animals Article Long Noncoding RNA LncPGCR Mediated by TCF7L2 Regulates Primordial Germ Cell Formation in Chickens Jingyi Jiang 1, Chen Chen 1, Shaoze Cheng 1, Xia Yuan 1, Jing Jin 1, Chen Zhang 1, Xiaolin Sun 1 , Jiuzhou Song 2 , Qisheng Zuo 1, Yani Zhang 1, Guohong Chen 1 and Bichun Li 1,* 1 Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu, China; [email protected] (J.J.); [email protected] (C.C.); [email protected] (S.C.); [email protected] (X.Y.); [email protected] (J.J.); [email protected] (C.Z.); [email protected] (X.S.); [email protected] (Q.Z.); [email protected] (Y.Z.); [email protected] (G.C.) 2 Animal & Avian Sciences, University of Maryland, College Park, MD 20741, USA; [email protected] * Correspondence: [email protected] Simple Summary: The potential of primordial germ cells (PGCs) for multidirectional differentiation, together with their unique regeneration ability, makes them one of the most promising seed cells in clinical medicine and tissue engineering research. However, not enough PGCs can be obtained to meet the demand, which limits their application. We defined a novel long noncoding RNA (lncRNA) mediated by epigenetics, which could activate the miR-6577-5p/Btrc pathway to promote the formation of PGCs. The technical system we have established is a useful tool to obtain sufficient PGCs for scientific research. Our study offers great theoretical and practical value in the production of transgenic animals or genomic imprinting in poultry. We believe that our study will help researchers Citation: Jiang, J.; Chen, C.; Cheng, in the fields of agricultural production, developmental biology, and cell biology. S.; Yuan, X.; Jin, J.; Zhang, C.; Sun, X.; Song, J.; Zuo, Q.; Zhang, Y.; et al. Abstract: Although lncRNAs have been identified as playing critical roles in the development of Long Noncoding RNA LncPGCR germ cells, their potential involvement in the development of PGCs in chickens remains poorly Mediated by TCF7L2 Regulates understood. Differentially expressed lncRNAs (DELs) from previous RNA-seq of embryonic stem Primordial Germ Cell Formation in Chickens. Animals 2021, 11, 292. cells (ESCs), PGCs, and spermatogonial stem cells (SSCs) were analyzed by K-means clustering, from https://doi.org/10.3390/ani11020292 which a key candidate, lncRNA (lncRNA PGC regulator, LncPGCR) was obtained. We confirmed that LncPGCR plays a positive role in the development of PGCs by increasing the expression of the PGC Academic Editors: Duy Ngoc Do and marker gene (Cvh and C-kit), while downregulating the pluripotency-associated gene (Nanog) in vitro Prashanth N Suravajhala and in vivo. The activation and expression of LncPGCR are regulated by histone acetylation, and Received: 28 December 2020 transcription factor TCF7L2. Mechanistically, a rescue assay was performed to further confirm that Accepted: 19 January 2021 LncPGCR contributed to the development of PGCs by regulating the gga-miR-6577-5p/Btrc signaling Published: 24 January 2021 pathway. Adsorption of gga-miR-6577-5p activated the WNT signaling cascade by relieving the gga-miR-6577-5p-dependent inhibition of Btrc expression. Taken together, our study discovered the Publisher’s Note: MDPI stays neutral growth-expedited role of LncPGCR in PGCs development, showing the potential LncPGCR/miR- with regard to jurisdictional claims in 6577-5p/Btrc pathway. The results and findings provide a novel insight into the development published maps and institutional affil- of PGCs. iations. Keywords: chicken (Gallus gallus); LncRNA; primordial germ cell; TCF7L2; Btrc; ceRNA Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and Chicken primordial germ cells (PGCs) would be invaluable for in vitro studies of conditions of the Creative Commons aspects of chicken embryogenesis. The multidirectional differentiation potential and unique Attribution (CC BY) license (https:// migration pathway are chicken PGCs’ major biological properties, which can be applied to creativecommons.org/licenses/by/ generate genetically modified poultry and conserve poultry’s genetic resources [1]. The 4.0/). induction of paracrine signaling [2,3], inhibition of somatic fate [4], alteration of epigenetic Animals 2021, 11, 292. https://doi.org/10.3390/ani11020292 https://www.mdpi.com/journal/animals Animals 2021, 11, x 2 of 14 [1]. The induction of paracrine signaling [2,3], inhibition of somatic fate [4], alteration of Animals 2021, 11, 292 epigenetic marks [5], and maintenance of pluripotency [6] are important to the2 of develop- 13 ment of PGCs. Considerable effort has been devoted to maintaining and propagating chicken PGCs in vitro. However, the limited number of PGCs which can be obtained greatlymarks [limits5], and their maintenance clinical application. of pluripotency Ther [6efore,] are importantin-depth exploration to the development is needed of of the developmentPGCs. Considerable mechanism effort of has PGC, been to devoted overcome to maintaining the limitations, and and propagating improve chicken our technical abilityPGCs into vitroeffectively. However, cultivate the limited or induce number PGCs of PGCs in vitro. which can be obtained greatly limits theirSince clinical the application. discovery Therefore,of PGCs, in-depthresearchers exploration have devoted is needed extensive of the development attention to the studymechanism of their of developmental PGC, to overcome mechanism, the limitations, which and is improvea complex our and technical sophisticated ability to regula- toryeffectively process, cultivate affected or induceby many PGCs factors in vitro. (Figure 1). Studies have shown that genes [4,7–9], Since the discovery of PGCs, researchers have devoted extensive attention to the signal pathways [10,11], and growth factors [12,13] all play important roles in the devel- study of their developmental mechanism, which is a complex and sophisticated regulatory opmentprocess, of affected PGCs by in manymice, factors and research (Figure1 ).into Studies their have mechanism shown that has genes gradually [ 4,7–9], turned signal to the fieldpathways of epigenetics [10,11], and [14]. growth The factors development [12,13] all playof PGCs important has been roles insubjected the development to an epigenetic of reprogrammingPGCs in mice, and process research that into is their genome-wide mechanism has[15,16], gradually including turned genome-wide to the field of epige-DNA meth- ylationnetics [ 14erasure,]. The development chromosome of inactivation, PGCs has been hi subjectedstone modification, to an epigenetic and reprogramming transposon silencing [17].process Current that is research genome-wide on chickens [15,16], includingshows only genome-wide that genes DNAsuch methylationas Wnt5A, and erasure, FGF8 may regulatechromosome the formation inactivation, of histonetheir primordial modification, germ and cells transposon [18–20]. silencing However, [17 ].the Current efficiency of inducingresearch onthe chickens production shows of onlyPGCs that in genesvitro ha suchs not as Wnt5Ayet achieved, and FGF8 the expectedmay regulate levels. the There- formation of their primordial germ cells [18–20]. However, the efficiency of inducing the fore, there is an urgent need for innovative exploration of the developmental mechanism production of PGCs in vitro has not yet achieved the expected levels. Therefore, there is an ofurgent chicken need PGCs for innovative from new exploration directions. of the developmental mechanism of chicken PGCs from new directions. Figure 1. Development of mouse and chicken primordial germ cells. FigureThe 1. Development recent release of of mouse an ultra-high-throughput and chicken primordial approach germ cells. to the sequencing of RNA (RNA-seq) has paved the way for identification and annotation of numerous long noncod- ing RNAsThe recent in the release chicken of genome an ultra-high-throughput [21]. Also, a quantity ofapproach binding sitesto the for sequencing PGC marker of RNA (RNA-seq)genes (Blimp1/Prdm1 has paved) werethe way found for to identification be related to lncRNAand annotation in mouse of PGCs numerous [7]. Therefore, long noncod- inglncRNAs RNAs havein the recently chicken come genome to the [21]. forefront Also, as a functionalquantity of molecules binding involvedsites for inPGC cell marker genesdifferentiation (Blimp1/Prdm1 by interacting) were withfound microRNA to be related [22], recruitingto lncRNA proteins in mouse as scaffolds PGCs [7]. to form Therefore, lncRNAscomplexes have [23], recently and trapping come transcription to the forefront factors as [ 24functional,25]. While molecules different expressioninvolved in po- cell dif- ferentiationsitions of LncRNA by interacting exhibit distinct with microRNA molecular mechanisms [22], recruiting [26,27 ],proteins the specific as scaffolds regulatory to form mechanism of LncRNA during the differentiation of ESCs into PGCs remains unclear. complexes [23], and trapping transcription factors [24,25]. While different expression po- Here, we provide evidence that LncRNAs intimately regulate PGC development sitionsthrough of a LncRNA novel network exhibit of functionaldistinct molecular interaction mechanisms between lncRNAs [26,27], and the miRNAs. specific In thisregulatory mechanismstudy, we have of LncRNA identified during a novel the PGC-specific differentiati