + neurons beside the caudal PVH nucleus in the mutant (Fig. S2B,D,F,H). The SON nucleus lies lateral to optic tracts and dorsal to pial surface of the brain. Neurons in the PVH nucleus and the SON nucleus originate from the same group of NPCs at E10.5 45 . During development, the PVH neurons are differentiated locally, while the SON neurons migrate ventro-laterally to their final destination 45 . Probably, those mis-located Brn2 + or Calbindin + neurons are related to the SON neurons. As expected, H&E staining results revealed that compared with the control (Fig. 5Ba,c), the number of magnocellular SON neurons in the mutant was reduced (Fig. 5Bb,d), and the reduction was significant (Fig. 5Be). Thus, the formation of the SON nucleus is also affected in the COUP-TFII mutant.
There are several possibilities for the loss of the Brn2 + PVH neurons in the COUP-TFII mutant embryos, such as apoptosis and proliferation defect. To investigate the mechanism for the reduction of the Brn2 + PVH neurons, we performed immunostaining assay to examine the expression of cleaved-Caspase-3, an apoptotic marker. Compared with the control at E15.5 (Fig. 5Ca,c,e,g, and inserts), there were more cleaved-Caspase-3 signals in the mutant PVH region (Fig. 5Cb,d,f,h, and inserts). Quantitative assays with samples from 3 pairs of animals at E15.5 confirmed that there were significantly more apoptotic cells in the mutant PVH than in the control (Fig. 5Ci). In addition, there were also more cleaved-Caspase-3 signals in the hypothalamus of the mutant than the control at E13.5 ( Fig. S3A-F), indicating that abnormal apoptosis occurs at earlier embryonic stages. Next, we assessed the expression of Ki67, a proliferation marker, at the PVH region at E12.5 and at E14.5. The expression of Ki67 was comparable between the control (Fig. 5Da,c,e,g) and the mutant (Fig. 5Db,d,f,h) at both stages, suggesting that proliferation was not altered in the mutant. Thus, our data show that increased apoptosis is one possible mechanism that leads to the reduction of the Brn2 + PVH neurons in the COUP-TFII mutant.
Bdnf gene is a direct downstream target of COUP-TFII. To investigate molecular mechanism responsible for the defects in the COUP-TFII mutant, real-time quantitative PCR (qPCR) assays were performed with total RNAs prepared from ventral forebrains of the control and the mutant at E14.5. As shown in Fig. 6A, the expression of COUP-TFII transcripts was reduced by approximately 50% in the mutant compared with the control. The expression of the COUP-TFI gene was not altered. Brn2, Sim1, Otp and Arnt2 genes are essential for the development of the PVH nucleus [17][18][19][20][21][22][23] . Compared with the control, the expression of these genes was slightly reduced in the mutant mouse at E14.5, but the difference was not significant (Fig. 6A), indicating that the function of COUP-TFII in the development of the PVH nucleus may be independent of these known regulatory genes.   There are several major secretory neurons in the PVH nucleus including the AVP, OT, CRH and TRH neurons 15,16 . Interestingly, the expression of Avp and Trh genes but not Ot and Crh genes was significantly reduced in the mutant at E14.5 (Fig. 6A), suggesting that COUP-TFII gene may be specifically required for the early differentiation of the AVP and TRH neurons. Nrp1 and Nrp2 genes, which are involved in cell migration and axon guidance, are regulated directly by COUP-TFII protein 39 . The expression of both Nrp1 and Nrp2 genes is reduced in the ventral forebrain of RXCre/+; COUP-TFII F/F mutant mouse at E12.5 39 . The expression of Nrp1 but not Nrp2 was decreased significantly in the mutant at E14.5 (Fig. 6A), suggesting that Nrp1 might specifically mediate the migration of the Brn2 + neurons in the hypothalamus.
Neurotrophin factors, such as NGF, BDNF, NT3 and CNTF, are essential for the survival and differentiation of neurons [46][47][48] . Since the increased apoptosis was detected in the mutant hypothalamus (Figs 5C and S3), the expression of neurotrophin factors was assessed by qPCR assays. The expression of the Ngf and Nt3 genes was undetectable in both the control and the mutant (data not shown). The expression of Cntf transcripts was not altered in the mutant, whereas the expression of Bdnf was significant lower in the mutant than the control (Fig. 6A). Bdnf transcripts are preferentially expressed at the ventro-lateral region of the VMH nucleus 49 . A specific BDNF antibody could detect the expression of BDNF protein in the cytoplasm of neurons at the ventro-lateral VMH nucleus in the adult mouse at 2 M (Fig. 6B,C). The expression of BDNF was readily detected in the rostral and the caudal part of the control hypothalamic areas including the PVH nucleus at E15.5 (Fig. 6D,E,H,I); in contrast, its expression was barely detectable in the mutant (Fig. 6F,G,J,K). Thus, the expression of Bdnf was reduced in the COUP-TFII mutant at both transcriptional and translational levels.
To determine the direct downstream targets of COUP-TFII among the Avp, Bdnf, Nrp1 and Trh genes, chromatin immuoprecipitation (ChIP) assay was performed with chromatin prepared from the hypothalamus of mouse embryos at E14.5. COUP-TFII positively regulates the expression of target genes through the Sp1 site by tethering to Sp1 protein 32,50,51 . COUP-TFII may promote the expression of Nrp1 gene in the ventral forebrain through a conserved Sp1 site in intron 12 39 . Our ChIP-qPCR assays showed that both COUP-TFII and Sp1 were recruited at the conserved Sp1 site of Nrp1 gene, but not at the control non-Sp1 site at 3′ UTR (Fig. S4). Next, an Sp1 site, which is evolutionarily conserved among human, chimpanzee, mouse, cow and opossum, was identified at the promoter region of the long form of the Bdnf gene (Fig. 6L). The binding of both COUP-TFII and Sp1 was enriched at the conserved Sp1 site of Bdnf gene, but not at a negative non-Sp1 control site, which was located 2 kb upstream (Fig. 6L). Unfortunately, no such evolutionarily conserved Sp1 site was identified in the genomic locus of either Avp or Trh. The findings above suggest that the Bdnf gene is a direct downstream target of COUP-TFII, and the reduced expression of Bdnf is a possible cause for the increased apoptosis in the COUP-TFII mutant.  (Fig. 7A). H&E staining results revealed that compared with the control (Fig. 7Aa,c,e,g,i,k), the posterior pituitary was noticeably smaller in the COUP-TFII mutant at P0 (Fig. 7Ab,d,f,h,j,l). Moreover, images at higher magnification revealed that there were a few isolated blood cells in the anterior pituitary of the control (Fig. 7Am,o,q); nevertheless, many blood cell clusters, indicated by white arrowheads, were observed in the anterior pituitary of the mutant (Fig. 7n,p,r), indicating a hypoplastic anterior pituitary. Most likely, the defective anterior and posterior pituitary is a cause of growth restriction of the COUP-TFII mutant.

The development of pituitary is compromised in the COUP-TFII mutant. In
Axon projections of AVP or OT magnocellular neurons target the posterior pituitary between E15.5 and E16.5 52 . The expression of AVP was readily detected in the control at E15.5 (Fig. 7Be,g); however, its expression was absent from the posterior pituitary of the mutant (Fig. 7Bf,h). In addition, compared with the control (Fig. S5A,C,E,G), the expression of both AVP and OT was barely detected in the mutant posterior pituitary at P0 (Fig. S5B,D,F,H). In the COUP-TFII mutant mouse, hypocellular PVH/SON nuclei, hypoplastic anterior pituitary, and shrunken posterior pituitary lead to the failure of formation of the HP axis, which could be a main reason for growth retardation of these mice.

Discussion
In the present study, we have observed that COUP-TFII is expressed in the developing embryonic hypothalamus. Similar to the phenotype of hemizygous deletion of COUP-TFII in both human and mouse, RXCre/+; COUP-TFII F/F mutant mice display noticeable growth restriction and poor postnatal viability. The development of the PVH and SON nuclei is affected in the COUP-TFII mutant mouse, with defects in both the anterior and the posterior pituitary. Moreover, increased apoptosis and mis-migration of the Brn2 + neurons are observed in the mutant. Mechanistic studies reveal that COUP-TFII may maintain or activate the expression of Bdnf and Nrp1 genes to ensure the appropriate morphogenesis and function of the HP axis, especially the PVH nucleus.
All patients with various 15q26 deletions generate growth retardation, associated with CDH and/or CHD [25][26][27][28][29][30][31] . The evidence from both clinical and mouse studies suggests that among the genes at the 15q26 locus, the loss of IGF1R at 15q26.3 is responsible for pre-and postnatal growth restriction 31,[53][54][55] . Interestingly, various CNVs of the COUP-TFII gene were also identified in patients with different 15q26 deletions 25,[27][28][29][30]33 . Moreover, the loss of an allele of the COUP-TFII gene leads to growth retardation and poor postnatal viability in pure-bred 129 Sv, C57BL/6 or ICR mouse and in 129 Sv/C57BL/6 mixture mouse 36 , indicating complete penetrance of the defect. Here, we find that the RXCre/+; COUP-TFII F/F mutant mice are half the size of the control mice at wean (Fig. 2), suggesting that the RXCre/+; COUP-TFII F/F mutant mouse phenocopies the COUP-TFII heterozygous null mouse. Therefore, other than IGF1R, haploinsufficiency of COUP-TFII is a possible cause for growth deficit in patients with 15q26 deletions.
The HP axis is an important neuroendocrine system coordinating the periphery and brain signals required for physiologic homeostasis and survival 16,56 . However, the detailed molecular and cellular mechanism responsible for the formation of the HP axis has not been fully clarified. Because the COUP-TFII gene is not expressed in the pituitary gland 57 , we mainly focused on its function in the hypothalamus. COUP-TFII is expressed in the hypothalamic NPCs and early differentiating PVH neurons, but not in the late differentiating and the mature PVH neurons (Figs 1 and 4), suggesting that COUP-TFII may play crucial roles in the early development of the hypothalamus. Indeed, the morphogenesis of the PVH nucleus is compromised in the COUP-TFII mutant with hypocellular SON nucleus, hypoplastic anterior pituitary with blood cell clusters, and shrunken posterior pituitary lacking AVP/OT projections (Figs 2, 5 and 7 and S5). The COUP-TFII mutant mouse phenocopies Brn2 −/− , Sim1 −/− and Arnt2 −/− mouse in terms of growth restriction and compromised posterior pituitary [17][18][19][20][21][22][23] . However, the development of the anterior pituitary is not affected in the Brn2 −/− , Sim1 −/− and Arnt2 −/− mouse [17][18][19][20][21][22][23] . In contrast, hypoplastic anterior pituitary is detected in the COUP-TFII mutant (Fig. 7), suggesting that the failure of formation of the HP axis, especially a defective anterior pituitary, is a possible cause for growth failure of the COUP-TFII mutant. The hypoplastic anterior pituitary could be caused by the reduction of GHRH neurons in the arcuate nucleus in the mutant (Fig. S1). The expression of Brn2, Sim1, Otp and Arnt2 transcripts is comparable between the control and the mutant (Fig. 6). The development of PVH NPCs remains normal in either Brn2 −/− or Sim1 −/− null mutant mouse at E15.5 [17][18][19] . Nonetheless, the Brn2 + early differentiating PVH neurons are noticeably reduced in the COUP-TFII mutant at E15.5 (Fig. 5). Therefore, distinct from Brn2 and Sim1, which participate in the terminal differentiation of PVH neurons, COUP-TFII is a novel key regulatory gene mediating the early morphogenesis of the HP axis, especially the PVH nucleus.
A hypocellular PVH nucleus is one of the most significant phenotypes in the COUP-TFII mutant (Fig. 2), which may be caused by several possibilities including abnormal apoptosis, lower proliferation, mis-migration or inappropriate differentiation. The Ki67 staining data show that proliferation is not altered in the mutant (Fig. 6). Both the PVH and SON neurons originate from the same population of NPCs at E10.5 45 . A few Brn2 + or Calbindin + neurons are mis-located laterally to the prospective mutant PVH nucleus (Figs 5 and S2), indicating that mis-migration may contribute to the hypocelluar PVH and SON nuclei in the mutant. As the direct targets of COUP-TFII protein, the Nrp1 and Nrp2 genes mediate the migration of Pax6 + neurons from the caudal ganglionic eminence to the basal medial amygdala nucleus 39 . Consistently, the expression of Nrp1 transcripts is reduced in the mutant at E14.5 (Fig. 6), suggesting that Nrp1 may specifically participate in the regulation of migration of the Brn2 + neuron in the hypothalamus. Furthermore, the number of cleaved-Caspase-3 + apoptotic cells is significantly increased in the mutant (Fig. 5). Neurotrophins, such as NGF and BDNF, are essential for the survival and growth of neurons during development 46,47,58 . Intriguingly, Bdnf −/− mouse displays postnatal growth retardation 59 ; however, the cause of growth restriction in Bdnf −/− mouse is not clear. In the COUP-TFII mutant embryo, the expression of Bdnf is reduced at both the transcriptional and translational levels (Fig. 6). ChIP assay in vivo data demonstrate further that the Bdnf gene is a direct downstream target of COUP-TFII (Fig. 6). RXCre recombinase deletes the COUP-TFII gene in the ventral forebrain including the hypothalamus and caudal ganglionic eminence (Fig. 1) 39 . The reduced expression of Bdnf protein is also observed in the ventral forebrain regions of the mutant (Fig. 6), which may explain the reduction of the Brn2 + PVH neurons, as well as MCH neurons and Orexin B neurons in the LHA in the adult mutant. Clearly, both abnormal apoptosis and mis-migration are responsible for the reduction of the Brn2 + neurons. Nonetheless, we cannot exclude the possibility that COUP-TFII gene may also regulate the differentiation of some specific subtypes of PVH neurons, since the expression of Avp and Trh transcripts is specifically reduced in the mutant (Fig. 6).
In summary, our data provide a functional validation that CNVs of the COUP-TFII gene are possible causes for growth retardation. In the control mouse embryonic hypothalamus, COUP-TFII is required to activate or maintain the expression of Bdnf and Nrp1, which ensure appropriate morphogenesis and function of the HP axis, especially the PVH nucleus, to coordinate the normal growth. In the mutant, the loss of COUP-TFII results in reduced expression of Bdnf, Nrp1 and Avp, which may cause apoptosis and mis-migration of the Brn2 + neurons, hypocelluar PVH nucleus, hypoplastic anterior pituitary with blood cell clusters and shrunken posterior pituitary lacking AVP/OT neural innervations. The defective formation of the HP axis, especially the anterior pituitary, leads to growth deficit (Fig. 7C).
Our findings together with other clinical and mouse studies, reveal that COUP-TFII gene mutations are strongly associated with growth retardation, CHD, CDH, congenital coloboma, and postnatal viability 27,28,[34][35][36][37]51,60,61 . Furthermore, enhanced expression of COUP-TFII is correlated with dilated cardiomyopathy 62 as well as the recurrence and progression of prostate cancer 63 . Thus, COUP-TFII mutations should be included in the differential diagnosis of birth defects, CHD, CDH, ocular defects and cancer. Our study will benefit the prediction, prevention and treatment of human diseases.

Methods
Ethics statement. All animal experiments were carried out following protocols approved by the Animal Ethics Committee of the Shanghai Institute of Biochemistry and Cell Biology. We confirm that all methods were performed in accordance with the relevant guidelines and regulations.
Animals. COUP-TFII-floxed mice and RXCre mice used in the study were of the C57B6/129 mixed background. Male mice were used in body weight study. Noon of the day of vaginal plugs was designated as embryonic day 0.5 (E0.5).
Hematoxylin and eosin (H&E) staining, Nissl staining, immunofluorescent staining, and immunohistochemical staining. For H&E staining, paraffin sections on slides were dewaxed in xylene for 3 min for three times, and rehydrated in 100% ethanol three times, 95% and 70% ethanol once, with 1 min each. The slides were rinsed in the distilled water, and were stained in hematoxylin solution for 30 sec. Then the slides were washed in running tap water for 2 min. The slides were immersed in acid alcohol for decolorizing, and then rinsed in distilled water. The slides were immersed in Lithium Carbonate solution, and also washed in distilled water. The slides were counterstained in eosin solution for 10 sec. And then, the slides were dehydrated with 95% ethanol once, and 100% ethanol three times with 1 min each. The slides were cleared in xylene twice with 2 min each. The slides were mounted in fume hood. Nissl staining was conducted with 0.1% Cresyl Violet for 10 minutes.
For immunofluorescence staining, the slides were dewaxed and rehydrated the same as H&E staining. Then the slides were washed in distilled water and 1X phosphate buffer solution (PBS) with 1 min each. The slides were treated with boiling 1X antigen retrieval solution (DAKO) for 15 min. After cooling down to room temperature (RT), the slides were rinsed with 1XPBS for 10 min for 3 times. The slides were treated with 3% H 2 O 2 in 1XPBS for 30 min, and then washed with 1XPBS for 10 min for 3 times. The sections were blocked with buffer containing 1% BSA, 5% Serum in 1XPBS for 1 h at RT, and then incubated with primary antibody in hybridization buffer overnight at 4 °C. After being washed with 1XPBS for 10 min for 3 times, sections were incubated with secondary antibody for 1 h at RT. The sections were washed and counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). After wash, the slides were mounted with mounting medium. In case, TSA kit (Invitrogen) was used. The samples were treated the same as the regular immunofluorescence staining till the completion of the primary antibody incubation. And then the processes were carried out with TSA kit by following the manufactory's protocol.
For immunohistochemical staining, the sections were treated the same as the immunofluorescence staining till the incubation with the second antibody. The slides were incubated with biotinylated secondary antibody for 1 h at RT. After being washed in 1XPBS for 5 min for 3 times, the slides were incubated with ABC Reagent (Vectorlabs) for 30 min. After being washed in 1XPBS for 5 min for 3 times, the slides were incubated with fresh prepared DAB substrate solution (Vectorlabs). The reaction was terminated till desire signals observed. The slides were counterstained with hematoxylin solution, and then dehydrated, cleared and mounted as described in the H&E staining.