RNA-Seq identifies genes whose proteins are upregulated during syncytia development in murine C2C12 myoblasts and human BeWo trophoblasts

Abstract

The fusion of villous cytotrophoblasts into the multinucleated syncytiotrophoblast is critical for the essential functions of the mammalian placenta. Using RNA-Seq gene expression, quantitative protein expression, and siRNA knockdown we identified genes and their cognate proteins which are similarly upregulated in two cellular models of mammalian syncytia development (human BeWo cytotrophoblast to syncytiotrophoblast and murine C2C12 myoblast to myotube). These include DYSF, PDE4DIP, SPIRE2, NDRG1, PLEC, GPR146, HSPB8, DHCR7, and HDAC5. These findings provide avenues for further understanding of the mechanisms underlying mammalian placental syncytiotrophoblast development.

Keywords: cell fusion; placenta; syncytialization.

Figure 1

RNA‐Seq gene expression heat maps of triplicate samples for C2C12 cells at 0d (mb) and 6d (mt) in differentiation media. In the plot, the arrowhead denotes the sample used to order the heat map

Figure 2

Principle components analysis of C2C12 cell gene expression data. Analysis was performed in R. The first and second principal components are plotted on the x‐ and y‐axis, respectively

Figure 3

The most highly expressed genes in (a) BeWo cells and (b) C2C12 cells. In each panel, the 50 most highly expressed genes in the indicated sample were selected and used to sort the heat map. The expression levels of those genes in each of the other samples is also shown

Figure 4

The set of genes that is differentially expressed upon fusion in both C2C12 and BeWo cells. Each gene that met our fold change and expression criteria was plotted by its fold change in C2C12 cells cultured in differentiation media from 0d (mb) to 6d (mt) on the x‐axis and the fold change in BeWo ‐forskolin (cyt) or +forskolin (syn) for 72h on the y‐axis. Only genes that were upregulated in both samples were retained

Figure 5

(a) RNA‐Seq analysis and (b) quantitative Western blot analysis of the three most upregulated genes of interest in BeWo cells ‐forskolin (cyt) or +forskolin (syn) and C2C12 cells in differentiation media at 0d (mb) or 6d (mt). RNA‐Seq results are expressed in transcripts per million (TPM). Immunoblots were quantified and normalized to levels of actin; note, multiple panels in Figures 5 and 6 used the same actin control

Figure 6

(a) RNA‐Seq analysis and (b) quantitative Western blot analysis of the six remaining upregulated genes of interest in BeWo cells –forskolin (cyt) or + forskolin (syn) and C2C12 cells in differentiation media at 0d (mb) or 6d (mt). RNA‐Seq results are expressed in transcripts per million (TPM). Immunoblots were quantified and normalized to levels of actin; note, multiple panels in Figures 5 and 6 used the same actin control

Figure 7

Effect of siRNA knockdown of hCGβ or DYSF expression on the syncytialization of BeWo cells. (a) Representative double‐label immunofluorescence of BeWo cytotrophoblasts (left) and syncytiotrophoblasts (right). BeWo cells ‐forskolin (cyt) or +forskolin (syn), were fixed, and nuclei were stained with DAPI (shown in magenta), while cell junctions were stained with anti‐ZO1 antibody (pseudo‐colored in yellow). Insets of syncyntia shown on the right were magnified 2‐fold. (b) Results of nuclei counts used to determine percent syncytialization and (c) representative Western blots of BeWo cells transfected with siRNA targeting the genes indicated (hCGβ and DYSF) and treated with either vehicle (cyt) or forskolin (syn) for 72h as described in Materials and Methods

Figure 8

Immunochemical staining of normal term human placental villi. Serial sections of normal term human placental villi samples were immunostained with antibodies to upregulated proteins and hematoxylin & eosin, as indicated. Robust HRP staining of syncytiotrophoblasts is clearly visible for upregulated proteins

Figure 9

Triplicate Western blots of upregulated genes of interest in BeWo cells ‐forskolin (cyt) or +forskolin (syn) and C2C12 cells in differentiation media for 0d (mb) or 6d (mt). Blots are grouped with the actin blot that was used for their normalization. hCGb and myosin heavy chain (MHC) are included as markers of syncytialization in BeWo cells and differentiation in C2C12 cells, respectively