Louis-Bruno Ruest

Peptide Elongation Factor eEF1A-2/S1 Expression in Cultured Differentiated Myotubes and Its Protective Effect against Caspase- 3-mediated Apoptosis

Peptide elongation factor eEF1A-2/S1, which shares 92% homology with eEF1A-1/EF-1α, is exclusively expressed in brain, heart, and skeletal muscle. In these tissues, eEF1A-2/S1 is the only type 1A elongation factor expressed in adulthood because a transition from eEF1A-1/EF-1α to eEF1A-2/S1 occurs in early postnatal development. In this article, we report that the expression of eEF1A-2/S1 protein is activated upon myogenic differentiation. Furthermore, we show that upon serum deprivation-induced apoptosis, eEF1A-2/S1 protein disappears and is replaced by its homolog eEF1A-1/EF-1α in dying myotubes; cell death is characterized by the activation of caspase-3. In addition, we show that the continuous expression of eEF1A-2/S1 resulting from adenoviral gene transfer protects differentiated myotubes from apoptosis by delaying their death, thus suggesting a prosurvival function for eEF1A-2/S1 in skeletal muscle. In contrast, myotube death is accelerated by the introduction of the homologous gene, eEF1A-1/EF-1α, whereas cells transfected with antisense eEF1A-1/EF-1αare protected from apoptosis. These results demonstrate that the two sister genes, eEF1A-1/EF-1α and eEF1A-2/S1, regulate myotube survival with the former exerting prodeath activity and the latter a prosurvival effect.

dHAND-Cre transgenic mice reveal specific potential functions of dHAND during craniofacial development

Most of the bone, cartilage, and connective tissue of the craniofacial region arise from cephalic neural crest cells. Presumably, patterning differences in crest cells are a result of regional action of transcription factors within the developing pharyngeal arches. The basic helix-loop-helix transcription factor dHAND/HAND2 is expressed throughout much of the neural crest-derived mesenchyme of the pharyngeal arches, suggesting that it plays a crucial role in craniofacial development. However, targeted inactivation of the dHAND gene results in embryonic lethality by E10.5 due to vascular defects, preventing further analysis of the role of dHAND in cephalic neural crest cell development. In order to examine putative roles of dHAND during later stages of embryogenesis, we have used a transgenic lineage marker approach, in which a portion of the dHAND upstream region containing an enhancer that directs dHAND expression to the pharyngeal arches is used to drive Cre recombinase expression. By crossing these dHAND-Cre transgenic mice with R26R mice, we can follow the fate of cells that expressed dHAND at any time during development by examining beta-galactosidase activity. We show that dHAND is first expressed in postmigratory cephalic neural crest cells within the pharyngeal arches. In older embryos, beta-galactosidase-labeled cells are observed in most of the neural crest-derived lower jaw skeleton and surrounding connective tissues. However, labeled cells only contribute to substructures within the middle ear, indicating that our transgene is not globally expressed in cephalic neural crest cells within the pharyngeal arches. Moreover, dHAND-Cre mice will provide a valuable tool for tissue-specific inactivation of gene expression in multiple tissue types of neural crest origin.

Regulation of epithelial-mesenchymal transition in palatal fusion.

During palatal fusion, the midline epithelial seam between the palatal shelves degrades to achieve mesenchymal confluence. Morphological and molecular evidence support the theory that the epithelial-mesenchymal transition is one mechanism that regulates palatal fusion. It appears that transforming growth factor (TGF)-β signaling plays a role in palatal EMT. TGFβ3 is the main inducer in palatal fusion and activates both Smad-dependent and -independent signaling pathways, including the key EMT transcription factors, Lef1, Twist, and Snail1, in the MEE prior to the palatal EMT program. The roles and interactions among these transcription factors will be discussed.

Development-dependent disappearance of caspase-3 in skeletal muscle is post-transcriptionally regulated.

Caspase‐3, a major player in apoptosis, engages apoptosis‐activated cells into an irreversible pathway leading to cell death. In this article, we report that caspase‐3 protein is absent from rat and mouse adult skeletal muscles, despite the abundant presence of its mRNA. During skeletal muscle development, caspase‐3 protein is present in neonatal animals, but its expression gradually decreases, and disappears completely by 1 month of age, when there is still abundant caspase‐3 mRNA. This discordance between caspase‐3 message and protein expression is unique to skeletal muscle, as in all other analyzed tissues the protein presence correlates with the presence of the mRNA. The only circumstance in which caspase‐3 protein appears in adults is in regenerating muscles; once regeneration is complete, however, it again becomes undetectable in repaired muscles. We conclude that caspase‐3 protein in skeletal muscle is uniquely regulated at the post‐transcriptional level, unseen in other tissues such as brain, heart, lung, kidney, thymus, spleen, liver, or testis. The post‐transcriptional regulation of caspase‐3 might serve as a fail‐safe mechanism to avoid accidental cell death. J. Cell. Biochem. 86: 21–28, 2002.

Deletion of the endothelin-A receptor gene within the developing mandible

Signaling from the endothelin-A (Ednra) receptor is responsible for initiating multiple signaling pathways within neural crest cells (NCCs). Loss of this initiation is presumably the basis for the craniofacial defects observed in Ednra−/− embryos. However, it is not known whether continued Ednra signaling in NCC derivatives is required for subsequent development of the lower jaw. To address this question, mice containing loxP recombination sequences flanking a portion of the Ednra gene were bred with transgenic mice that express Cre recombinase under control of a Dlx5/6 enhancer element. We find that while Ednra gene inactivation within the mandibular arch of these Ednra conditional knockout embryos is detectable by embryonic day (E) 10.5, mandibular arch-specific gene expression is normal, as is overall mandible development. These results suggest that while Ednra receptor signaling is crucial for early NCC patterning, subsequent Ednra signaling is not essential for mandible bone development.

Differential Programming of p53-Deficient Embryonic Cells During Rotenone Block

Mitochondrial dysfunction has been implicated in chemical toxicities. The present study used an in vitro model to investigate the differential expression of metabolic pathways during cellular stress in p53-efficient embryonic fibroblasts compared to p53-deficient cells. These cell lines differed with respect to NADH/NAD(+) balance. This ratio constitutes a driving force for NAD- and NADH-dependent reactions and is inversed upon exposure to Rotenone (complex I inhibitor). Rotenone perturbed the structure of the elongated fibrillar tubulin network and decreased mRNA expression of tubulin genes both suggesting reprogramming and reorganization of the cytoskeleton in both cell lines. These changes were reflected in the abundance of specific mRNA and microRNA (miRNA) species as determined from genome-based analysis. Changes in mRNA and miRNA expression profiles reflected differences in energy utilizing pathways, consistent with the notion that the p53 pathway influences the cellular response to mitochondrial dysfunction and that at least some control may be embedded within specific mRNA/miRNA networks in embryonic cells.