Yamei Xiao

Genetic modification and screening in rat using haploid embryonic stem cells.

The rat is an important animal model in biomedical research, but practical limitations to genetic manipulation have restricted the application of genetic analysis. Here we report the derivation of rat androgenetic haploid embryonic stem cells (RahESCs) as a tool to facilitate such studies. Our approach is based on removal of the maternal pronucleus from zygotes to generate androgenetic embryos followed by derivation of ESCs. The resulting RahESCs have 21 chromosomes, express pluripotency markers, differentiate into three germ layer cells, and contribute to the germline. Homozygous mutations can be introduced by both large-scale gene trapping and precise gene targeting via homologous recombination or the CRISPR-Cas system. RahESCs can also produce fertile rats after intracytoplasmic injection into oocytes and are therefore able to transmit genetic modifications to offspring. Overall, RahESCs represent a practical tool for functional genetic studies and production of transgenic lines in rat.

Protein phosphatase-1 regulates Akt1 signal transduction pathway to control gene expression, cell survival and differentiation

AKT pathway has a critical role in mediating signaling transductions for cell proliferation, differentiation and survival. Previous studies have shown that AKT activation is achieved through a series of phosphorylation steps: first, AKT is phosphorylated at Thr-450 by JNK kinases to prime its activation; then, phosphoinositide-dependent kinase 1 phosphorylates AKT at Thr-308 to expose the Ser-473 residue; and finally, AKT is phosphorylated at Ser-473 by several kinases (PKD2 and others) to achieve its full activation. For its inactivation, the PH-domain containing phosphatases dephosphorylate AKT at Ser-473, and protein serine/threonine phosphatase-2A (PP-2A) dephosphorylates it at Thr-308. However, it remains unknown regarding which phosphatase dephosphorylates AKT at Thr-450 during its inactivation. In this study, we present both in vitro and in vivo evidence to show that protein serine/threonine phosphatase-1 (PP-1) is a major phosphatase that directly dephosphorylates AKT to modulate its activation. First, purified PP-1 directly dephosphorylates AKT in vitro. Second, immunoprecipitation and immunocolocalization showed that PP-1 interacts with AKT. Third, stable knock down of PP-1α or PP-1β but not PP-1γ, PP-2Aα or PP-2Aβ by shRNA leads to enhanced phosphorylation of AKT at Thr-450. Finally, overexpression of PP-1α or PP-1β but not PP-1γ, PP-2Aα or PP-2Aβ results in attenuated phosphorylation of AKT at Thr-450. Moreover, our results also show that dephosphorylation of AKT by PP-1 significantly modulates its functions in regulating the expression of downstream genes, promoting cell survival and modulating differentiation. These results show that PP-1 acts as a major phosphatase to dephosphorylate AKT at Thr-450 and thus modulate its functions.

Protein Phosphatase-1 Modulates the Function of Pax-6, a Transcription Factor Controlling Brain and Eye Development

Pax-6 is an evolutionarily conserved transcription factor and acts high up in the regulatory hierarchy controlling eye and brain development in humans, mice, zebrafish, and Drosophila. Previous studies have shown that Pax-6 is a phosphoprotein, and its phosphorylation by ERK, p38, and homeodomain-interacting protein kinase 2 greatly enhances its transactivation activity. However, the protein phosphatases responsible for the dephosphorylation of Pax-6 remain unknown. Here, we present both in vitro and in vivo evidence to show that protein serine/threonine phosphatase-1 is a major phosphatase that directly dephosphorylates Pax-6. First, purified protein phosphatase-1 directly dephosphorylates Pax-6 in vitro. Second, immunoprecipitation-linked Western blot revealed that both protein phosphatase-1α and protein phosphatase-1β interact with Pax-6. Third, overexpression of protein phosphatase-1 in human lens epithelial cells leads to dephosphorylation of Pax-6. Finally, inhibition of protein phosphatase-1 activity by calyculin A or knockdown of protein phosphatase-1α and protein phosphatase-1β by RNA interference leads to enhanced phosphorylation of Pax-6. Moreover, our results also demonstrate that dephosphorylation of Pax-6 by protein phosphatase-1 significantly modulates its function in regulating expression of both exogenous and endogenous genes. These results demonstrate that protein phosphatase 1 acts as a major phosphatase to dephosphorylate Pax-6 and modulate its function.

The Goldfish SG2NA Gene Encodes Two α-Type Regulatory Subunits for PP-2A and Displays Distinct Developmental Expression Pattern

SG2NA is a member of the striatin protein family. In human and mouse, the SG2NA gene encodes two major protein isoforms: SG2NAα and SG2NAβ. The functions of these proteins, except for acting as the regulatory subunits for PP-2A, remain largely unknown. To explore the possible functions of SG2NA in lower vertebrates, we have isolated two SG2NA cDNAs from goldfish, Carassius auratus. Our results reveal that the first cDNA contains an ORF of 2118 bp encoding a deduced protein with 705 amino acids, and the second one 2148 bp coding for a deduced protein of 715 amino acids. Comparative analysis reveals that both isoforms belong to the α-type, and are named SG2NAα and SG2NAα+. RT-PCR and western blot analysis reveal that the SG2NA gene is differentially expressed in 9 tissues examined. During goldfish development, while the SG2NA mRNAs remain relatively constant in the first 3 stages and then become decreased and fluctuated from gastrula to larval hatching, the SG2NA proteins are fluctuated, displaying a peak every 3 to 4 stages. Each later peak is higher than the earlier one and the protein expression level becomes maximal at hatching stage. Together, our results reveal that SG2NA may play an important role during goldfish development and also in homeostasis of most adult tissues.

Involvement of JNK in the Embryonic Development and Organogenesis in Zebrafish

Abstractc-Jun N-terminal kinase (JNK) is one of the mitogen-activated protein kinases. Previous studies showed that the JNK is involved in signaling pathways initiating cell cycle, and eventually, causing apoptosis through persistent activation in mammals. In this article, it is further revealed that the jnk1 gene is closely related with the embryonic development and organogenesis in zebrafish. RT-PCR and Western blot analysis show that there were distinct expression patterns of JNK at the different developmental stages as well as in the various tissues in zebrafish. Knockdown of jnk1 by RNA interference (RNAi) resulted in high lethal, serious retardation and malformations of embryos in zebrafish. SP600125, a JNK-specific inhibitor, gives rise to high mortality in zebrafish, similar to that caused by the jnk1 RNA interference. SP600125 is also responsible for the severe abnormality of organs, especially the skeletal system, such as skull, mandible deficiency, and cyrtosis heterauxesis. The results also indicate that the inhibition of JNK by SP600125 suppresses the ovarian differentiation during the embryo development in zebrafish. Overall, our study demonstrates that the jnk1 gene is required for ovary differentiation and development in the zebrafish, and down-regulated JNK directly inhibits ovary differentiation during early ontogenetic stages.

Generation of Transgenic Rats through Induced Pluripotent Stem Cells

Background: Rat induced pluripotent stem cells (riPSCs) failed to produce transgenic rats. Results: We found that an optimized induction medium improved the efficiency of iPSC generation from rat somatic cells. The riPSCs could successfully generate transgenic rats. Conclusion: We could generate high quality riPSCs that could be used to produce transgenic rats. Significance: RiPSCs can be used as a novel tool in genetic and genomic studies of the rat. The rat is an important animal model for human disease research. Using inhibitors of glycogen synthase kinase 3 and MAPK signaling pathways, rat embryonic stem cells and rat induced pluripotent stem cells (riPSCs) have been derived. However, unlike rat embryonic stem cells, germ line competent riPSCs have only been derived from Wistar rats at low efficiency. Here, we found that an optimized induction medium containing knock-out serum replacement and vitamin C improved the rate and efficiency of riPSCs generation from Dark Agouti rat fibroblasts and Sertoli cells. riPSCs maintained an undifferentiated status for >30 passages and could differentiate into various cells types including germ cells when injected into rat blastocysts. Moreover, transgenic riPSCs could be generated through the PiggyBac transposon, which could be used to generate transgenic rats through germ line transmission. riPSCs can be used as a novel tool in genetic and genomic studies of the rat.

The p53-Bak apoptotic signaling axis plays an essential role in regulating differentiation of the ocular lens.

The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle checking. In our previous studies, we demonstrated that p53 directly regulates Bak in mouse JB6 cells (Qin et al. 2008. Cancer Research. 68(11):4150) and that p53-Bak signaling axis plays an important role in mediating EGCG-induced apoptosis. Here, we demonstrate that the same p53-Bak apoptotic signaling axis executes an essential role in regulating lens cell differentiation. First, during mouse lens development, p53 is expressed and differentially phosphorylated at different residues. Associated with p53 expression, Bak is also significantly expressed during mouse lens development. Second, human p53 directly regulates Bak promoter and Bak expression in p53 knockout mice (p53-/-) was significantly downregulated. Third, during in vitro bFGF-induced lens cell differentiation, knockdown of p53 or Bak leads to significant inhibition of lens cell differentiation. Fourth, besides the major distribution of Bak in cytoplasm, it is also localized in the nucleus in normal lens or bFGF-induced differentiating lens cells. Finally, p53 and Bak are co-localized in both cytoplasm and nucleus, and their interaction regulates the stability of p53. Together, these results demonstrate for the first time that the p53-Bak apoptotic signaling axis plays an essential role in regulating lens differentiation.

The PP2A-Aβ gene is regulated by multiple transcriptional factors including Ets-1, SP1/SP3, and RXRα/β

Protein phosphatase-2A (PP-2A) is a major serine/threonine phosphatase abundantly expressed in eukaryotes. PP-2A is a heterotrimer that contains a 65 kD scaffold A subunit, a 36 kD catalytic C subunit, and a regulatory B subunit of variable isoforms ranging from 54-130 kDs. The scaffold subunits, PP2A-Aα/β, act as platforms for both the C and B subunits to bind, and thus are key structural components for PP-2A activity. Mutations in both genes encoding PP2A-Aα and PP2A-Aβ lead to carcinogenesis and likely other human diseases. Our previous work showed that the gene coding for PP2A-Aα is positively regulated by multiple transcription factors including Ets-1, CREB, and AP-2α but negatively regulated by SP-1/SP-3. In the present study, we have functionally dissected the promoter of the mouse PP2A-Aβ gene. Our results demonstrate that three major cis-elements, including the binding sites for Ets-1, SP1/SP3, and RXRα/β, are present in the proximal promoter of the mouse PP2A-Aβ gene. Gel mobility shifting assays reveal that Ets-1, SP1/SP3, and RXRα/β all bind to PP2A-Aβ gene promoter. In vitro mutagenesis and reporter gene activity assays demonstrate that while Ets-1 displays negative regulation, SP1/SP3 and RXRα/β positively regulate the promoter of the PP2A-Aβ gene. Co-expression of the cDNAs encoding Ets-1, SP1/SP3, or RXRα/β and the luciferase reporter gene driven by PP2A-Aβ promoter further confirm their control over the PP2A-Aβ promoter. Finally, ChIP assays demonstrate that Ets-1, SP1/SP3, and RXRα/β can all bind to the PP2A-Aβ gene promoter. Together, our results reveal that multiple transcription factors regulate the PP2A-Aβ gene. Moreover, our results provide important information explaining why PP2A-Aα and PP2A-Aβ display distinct expression levels.

Comparative transcriptome analysis of molecular mechanism underlying gray-to-red body color formation in red crucian carp ( Carassius auratus , red var.)

Red crucian carp (Carassius auratus red var.) is an ornamental fish with vivid red/orange color. It has been found that the adult body color of this strain forms a gray-to-red change. In this study, skin transcriptomes of red crucian carp are first obtained for three different stages of body color development, named by gray-color (GC), color-variation (CV), and red-color (RC) stages, respectively. From the skins of GC, CV, and RC, 103,229; 108,208; and 120,184 transcripts have been identified, respectively. Bioinformatics analysis reveals that 2483, 2967, and 4473 unigenes are differentially expressed between CV and GC, RC and CV, and RC and GC, respectively. A part of the differentially expressed genes (DEGs) are involved in the signaling pathway of pigment synthesis, such as the melanogenesis genes (Mitfa, Pax3a, Foxd3, Mc1r, Asip); tyrosine metabolism genes (Tyr, Dct, Tyrp1, Silva, Tat, Hpda); and pteridine metabolism genes (Gch, Xdh, Ptps, Tc). According to the data of transcriptome and quantitative PCR, the expression of Mitfa and its regulated genes which include the genes of Tyr, Tyrp1, Dct, Tfe3a, and Baxα, decreases with gray-to-red change. It is suggested that Mitfa and some genes, being related to melanin synthesis or melanophore development, are closely related to the gray-to-red body color transformation in the red crucian carp. Furthermore, the DEGs of cell apoptosis and autophagy pathway, such as Tfe3a, Baxα, Hsp70, Beclin1, Lc3, Atg9a, and Atg4a, might be involved in the melanocytes fade away of juvenile fish. These results shed light on the regulation mechanism of gray-to-red body color transformation in red crucian carp, and are helpful to the selective breeding of ornamental fish strains.

Comparative Transcriptome and DNA methylation analyses of the molecular mechanisms underlying skin color variations in Crucian carp ( Carassius carassius L. )

BackgroundCrucian carp is a popular ornamental strain in Asia with variants in body color. To further explore the genetic mechanisms underlying gray and red body color formation in crucian carp, the skin transcriptomes and partial DNA methylation sites were obtained from red crucian carp (RCC) and white crucian carp (WCC). Here, we show significant differences in mRNA expression and DNA methylation sites between skin tissues of RCC and WCC.ResultsTotals of 3434 and 3683 unigenes had significantly lower and higher expression in WCC, respectively, compared with unigenes expressed in RCC. Some potential genes for body color development were further identified by quantitative polymerase chain reaction, such as mitfa, tyr, tyrp1, and dct, which were down-regulated, and foxd3, hpda, ptps, and gch1, which were up-regulated. A KEGG pathway analysis indicated that the differentially expressed genes were mainly related to mitogen activated protein kinase (MAPK), Wnt, cell cycle, and endocytosis signaling pathways, as well as variations in melanogenesis in crucian carp. In addition, some differentially expressed DNA methylation site genes were related to pigmentation, including mitfa, tyr, dct, foxd3, and hpda. The differentially expressed DNA methylation sites were mainly involved in signaling pathways, including MAPK, cAMP, endocytosis, melanogenesis, and Hippo.ConclusionsOur study provides the results of comparative transcriptome and DNA methylation analyses between RCC and WCC skin tissues and reveals that the molecular mechanism of body color variation in crucian carp is strongly related to disruptions in gene expression and DNA methylation during pigmentation.