Knockdown of Lmo7 inhibits chick myogenesis

Abstract

Edited by Dietmar MansteinThe multifunctional protein Lmo7 has been implicated in some aspects of myogenesis in mammals. Here we studied the distribution and expression of Lmo7 and the effects of Lmo7 knockdown in primary cultures of chick skeletal muscle cells. Lmo7 was localized within the nuclei of myoblasts and at the perinuclear region of myotubes. Knockdown of Lmo7 using siRNA specific to chick reduces the number and width of myotubes and the number of MyoD positive-myoblasts. Both Wnt3a enriched medium and Bio, activators of the Wnt/beta-catenin pathway, could rescue the effects of the Lmo7 knockdown suggesting a crosstalk between the Wnt/beta-catenin and Lmo7-mediated signaling pathways. Our data shows a role of Lmo7 during the initial events of chick skeletal myogenesis, particularly in myoblast survival.

The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis

“…A transcriptome analysis of MbCD-treated chick muscle cells revealed multiple changes, involving cell proliferation (cell cycle and p53 signaling), cell adhesion and cytoskeleton (focal adhesion, tight junctions, adherens junctions, gap junctions, and actin cytoskeleton), membrane trafficking-related processes (phagosome, lysosome, and endocytosis), and cell death (apoptosis and autophagy). Among all transcripts that were affected by cholesterol depletion, the levels of Lim domain only protein 7 (Lmo7) mRNA were the most upregulated (Possidonio et al, 2014a(Possidonio et al, , 2016. Lmo7 is a multifunctional protein that can be found in the nucleus, cytoplasm and/or at adhesion junctions in many tissues (Figure 1), with high levels of expression in skeletal muscle, where it has a role as a transcription factor regulating the expression of many skeletal muscle genes, including Pax3, Pax7, MyoD, and Myf5 (Holaska et al, 2006;Possidonio et al, 2016).…”

“…Among all transcripts that were affected by cholesterol depletion, the levels of Lim domain only protein 7 (Lmo7) mRNA were the most upregulated (Possidonio et al, 2014a(Possidonio et al, , 2016. Lmo7 is a multifunctional protein that can be found in the nucleus, cytoplasm and/or at adhesion junctions in many tissues (Figure 1), with high levels of expression in skeletal muscle, where it has a role as a transcription factor regulating the expression of many skeletal muscle genes, including Pax3, Pax7, MyoD, and Myf5 (Holaska et al, 2006;Possidonio et al, 2016).…”

“…The meaning of these nuclear movements is still elusive during skeletal myogenesis, but the perinuclear region is becoming a focus of studies because of its highly dynamic and specialized concentration of molecules. The juxtanuclear area of muscle cells preferentially harbor specific organelles, such as centrosomes and lysosomes, as well as a number of structural and signaling proteins, such as desmin, Gli-1, and Lmo7 (Figure 1; Mermelstein et al, 2006;Possidonio et al, 2016;Teixeira et al, 2018;Bagri et al, 2020). Interestingly, at the end of skeletal muscle differentiation in primary chick muscle cell cultures, a single myotube may exhibit hundreds of nuclei within its cytoplasm (Figure 3), whereas myotubes formed in cultures of widely used skeletal muscle cell lines, such as mouse C2C12, rat L6 and L8 cells, and human CL25, are usually composed by only a few (2-20) nuclei.…”

Acidic Compartment Size, Positioning, and Function during Myogenesis and Their Modulation by the Wnt/Beta-Catenin Pathway

“…siRNA sequences specific for LAMP-2 of Gallus gallus (mRNA sequence obtained from NCBI reference sequence NM_001030295) or enhanced green fluorescent protein (eGFP) were from Integrated DNA Technologies (USA), as previously described [ 12 ]. The Gallus gallus sequences of siRNA that were selected to target LAMP-2 or eGFP are the following: eGFP from Gallus gallus : 5′rArArGrCrUrGrArCrCrCrUrGrArArGrUrUrCrArUrCrUrGCA 3′ (strand) 5′rUrGrCrArGrArUrGrArArCrUrUrCrArGrGrGrUrCrArGrCrUrUrGrC 3′ (passenger) LAMP-2 from Gallus gallus (seq 1467-1492): Sense 5′ rGrArArUrUrGrGrArArArUrUrUrArCrUrUrGrArArGrCrUAC 3′ Antisense 5′ rGrUrArGrCrUrUrCrArArGrUrArArArUrUrUrCrCrArArUrUrCrArU 3′ …”

γ‐Secretase Inhibition Induces Muscle Hypertrophy in a Notch‐Independent Mechanism

“…Design and selection of all siRNA sequences, specific for Notch‐1 of Gallus gallus (mRNA sequence obtained from NCBI reference sequence NM_001030295) were performed as described by Possidonio et al . For control siRNA (nonspecific), we decided to target the enhanced green fluorescent protein (eGFP), also used in Possidonio et al . The three sequences of siRNA that were selected to target Notch‐1 are shown in Figures and .…”