Gbx2 Is Required for the Migration and Survival of a Subpopulation of Trigeminal Cranial Neural Crest Cells.

The development of key structures within the mature vertebrate hindbrain requires the migration of neural crest (NC) cells and motor neurons to their appropriate target sites. Functional analyses in multiple species have revealed a requirement for the transcription factor gastrulation-brain-homeobox 2 (Gbx2) in NC cell migration and positioning of motor neurons in the developing hindbrain. In addition, loss of Gbx2 function studies in mutant mouse embryos, Gbx2neo, demonstrate a requirement for Gbx2 for the development of NC-derived sensory neurons and axons constituting the mandibular branch of the trigeminal nerve (CNV). Our recent GBX2 target gene identification study identified multiple genes required for the migration and survival of NC cells (e.g., Robo1, Slit3, Nrp1). In this report, we performed loss-of-function analyses using Gbx2neo mutant embryos, to improve our understanding of the molecular and genetic mechanisms regulated by Gbx2 during anterior hindbrain and CNV development. Analysis of Tbx20 expression in the hindbrain of Gbx2neo homozygotes revealed a severely truncated rhombomere (r)2. Our data also provide evidence demonstrating a requirement for Gbx2 in the temporal regulation of Krox20 expression in r3. Lastly, we show that Gbx2 is required for the expression of Nrp1 in a subpopulation of trigeminal NC cells, and correct migration and survival of cranial NC cells that populate the trigeminal ganglion. Taken together, these findings provide additional insight into molecular and genetic mechanisms regulated by Gbx2 that underlie NC migration, trigeminal ganglion assembly, and, more broadly, anterior hindbrain development.

Muscle Protein Signaling in C2C12 Cells Is Stimulated to Similar Degrees by Diverse Commercial Food Protein Sources and Experimental Soy Protein Hydrolysates.

Dietary protein stimulates muscle protein synthesis and is essential for muscle health. We developed a screening assay using C2C12 mouse muscle cells to assess the relative abilities of diverse commercial protein sources and experimental soy protein hydrolysates (ESH), after simulated gut digestion (SGD), to activate the mechanistic target of rapamycin complex I (mTORC1) muscle protein synthesis signaling pathway (p70S6K(Thr389) phosphorylation). Activation of mTORC1 was expressed as a percentage of a maximal insulin response. The bioactivities of proteins grouped by source including fish (81.3 ± 10.6%), soy (66.2 ± 4.7%), dairy (61.8 ± 4.3%), beef (53.7 ± 8.6%), egg (52.3 ± 10.6%), soy whey (43.4 ± 8.6%), and pea (31.4 ± 10.6%) were not significantly different from each other. Bioactivity for ESH ranged from 28.0 ± 7.5 to 98.2 ± 6.6%. The results indicate that both the protein source and processing conditions are key determinants for mTORC1 activation. Regression analyses demonstrated that neither leucine nor total branched-chain amino acid content of proteins is the sole predictor of mTORC1 activity and that additional factors are necessary.

Elongation factor 1 alpha1 and genes associated with Usher syndromes are downstream targets of GBX2.

Gbx2 encodes a DNA-binding transcription factor that plays pivotal roles during embryogenesis. Gain-and loss-of-function studies in several vertebrate species have demonstrated a requirement for Gbx2 in development of the anterior hindbrain, spinal cord, inner ear, heart, and neural crest cells. However, the target genes through which GBX2 exerts its effects remain obscure. Using chromatin immunoprecipitation coupled with direct sequencing (ChIP-Seq) analysis in a human prostate cancer cell line, we identified cis-regulatory elements bound by GBX2 to provide insight into its direct downstream targets. The analysis revealed more than 286 highly significant candidate target genes, falling into various functional groups, of which 51% are expressed in the nervous system. Several of the top candidate genes include EEF1A1, ROBO1, PLXNA4, SLIT3, NRP1, and NOTCH2, as well as genes associated with the Usher syndrome, PCDH15 and USH2A, and are plausible candidates contributing to the developmental defects in Gbx2(-/-) mice. We show through gel shift analyses that sequences within the promoter or introns of EEF1A1, ROBO1, PCDH15, USH2A and NOTCH2, are directly bound by GBX2. Consistent with these in vitro results, analyses of Gbx2(-/-) embryos indicate that Gbx2 function is required for migration of Robo1-expressing neural crest cells out of the hindbrain. Furthermore, we show that GBX2 activates transcriptional activity through the promoter of EEF1A1, suggesting that GBX2 could also regulate gene expression indirectly via EEF1A. Taken together, our studies show that GBX2 plays a dynamic role in development and diseases.