Study on the Function and Mechanism of Lin28b in the Formation of Chicken Primordial Germ Cells

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Article Study on the Function and Mechanism of Lin28B in the Formation of Chicken Primordial Germ Cells

Qisheng Zuo 1,2, Jing Zhou 1,2, Man Wang 1,2, Yani Zhang 1,2, Guohong Chen 1,2 and Bichun Li 1,2,*

1 Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China; [email protected] (Q.Z.); [email protected] (J.Z.); [email protected] (M.W.); [email protected] (Y.Z.); [email protected] (G.C.) 2 Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China * Correspondence: [email protected]; Tel.: +86-0514-87997207

Simple Summary: In this study, we explored the function and molecular mechanism of Lin28B in the formation of chicken primordial germ cells (PGCs) in detail. Our results indicate that Lin28B participates in the formation of PGCs through let-7a-3p, which set a theoretical foundation for improving the function and mechanism of the Lin28 family in the formation of PGCs.

Abstract: Lin28A and Lin28B are two homologues of the same family of RNA binding proteins (RBPs). The function and molecular mechanism of Lin28A in the formation of primordial germ cells (PGCs) are very clear, but the related research on Lin28B is rarely reported. Here, we found that the overexpression of Lin28B can promote the formation of PGC in vivo. Furthermore, the overexpression of Lin28B also resulted in the inhibition of totipotency gene expression and upregulated the PGCs marker genes, and a signiﬁcant increase in the number of PGCs in genital ridge, as detected by Periodic Acid-Schiff(PAS) staining. However, the inhibited Lin28B expression showed completely opposite results, which were conﬁrmed on the PGC induction model in vitro. Mechanistically, we found that the overexpression of Lin28B can inhibit the maturation of let-7a-3p, and the results Citation: Zuo, Q.; Zhou, J.; Wang, M.; of high-throughput sequencing indicated that let-7a-3p was a negative regulator of the formation Zhang, Y.; Chen, G.; Li, B. Study on process of PGCs. Therefore, we conclude that our results determine that Lin28B participates in the the Function and Mechanism of formation of PGCs through let-7a-3p, which set a theoretical foundation for improving the function Lin28B in the Formation of Chicken and mechanism of Lin28 family in the formation of PGCs. Primordial Germ Cells. Animals 2021, 11, 43. https://doi.org/10.3390/ Keywords: primordial germ cells; Lin28B; gene function; let-7a; miRNA ani11010043

Received: 13 October 2020 Accepted: 24 December 2020 1. Introduction Published: 28 December 2020 The function of Lin28 is primarily concentrated on embryo development [1,2] and cell Publisher’s Note: MDPI stays neu- reprogramming [3,4], which could code two RNA-binding proteins, Lin28A and Lin28B [5]. tral with regard to jurisdictional claims As early as 2009, it was shown that Lin28A played an important role in the cytogenesis of in published maps and institutional primary germ cells (PGCs) [6]. Particularly, Lin28A inhibits the maturation of let-7, thereby afﬁliations. activating the expression of the Blimp1 gene (key gene for the formation of PGCs) [7,8]. However, the function of Lin28B in the formation of PGCs remains unknown. Recently, several research groups have found that the SNP locus in Lin28B is closely related to the age of female menarche [9–11], indicating the close correlation of Lin28B in animal reproduction. Copyright: © 2020 by the authors. Li- Subsequently, increasing studies observed the function of Lin28A and Lin28B in the censee MDPI, Basel, Switzerland. This PGCs development of mammals. The proliferation of PGCs in mice with Lin28A knockout article is an open access article distributed was impaired, leading to decreased number of germ cells during embryo development [12]. under the terms and conditions of the Lin28A knockdown during the differentiation of ESCs into PGCs could signiﬁcantly reduce Creative Commons Attribution (CC BY) license (https://creativecommons.org/ the expression of PGCs-formation related genes, such as Blimp1, Prdm14, and Stella [13]. licenses/by/4.0/). Similar to Lin28A, the knockdown of Lin28B could impair the formation of PGCs that are

Animals 2021, 11, 43. https://doi.org/10.3390/ani11010043 https://www.mdpi.com/journal/animals Animals 2021, 11, 43 2 of 12

derived from Embryoid Bodies (EBs). The knockdown of Lin28B in ESCs could inhibit the differentiation of ESCs into PGCs [8]. Contrastingly, the expression pattern of Lin28A and Lin28B in the reproductive system is not always consistent. When there are only PGCs in genital ridge, the expression level of Lin28A is the highest. With the progress of pregnancy, the expression level of Lin28 decreased significantly, but that of Lin28B did not change markedly [14,15], which makes the function of Lin28B in PGCs uncertain. Although Lin28A and Lin28B are homologies, they bind to target miRNA and play a major role in post-transcriptional control [16,17]. Lin28A avoids Dicer processing by inducing the 30 end of pre-let-7 into uraci, and, finally, degrades it [18]. Contrastingly, Lin28B is a posttranscriptional inhibitor of pre-let-7, it binds to the end of pre-let-7 through its cold shock domain(CSD) and to the GGAG motif of pre-let-7 through its zinc finger domains(ZFD) [19,20]. This raises a question regarding whether totally different mechanism will lead to totally different function. For this reason, the function and underlying molecular mechanisms of Lin28B in the formation of PGCs were comprehensively studied while using chicken PGCs as the study objects. The results of the study may lay foundations for analyzing the function and mechanism of Lin28B in the formation of chicken PGCs.

2. Materials and Methods 2.1. Ethics Statement All of the procedures involving the care and use of animals conformed to the U.S. National Institute of Health guidelines (NIH Pub. No. 85-23, revised 1996) and they were approved by the Laboratory Animal Management and Experimental Animal Ethics Committee of Yangzhou University.

2.2. Reagents BMP4 (cyt-361) was from PROSPEC (Beijing, China). Dulbecco’s modiﬁed eagle medium (DMEM, 41965062) and fetal bovine serum (FBS, 10100-147) were supplied by Gibco (Carlsbad, CA, USA). The transfection reagents FuGENE®®HD (E2311) and Dual-Luciferase®® reporter assay system were from Promega (Madison, WI, USA). The PrimeSTAR®® Max DNA polymerase, the reverse-transcription kit (RR036A), the quantiﬁ- cation kit for qRT-PCR(RR820A), and the restriction endonucleases SnaB I, Kpn I, and Xho I were supplied by Takara (Takara, Dianlian, China). The CVH (DDX4) antibody (ab27591) and the CKIT antibody (ab5634) were from abcam (San Francisco, CA, USA).

2.3. Cell Treatments and Grouping We studied the role of Lin28B in the formation of PGCs in vitro while using the BMP4 induction model that was established previously. The isolation and cultivation of ESCs are based on previous study [21]. Well-grown ESCs were transferred to 24-well plates and then treated/grouped, as follows. The routinely induced BMP4 was used as a control. The ESCs that were transfected with oeLin28B and siLin28B vector then induced by BMP4 were the treatment group. During induction, the culture media were replaced every two days. Cell morphology was checked with a ﬂuorescence inversion microscope system. The zero-day- old, two-day-old, four-day-old, and six-day-old cells were collected for later analysis. The in vivo experiment was performed, as follows. The in vivo experiment was performed, as follows: the vectors of oeLin28B and siLin28B were mixed with PEI (M:V = 1:1), respectively, and then the mixture was injected into the blood vessels of 2.5 day-old chicken embryos with 1µg. The oeLin28B group and siLin28B group were the treatment group, and chicken embryos were checked every two days. The genital ridges of 4.5-day-old chicken embryos were collected for later analysis.

2.4. Construction of Lin28B Overexpression Vector The forward and reverse primers were designed while using the Primer 5.0 software according to the sequence of chicken Lin28B in GenBank (accession no.: NM_001034818.1). The forward primer was (F):50-cgggatccATGGCCGAAGCAGGGGC-30 and the reverse Animals 2021, 11, 43 3 of 12

primer was (R):50-cggaattcCGCACA TGACACA-30. The enzyme digestion sites were BamH I and EcoR I. The total RNA of 4.5-day-old reproductive ridge was extracted and then reverse-transcripted to cDNA. With the cDNA as a template, clonal fragments of the target gene Lin28B were obtained through the use of PCR. The conditions for PCR were three min. at 98 ◦C, 25 s at 98 ◦C, 30 s at 64 ◦C, and 1 min. at 72 ◦C. The PCR cycles numbered 35 with an extension time for 7 min. at 72 ◦C. The linear plasmid pcDNA3.1 after dual-enzyme digestion and ampliﬁed target gene fragments of Lin28B were ligated in order to construct the Lin28B overexpression vector, oeLin28B.

2.5. Construction of the Lin28B Interference Vector We designed three targets according to the sequence of Lin28B and synthesized a single-strand RNA (Supplementary Table S1). After being annealed, the RNA was ligated to the lentivirus interference vector skeleton of piLenti-siRNA-GFP to construct four overexpression interference vectors of Lin28B, called siLin28B-1, siLin28B-2, siLin28B-3, and siLin28B-4. These four vectors were then transfected to confluent DF-1 cells, according to the mixture of plasmids and FuGENE HD at a ratio of 1:3 (M/V). Forty-eight hours later, the transfected DF-1 cells were selected with 10 ng/µL of puromycin for 24 h, after which the expression of green fluorescence proteins was observed with a fluorescence microscope. The DF-1 cells were collected from every group, and their total RNA was extracted with the Trizol method. The cDNA was synthesized through the use of a reverse-transcription kit, and the relative expression level of Lin28B was assayed with qRT-PCR. The amount of expression was calculated according to the 2−∆∆Ct method, and β-actin was the internal reference gene.

2.6. qRT-PCR Zero-day-old, two-day-old, four-day-old, and six-day-old cells during in vitro induction as well as the zero-day and 4.5-day-old in vivo induction cells from each group were collected. The total RNA was extracted with the Trizol method and it was transcribed to cDNA. The expression of the NANOG and the marker genes of PGCs genesis, such as DEAD-box helicase 4(DDX4, aslo called Cvh), Chicken tyrosine kinase receptor(C-kit), and PR domain 1(Prdm1, aslo called Blimp1), was determined with β-actin as the internal reference gene. qRT-PCR assay was based on the reverse-transcription kit from Tiangen (production no.: FP215). The 20 µl of reaction system includes 2 µl of cDNA (50 ng), 10 µl of 2 ×SuperReal Color PreMix, 0.6 µl of the forward and reverse primers (10 µM), and ddH2O. The reaction procedure of PCR is 15 min. at 95 ◦C, 10 s at 95 ◦C, and 32 s at 62 ◦C for 40 cycles. The expression was analyzed with the Ct values, and the primes are supplied in Supplementary Table S2.

2.7. PAS Staining 4.5-day-old chicken embryos from each group were collected and ﬁxed for 24 h. The ﬁxed chicken embryos were then treated with 79% alcohol overnight, 70% alcohol for 1 h, 80% alcohol for 1 h, 90% alcohol for 1 h, 100% alcohol for 1 h, xylene for 10 min., and then xylene for an additional period of 10 min. The resulting chicken embryos were then immersed into 65 ◦C wax for 1 h and they were cooled down. Subsequently, the chicken embryos were sectioned at 8 µm, dewaxed, hydrated, and then subjected to glycogen staining with the PAS kit (Solarbio, Beijing, Chinas; product no.: G1281).

2.8. Screening and Testing of Lin28B-Binding micRNA The online software (http://mirdb.org/cgi-bin/search.cgi) is used in order to predict chicken micRNA Let7s. Well-grown DF-1 cells were transfected with oeLin28B and siLin28B, respectively. 48 h later, the transfected cells were collected and the total RNAs were extracted according to the miRNA isolation kits (thermoﬁsher, Shanghai, Chinas) and then transcribed into cDNA, according to the manufacturer’s recommendations. The relative expression level of micRNA-let7s was assayed while using U6 as an internal reference. Animals 2021, 11, 43 4 of 12

Supplementary Table S3 lists the primers for qRT-PCR of micRNA-let7s. The condition for qRT-PCR was, as follows, according to the reagent kit. The reaction system includes 50 ng cDNA, 10 µL of 2 × miRucte Plus miRNA PreMix, 0.4 µL of forward primer, 0.4 µL of reverse primer (10 µM), and ddH2O. The entire reaction volume is 20 µl. The procedure for PCR is 15 min. at 95 ◦C, 20 s at 94 ◦C, 30 s at 63 ◦C, and 34 s at 72 ◦C for ﬁve cycles, followed by 20 s at 94 ◦C and an extension at 60 ◦C for 34 s. The total number of cycles was 40. The expression was analyzed with the Ct values.

2.9. Immunocytochemical Detection of Reproductive Marker Protein All collected groups of ESCs were cultured for six days, washed twice with PBS, ﬁxed with 4% paraformaldehyde for 30 min., washed three times with PBS, treated with 0.1% Triton for 15 min., washed three times with PBS, and then added 10% FBS-PBS. After blocking for 2 h, added primary antibody CVH, CKIT, incubated for 2 h at 37 ◦C and overnight at 4 ◦C; washed primary antibody with PBS, added secondary antibody, incubated for 2 h at 37 ◦C in the dark; after that, washed secondary antibody with PBS, incubate for 15 min., stained with 5 ng/µL DAPI, blocked with glycerol (50% glycerol, 50% PBS).

2.10. Data Analysis qRT-PCR were replicated three times. The data were analyzed by ANOVA with SPSS 19.0 software package (SPSS, Chicago, IL, USA). The means were compared by the least significant difference (LSD) test. Each replication was an experiment unit. p < 0.05 was considered to be significant, and p < 0.01 was highly significant. The charts were prepared in GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA).

3. The Results 3.1. Subsection Construction of Overexpression and Interference Vector of Lin28B The CDS region (979 bp long) of Lin28B(NM_001034818.1) was successfully amplified while using PCR. The results of agar gel electrophoresis showed there were specific bands at approximately 1000 bp (Figure1A, Left). The amplified fragment was ligated to pcDNA3.1 in order to construct an overexpression vector of Lin28B, and the results of dual-enzyme digestion showed that there were two bands at 979 bp and 5400 bp (Figure1A, Right ). The sequencing results showed that the amplified product of 979 bp had a 99% similarity to the coding area of Lin28B, thus indicating that the Lin28B vector was successfully constructed, and the vector was named oeLin28B (Figure1B). The interference vector of Lin28B was constructed while using piLenti-siRNA-GFP as the skeleton (Figure1C). The sequencing results confirmed that the expression interference vector of Lin28B was successfully constructed (Figure1C), and the vectors were named si Lin28B-1, siLin28B-2, siLin28B-3, and siLin28B-4. The expression interference vectors were transfected into DF1 cells (Figure2A ). The results showed that the transfection efficiency of these expression interference vectors in DF1 was greater than 70%. The results of qRT-PCR showed that siLin28B-1, siLin28B-2, and siLin28B-3 could significantly reduce the expression of Lin28B (p < 0.01), and the expression levels were reduced by 88%, 62%, and 31%, respectively (Figure2B). Moreover, siLin28B-1 had the highest efficiency in interfering with the expression of Lin28B, whereby it was named siLin28B. Meanwhile, the rescue experiment further showed that oeLin28B overexpression in DF-1 markedly rescued the expression of Lin28B following siLin28B interference (Figure2C). The results suggest that oe Lin28B and siLin28B could both overexpress and interfere with the activity of Lin28B. Animals 2021, 11, x 5 of 12 Animals 2021, 11, x 5 of 12

Animals 2021, 11, 43 following siLin28B interference (Figure 2C). The results suggest that oeLin28B 5and of 12 following siLin28B interference (Figure 2C). The results suggest that oeLin28B and siLin28B could both overexpress and interfere with the activity of Lin28B. siLin28B could both overexpress and interfere with the activity of Lin28B.

Figure 1. Construction of overexpression and interference vector of Lin28B. (A) Left: cloning re- Figure 1. ConstructionFigure 1. Construction of overexpression of overexpression and interference and interference vector of vectorLin28B of.( ALin28B) Left:. ( cloningA) Left: resultscloning of re-Lin28B,1: Lin28B sults of Lin28B,1: Lin28B amplification bands,M: DL5000 marker; Right: double digestion results of sults of Lin28B,1: Lin28B amplification bands,M: DL5000 marker; Right: double digestion results of ampliﬁcation bands, M: DL5000Lin28B marker; overexpression Right: double vector, digestionM: DL 5000 results marker; of Lin28B1: no enzymeoverexpression digestion; vector, 2: single M: enzyme DL 5000 diges- Lin28B overexpression vector, M: DL 5000 marker; 1: no enzyme digestion; 2: single enzyme diges- marker; 1: no enzyme digestion;tion; 3: 2: d singleouble enzymeenzyme; digestion;4: ddH2O. 3:(B double,C) Construction enzyme;4: schematic ddH2O. (diagramB,C) Construction of Lin28B overexpression schematic tion; 3: double enzyme; 4: ddH2O. (B,C) Construction schematic diagram of Lin28B overexpression diagram of Lin28B overexpressionand interference and interference vector. vector. and interference vector.

FigureFigure 2. 2. ActivityActivity detection detection of of overexpression overexpression and and interference interference vector vector of ofLin28B.Lin28B. (A()A Lin28B) Lin28B inter-inter- Figure 2. Activity detection of overexpression and interference vector of Lin28B. (A) Lin28B inter- ferenceference vector vector transfected transfected into into DF DF-1-1 cells, cells, non non-transfected-transfected cells cells were were blank control, Scale bar: 50µm. ference vector transfected into DF-1 cells, non-transfected cells were blank control, Scale bar: 50μm.(B) qRT-PCR (B) qRT was-PCR used was toused detect to detect the relative the relativeLin28B Lin28Bgene gene expression expression following followingsiLin28B siLin28B1-4 vector1-4 50μm. (B) qRT-PCR was used to detect the relative Lin28B gene expression following siLin28B1-4 vectortransfection. transfection. (C) Activity (C) Activity detection detection of Lin28B of Lin28Boverexpression overexpression and interference and interference vector vector by the by rescue the vector transfection. (C) Activity detection of Lin28B overexpression and interference vector by the rescue experiment. Different uppercase letters represent highly significant, and the same letters rescue experiment.experiment. Different uppercase Different uppercaseletters represent letters highly represent significant, highlysigniﬁcant, and the same and letters the same letters represent represent no significant. represent no significant.no signiﬁcant.

3.2. Lin28B Overexpression/Interference Could Promote/Inhibit PGCs Formation In Vivo

In order to investigate the role of Lin28B in the formation of PGCs, oeLin28B and siLin28B were injected into chicken embryos incubated for 2.5 days (E2.5, HH14) through embryo blood vessels, and were then incubated for 4.5 days after injection. qRT-PCR detected the expression of PGCs related genes in the genital ridges of chicken embryos that were incubated for 4.5 days. The results showed that, when oeLin28B was injected into the Animals 2021, 11, x 6 of 12 Animals 2021, 11, x 6 of 12

3.2. Lin28B Overexpression/Interference Could Promote/Inhibit PGCs Formation In Vivo 3.2. Lin28B Overexpression/Interference Could Promote/Inhibit PGCs Formation In Vivo In order to investigate the role of Lin28B in the formation of PGCs, oeLin28B and siLin28BIn wereorder injected to investigate into chicken the role embryos of Lin28B incubated in the forformation 2.5 days of(E2.5, PGCs, HH14) oeLin28B through and Animals 2021, 11, 43 embryosiLin28B blood were vessels, injected and into were chicken then embryos incubated incubated for 4.5 days for 2.5after days injection. (E2.5, HH14)qRT-PCR through6 of de- 12 tectedembryo the bloodexpression vessels, of PGCsand were related then genes incubated in the forgenital 4.5 days ridges after of chickeninjection. embryos qRT-PCR that de- weretected incubated the expression for 4.5 days of PGCs. The relatedresults showedgenes in thatthe ,genital when oe ridgesLin28B of waschicken injected embryos into the that were incubated for 4.5 days. The results showed that, when oeLin28B was injected into the genital ridgeridge ofof the the chicken chicken embryo, embryo,Lin28B Lin28Bgene gene expression expression was was significantly significantly up-regulated. up-regu- genital ridge of the chicken embryo, Lin28B gene expression was significantly up-regu- Contrastingly,lated. Contrastingly, when when siLin28B siLin28Bwas injected was injected into the into genital the genital ridge ridge of the of chicken the chicken embryo, em- lated. Contrastingly, when siLin28B was injected into the genital ridge of the chicken em- Lin28Bbryo, Lin28Bgene expression gene expression was significantly was significantly down-regulated. down This-regulated. suggests This that suggestsoeLin28B and that bryo, Lin28B gene expression was significantly down-regulated. This suggests that sioeLin28BLin28Bcould and si bothLin28B be expressedcould both in be chicken expressed embryo in chicken (Figure 3embryoA). Further (Figure gene 3A). expression Further oeLin28B and siLin28B could both be expressed in chicken embryo (Figure 3A). Further analysisgene expression showed analysis that, when showedLin28B thatwas, when overexpressed, Lin28B was theoverexpressed, PGC makers theCvh PGC, C-kit make, andrs gene expression analysis showed that, when Lin28B was overexpressed, the PGC makers Blimp1Cvh, C-werekit, and significantly Blimp1 were up-regulated, significantly whileup-regulated, the totipotency while the marker totipotency gene NANOG marker genewas Cvh, C-kit, and Blimp1 were significantly up-regulated, while the totipotency marker gene significantlyNANOG was down-regulated significantly down (Figure-regulated3B,C). Contrastingly,(Figure 3B,C). aContrastingly, contrary result a contrary was observed result NANOG was significantly down-regulated (Figure 3B,C). Contrastingly, a contrary result whenwas observedLin28B was when inhibited. Lin28B was In order inhibited to further. In order confirm to further the regulating confirm the role regulating of Lin28B rolein was observed when Lin28B was inhibited. In order to further confirm the regulating role theof Lin28B formation in the of formation PGCs, we of collected PGCs, we 4.5-day-old collected chicken 4.5-day- embryosold chicken with embryos different withLin28B dif- of Lin28B in the formation of PGCs, we collected 4.5-day-old chicken embryos with dif- treatments,ferent Lin28B prepared treatments, paraffin prepared slices, paraffin and quantified slices, and the changequantified in numbers the change of PGCsin numbers in the genitalofferent PGCsridges Lin28B in the through genitaltreatments, ridges PAS prepared staining. through Theparaffin PAS results sta slicesining. showed, andThe quantifiedresults that the showed number the change that of PGCsthe in number numbers in the genitalofof PGCs PGCs ridge in in the ofthe thegenital genital siLin28B ridge ridgesgroup of throughthe was siLin28B significantly PAS groupstaining. reducedwas The significantly results when comparedshowed reduced that to whenthe the control number com- groupparedof PGCs (25to the ±in 1.25 controlthe vs.genital 38group± ridge1.53, (25 ofp± < 1.25the 0.01). sivs.Lin28B Contrastingly,38 ± 1.53, group p < was 0.01). the significantly number Contrastingly, of PGCs reduced the in thenumber when genital com- of ridgesPGCspared in of to the the the genital oe controlLin28B ridges groupgroup of (25 the was ± oe 1.25 significantlyLin28B vs. 38 group ± 1.53, increased was p

FigureFigure 3.3. Lin28BLin28B overexpression/interferenceoverexpression/interference could promote/inhibit the the formation formation of PGCs in vivovivo..( (A–C) qRT-PCRqRT-PCR waswas usedusedFigure toto detectdetect 3. Lin28B thethe expressionexpression overexpression/interference ofof Lin28B,Lin28B, primaryprimary could germgerm cellspromote/inhibitcells (PGCs)(PGCs) markermarker the genesformationgenes (Cvh,(Cvh, of Ckit,Ckit PGCs, andand in Blimp1),Blimp1)vivo. (A,– andandC) qRT totipotency-PCR was markermarkerused to genegene detect (NANOG(NANOG the expression))in in vivo vivo.. of Different Different Lin28B, uppercase primaryuppercase germ letters letters cells represent represent (PGCs) highly marker highly significant, genessignificant, (Cvh, different differentCkit, and lowercase lowercase Blimp1) letters, and letters totipotency represent repre- significant,sentmarker significant, gene and ( the NANOGand same the )same letters in vivo. letters represent Different represent not uppercase significant. not significant. letters represent highly significant, different lowercase letters represent significant, and the same letters represent not significant.

Figure 4. Quantity detection of PGCs by Periodic Acid-Schiff(PAS) staining; up-scale bar: 200 μm; FiguredownFigure-scale 4. 4.Quantity Quantity bar: 40 detectionμm. detection Left: t ofhe of PGCs PGCs PGCs by were by Periodic Periodic marked Acid-Schiff(PAS) Acid by arrow,-Schiff(PAS) Right: staining; stainingstatistical; up-scale up analysis-scale bar: bar: of 200the 200 µ μm;m; down-scaledown-scale bar: bar: 40 40µ μm.m. Left:Left: t thehe PGCs PGCs were were marked marked by by arrow, arrow, Right: Right: statistical statistical analysis analysis of ofthe the number of PGCs in genital ridge after overexpression or interference of Lin28B. Different uppercase letters represent highly signiﬁcant, and the same letters represent no signiﬁcant.

3.3. Lin28B Could Promote Formation of EB in Bmp4-Induced Model In Vitro It is well known that ESCs could be induced into PGCs in vitro [22]. In order to further conﬁrm the function of Lin28B in the formation of PGCs, we transfected oeLin28B and Animals 20202121,, 1111,, xx 77 of 1212

number of PGCs in genital ridge after overexpression or interference of Lin28B.. DifferentDifferent upper-upper- casecase lettersletters representrepresent highlyhighly significant,significant, andand thethe samesame lettersletters representrepresent nono significant.significant. Animals 2021, 11, 43 7 of 12 3.3. Lin28B Could Promote Formation of EB in Bmp4--InducedInduced ModelModel IIn Vitro ItIt isis wellwell knownknown thatthat ESCsESCs couldcould bebe inducedinduced intointo PGCsPGCs inin vitrovitro [22][22].. InIn orderorder toto fur-fur- therther confirmconfirm thethe functionfunction ofof Lin28B inin thethe formationformation ofof PGCs,PGCs, wewe transfectedtransfected oeLin28B and siLin28B into ESCs, respectively, and then induced by BMP4 (Figure5A). The results of siLin28B intointo ESCsESCs,, respectivelyrespectively,, and then induced by BMP4 (Figure 5A). The results of qRT-PCR showed that oeLin28B and siLin28B could overexpress/inhibit the expression of qRT--PCR showed that oeLin28B and siLin28B could overexpress/inhibitinhibit thethe expressionexpression ofof Lin28B during PGCs formation (Figure5B). The results of morphological statistics showed Lin28B during PGCs formationformation (Figure(Figure 5B).5B). TheThe resultsresults ofof morphologicalmorphological statisticsstatistics showedshowed that cells expanded at day 2 after BMP4 induction (control group), a small number of EBs thatthat cellscells expandedexpanded atat dayday 22 afterafter BMP4BMP4 inductioninduction (control(control group),group), aa smallsmall numbernumber ofof EBsEBs appeared at day 4, and more EBs appeared at day 6. However, EB was not observed from appeared at day 4, and more EBs appeared at day 6. However, EB was not observed from day 2 to day 6 after Lin28B interference during BMP4 induction. Contrastingly, small EBs day 2 to day 6 after Lin28B interferenceinterference duringduring BMP4BMP4 induction.induction. Contrastingly,Contrastingly, smallsmall EBsEBs were observed at day 2, more EBs were observed and began to break at day 4, and, at day were observed at day 2, more EBs were observed and began to break at day 4, and, at day were6, the observed edge of EBs at day began 2, more to break EBs andwere small observed amounts and began of cells to were break released at day 4, from and the, at EBsday 6, the edge of EBs began to break and small amounts of cells were released from the EBs (Figure6, the edge6). This of EBs suggests began that to breakLin28 andcan small regulate amounts the formation of cells were of EBs. released from the EBs (Figure(Figure 6).6). ThisThis suggestssuggests thatthat Lin28 can regulate the formation of EBs.

Figure 5. Lin28B could promote the formation of embryoid bodies(EBs) in BMP4-induced model Figure 5. Lin28B could promote the formation of embryoid bodies(EBs) inin BMP4BMP4--inducedinduced modelmodel in vitro.(A) Schematic diagram of illustrating the Lin28B function via PGCs induction model in vitro. inin vitro.vitro. ((A)) SchematicSchematic diagramdiagram ofof illustratingillustrating thethe Lin28B function via PGCs induction model in vitro.(B) Expression ((B)) ExpressionExpression detection detectiondetection of Lin28B ofof Lin28Bduring during the induction the induction of PGCs. of Different PGCs. Different uppercase uppercase letters represent letters representrepresenthighly signiﬁcant highlyhighly significantsignificant and the same andand letters thethe ssame represent letters norepresent signiﬁcant. no significant.

Figure 6. Observation (Left) and statistics (Right) of the number of EBs during induction after Figure 6. ObservationObservation (Left (Left) and) and statistics statistics (Right (Right) of) the of the number number of EBs of EBsduring during induction induction after after different treatments of Lin28B.. ScaleScale bar:bar: 6060 μm. different treatments of Lin28B. Scale bar: 60 µm. 3.4. Lin28B Could Positively Modulate Formation of PGCs in Bmp4-Induced Model In Vitro In order to conﬁrm the effects of Lin28B on BMP4 induction, cells in different induction period were collected to detect the expression of NANOG, and marker genes of PGCs, such as Cvh, C-kit, and Blimp1 (Figure7A,B). The results showed that NANOG expression Animals 2021, 11, x 8 of 12

3.4. Lin28B Could Positively Modulate Formation of PGCs in Bmp4-Induced Model In Vitro Animals 2021, 11, 43 8 of 12 In order to confirm the effects of Lin28B on BMP4 induction, cells in different induction period were collected to detect the expression of NANOG, and marker genes of PGCs, such as Cvh, C-kit, and Blimp1 (Figure 7A,B). The results showed that NANOG expression did not differ didsignificantly not differ from significantly the normal from BMP4 the induction normal BMP4 (control) induction at day 2, (control) but it was at day 2, but it significantly upwas-regulated significantly at day up-regulated 4 and day 6 ( atp < day 0.01) 4 andafter day Lin28B 6 (p inhibition.< 0.01) after TheLin28B expres-inhibition. The sion of markerexpression genes of PGCs of marker was markedly genes of PGCs reduced was at markedly day 4 and reduced day 6 ( atp < day 0.01). 4 and Con- day 6 (p < 0.01). trastingly, althoughContrastingly, NANOGalthough was downNANOG-regulatedwas after down-regulated Lin28B overexpression, after Lin28B no signif-overexpression, no icant differencesignificant was observed difference when was compared observed to whenthe control. compared The marker to the control. genes of The PGCs marker genes of were not significantlyPGCs were expressed not significantly at day 2 after expressed Lin28B at overexpression day 2 after Lin28B as comparedoverexpression to the as compared control, but theyto the were control, markedly but theyup-regulated were markedly at day 4 up-regulated and day 6 (p at < day0.01). 4 T andhe six day-day 6 (-p < 0.01). The old cells weresix-day-old subjected to cells an wereindirect subjected immunofluorescence to an indirect immunofluorescencetest (Figure 7C). The testresults (Figure 7C). The showed that theresults proport showedion of thatCVH the + CKIT+ proportion was significantly of CVH + increased CKIT+ was after significantly Lin28B over- increased after expression as Lin28Bcomparedoverexpression to the control. as Contrastingly, compared to the the control.proportion Contrastingly, of CVH + CKIT+ the proportion was of CVH reduced significantly+ CKIT+ after was Lin28B reduced inhibition. significantly In conclusion, after Lin28B theinhibition. inhibition of In Lin28B conclusion, could the inhibition significantly inhibitof Lin28B thecould formation significantly of PGCs, inhibit while the Lin28B formation overexpression of PGCs, while couldLin28B signifi-overexpression cantly promotecould the formation significantly of PGCs. promote the formation of PGCs.

Figure 7. Lin28BFigure could 7. Lin28B positively could modulate positively the modulate formation the of formation PGCs in BMP4-inducedof PGCs in BMP4 model-inducedin vitro model.(A, Bin) qRT-PCR was used to detectvitro. the expression(A,B) qRT- ofPCRNANOG was used, Cvh to, Ckitdetect, and the Blimp1 expression during of theNANOG induction, Cvh, of Ckit PGCs, andin Blimp1 vitro. Different during uppercase the induction of PGCs in vitro. Different uppercase letters represent highly significant, different letters represent highly significant, different lowercase letters represent significant, and the same letters represent no lowercase letters represent significant, and the same letters represent no significant. (C) Indirect significant. (C) Indirect immunofluorescence detection of the efficiency of the formation of PGCs at day 6 in the induced immunofluorescence detection of the efficiency of the formation of PGCs at day 6 in the induced µ model after treatmentmodel after with treatment different with Lin28B differentin vitro Lin28B. Scale in bar: vitro 60. Scalem. bar: 60 μm.

3.5. Lin28B Promotes3.5. Lin28B Formation Promotes of PGCs Formation Through of PGCs Inhibition Through of gga Inhibition-Let-7a-3p of gga-Let-7a-3p The study resultsThe showed study results that Lin28B showed functions that Lin28B by bindingfunctions the by let7 binding family the [8]let7. A totalfamily [8]. A total of 17 gga-let7sof were 17 gga-let7s predicted were in chicken predicted by in online chicken software by online (method software for(method details) in for order details) in order to to investigateinvestigate the key micRNA the key micRNAlet7s targetedlet7s targeted by Lin28B by Lin28B(Figure(Figure 8A). 8 WeA). transfected We transfected si Lin28B siLin28B and oeandLin28B oeLin28B in DFin-1 DF-1 cells cells,, and andthen then detected detected the expression the expression of these of these micRNAs micRNAs with qRT- PCR. We found that only let-7a-2-3p, let-7a-3p, let-7b, and let-7k-5p of the 17 gga-let7s were signiﬁcantly regulated by Lin28B (the expression trend is opposite to Lin28B) (Figure8A). Additionally, we collected zero-day and four-day-old cells in the PGC induction model in vitro [22] and performed transcriptome sequencing (data not published), and found that Animals 2021, 11, x 9 of 12

with qRT-PCR. We found that only let-7a-2-3p, let-7a-3p, let-7b, and let-7k-5p of the 17 gga- Animals 2021, 11, 43 let7s were significantly regulated by Lin28B (the expression trend is opposite to Lin28B9 of 12) (Figure 8A). Additionally, we collected zero-day and four-day-old cells in the PGC induction model in vitro [22] and performed transcriptome sequencing (data not published), and found that there are five gga-let7s(let-7a-3p, let-7g-5p, let-7f-5p, let-7i, and let-7c-5p) in thethere process are ﬁve ofgga-let7s the formation(let-7a-3p of, let-7g-5pPGCs in, vitrolet-7f-5p, of ,whichlet-7i, andthe expressionlet-7c-5p) in of the let process-7a-3p, let of- the7g- 5pformation, let-7f-3p of, and PGCs letin-7i vitro showed, of which a downward the expression trend, ofwhichlet-7a-3p was, let-7g-5pconsistent, let-7f-3p with the, and expres-let-7i sionshowed rule aof downward gga-let7s during trend, the which formation was consistent of PGCs with(Figure the 8B) expression. However, rule we of found gga-let7s that afterduring the the overexpression formation of PGCs of Lin28B (Figure, let8-B).7g- However,5p, let-7f-3p we, and found let- that7i showed after the an overexpression upward trend, whichof Lin28B means, let-7g-5p that, ,inlet-7f-3p addition, and to let-7ilet-7a-showed3p, the maturation an upward process trend, whichof let-7g means-5p, let that,-7f-3p in, andaddition let-7i to islet-7a-3p not regulated, the maturation by Lin28B process. Moreover, of let-7g-5p we examined, let-7f-3p ,the and expressionlet-7i is not of regulated let-7a-3p inby ESCsLin28B and. Moreover, PGCs, andwe examined found that the let expression-7a-3p was of let-7a-3p significantlyin ESCs down and-regulated, PGCs, and whichfound showethat let-7a-3pd a completelywas signiﬁcantly opposite down-regulated, trend to the expression which showed of Lin28B a completely (Figure 8C) opposite. Combining trend Lin28B theseto the results, expression we therefore of conclude(Figure8 C).that Combining Lin28B regulates these results,the formation we therefore of chicken conclude PGCs that Lin28B regulates the formation of chicken PGCs through let-7a-3p. through let-7a-3p.

Figure 8. Lin28B promotes the formation of PGCs through the inhibition of gga-let-7a-3pgga-let-7a-3p.(. (A) qRT-PCRqRT-PCR was used to detect thethe expressionexpression ofof let-7let-7 familyfamily afterafter overexpressionoverexpression andand inhibitioninhibition ofof Lin28B,Lin28B, onlyonly let-7a-2-3plet-7a-2-3p,, let-7a-3plet-7a-3p,, let-7b,let-7b, andand let-7k-5plet-7k-5p showedshowed opposite trend to Lin28B.Lin28B. ((B)) miRNAmiRNA sequencingsequencing to screenscreen let-7let-7 ((let-7a-3plet-7a-3p, let-7g-5plet-7g-5p, let-7f-5plet-7f-5p, let-7ilet-7i, and let-7c-5plet-7c-5p) during the formation of PGCs in vitrovitro..( (C) qRT-PCRqRT-PCR waswas usedused toto detectdetect thethe expressionexpression ofof let-7a-3plet-7a-3p andand Lin28BLin28B duringduring thethe generation of PGCs in vivo. “*” means p < 0.05, and “**” means p < 0.01. generation of PGCs in vivo. “*” means p < 0.05, and “**” means p < 0.01. 4. Discussion 4. DiscussionThe research study deeply examined the function of Lin28B in the formation of PGCs, beingThe screened research and study obtained deeply the examined micRNA the modulated function ofbyLin28B Lin28Bin, and the preliminarily formation of PGCs, clari- fiedbeing the screened molecular and mechanism obtained the by micRNA which L modulatedin28B participates by Lin28B in ,the and process preliminarily of PGC clariﬁed genesis bythe inhibiting molecular the mechanism maturation by of which gga-letLin28B-7a-3p.participates in the process of PGC genesis by inhibitingDespite the that maturation both Lin28A of gga-let-7a-3p and Lin28B. are from the same RBP family, more research has focusedDespite on that Lin28A both Lin28A. As earlyand asLin28B 2009 [6]are, it from was the shown same that RBP Lin28A family, could more researchregulate hasthe PGCfocused genesis on Lin28A of mammals.. As early The as 2009underlying [6], it was molecular shown mechanism that Lin28A iscould that regulateLin28A could the PGC ac- tivategenesis the of expression mammals. of The Blimp1 underlying to participate molecular in mechanismthe formation is thatof PGCsLin28A by couldinhibiting activate the maturationthe expression of ofletBlimp1-7’s precursorto participate [18]. However, in the formation no relevant of PGCs study by inhibiting concerning the maturationLin28B has everof let-7 been’s precursor reported. [The18]. function However, of Lin28B no relevant in PGC study genesis concerning was onlyLin28B putativelyhas everpredicted been reported. The function of Lin28B in PGC genesis was only putatively predicted by in vitro induction experiment or by addressing the association between SNP loci and menstrual cycle, because there are SNP loci in Lin28B [14,23]. However, no deﬁnite conclusion has been drawn. In the subject study, we systematically examined the function of Lin28B in Animals 2021, 11, 43 10 of 12

the genesis of PGCs while using the chicken as a model animal. The results showed that, similar to Lin28A, Lin28B could also regulate PGCs genesis. Although the study object of the study is chicken, the results could serve as the foundation for understanding the specific function and mechanism of Lin28B gene family in PGCs genesis. Based on the results of this study, we can improve the regulatory role of Lin28 family in the formation of chicken PGCs, laying a foundation for the application of chicken PGCs in the production of transgenic animals, resource protection, and other fields With the in-depth study in Lin28, increasing numbers of scholars have started to examine the functioning mechanism of Lin28 in totipotency, genesis of germ cells, and tumorigenesis [1,4,24]. The results showed that Lin28A and its collateral derivative Lin28B are the key participants for Let-7 processing [25], which functions by modifying its processing or stability through binding to Let-7 pre-miRNA or pri-miRNA. [26] Nie et al. [27] found the link between Blimp1 and Let7a in Hodgkin’s lymphoma cells. They found that Let-7 was highly expressed in Reed–Sternberg cells, whereas Blimp1, a primary regulator for the differentiation of B cells, was significantly inhibited. The binding of Let-7a miRNA to the target site in 30UTR of Blimp1 was further observed, which could inhibit Blimp1 expression. This undoubtedly proven that Lin28 indirectly regulates Blimp1. It was not until 2009 when West et al. [6] confirmed that link between Lin28 and PGCs. West et al. knocked out Lin28A or Lin28B in the ESCs of Stella-GFP, which led to a small number of positive TNAP communities (an early marker of PGCs). They assumed that Lin28A and Lin28B could inhibit the expression of Blimp1 by inhibiting the expression of Let-7. In fact, the deletion of the let-7 locus in Blimp1 30UTR can rescue the phenotypic loss of PGCs due to Lin28 knockdown. In addition, an overexpression of Lin28 could induce the expression of more marker genes of TNAP-positive PGCs. Such findings confirmed the importance of Lin28 in PGCs genesis; our results filled the gap in the study of Lin28B in the formation of PGCs in chickens. Early studies of Lin28 and Let-7 focused on mammalian PGCs formation, with few reports on chickens. Therefore, it is the first time we demonstrated the key role of Let-7 miRNA biosynthesis in PGCs. We expanded this finding from mammalian to the poultry area, studied and analyzed the function of Lin28B in PGC genesis, and targeted it to gga-let-7a-3p. Our study may provide a constructive basis for future in-depth research.

5. Conclusions In this study, we confirmed that Lin28B promoted the formation of chicken PGCs through in vivo and in vitro experiments. The let-miRNA family that was opposite the expression of Lin28B was identified by combining the results of high-throughput sequencing, and it confirmed that Lin28B promoted the formation of chicken PGCs by inhibiting the maturation of gga-let-7a-3p.

Supplementary Materials: The following are available online at https://www.mdpi.com/2076-261 5/11/1/43/s1, Table S1, Target sites sequence of Lin28B gene; Table S2 qRT-PCR primers sequence of related genes; Table S3, qRT-PCR primers sequence of related genes. Author Contributions: B.L. conceived and designed the experiments; Q.Z. performed the experiments; Q.Z. analyzed the data; Q.Z., J.Z. wrote the manuscript; G.C., Y.Z. edited the manuscript; M.W. revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Jiangsu Science and Technology Project (Youth Fund): BK20180918; Natural Science Research Project of Jiangsu Higher Education Institutions: 18KJB230008; Key Research and Development Program); National Natural Science Foundation of China (31872341, 31572390). Institutional Review Board Statement: All experimental procedures in the present study were reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University (approval number: 151-2014). Procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (Yangzhou University, China, 2012) and the Standards for the Administration of Experimental Practices (Jiangsu, China, 2008). We also conﬁrm that the ﬁeld studies did not involve endangered or protected species. Informed Consent Statement: Not applicable. Animals 2021, 11, 43 11 of 12

Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors thank the Poultry Institute of the Chinese Academy of Agricultural Sciences Experimental Poultry Farm for providing experimental materials. Conﬂicts of Interest: The authors declare that there is no conﬂict of interest.

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