Cell lineage-guided mass spectrometry reveals increased energy metabolism and reactive oxygen species in the vertebrate organizer.

Molecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been mostly limited to transcripts and a few proteins, the latter due to limitations in detection and sensitivity. The Spemann-Mangold Organizer (SMO) in the South African Clawed Frog (), a popular model of development, has long been known to be the origin of signals that pattern the mesoderm and central nervous system. Molecular screens of the SMO have identified several genes responsible for the ability of the SMO to establish the body axis.

Time-resolved quantitative proteomic analysis of the developing otic vesicle reveals putative congenital hearing loss candidates.

Over 200 genes are known to underlie human congenital hearing loss (CHL). Although transcriptomic approaches have identified candidate regulators of otic development, little is known about the abundance of their protein products. We used a multiplexed quantitative mass spectrometry-based proteomic approach to determine protein abundances over key stages of otic morphogenesis to reveal a dynamic expression of cytoskeletal, integrin signaling, and extracellular matrix proteins.

Cell Lineage-Guided Microanalytical Mass Spectrometry Reveals Increased Energy Metabolism and Reactive Oxygen Species in the Vertebrate Organizer.

Molecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been limited to transcripts and some proteins due to limitations in detection and sensitivity. The Spemann-Mangold Organizer (SMO) in the South African Clawed Frog ( ), a popular model of development, has long been discovered to induce the patterning of the central nervous system. Molecular screens on the tissue have identified several genes, such as goosecoid, chordin, and noggin, with independent ability to establish a body axis.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo.

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos.

Mass spectrometry based proteomics for developmental neurobiology in the amphibian Xenopus laevis.

The South African clawed frog (Xenopus laevis), a prominent vertebrate model in cell and developmental biology, has been instrumental in studying molecular mechanisms of neural development and disease. Recently, high-resolution mass spectrometry (HRMS), a bioanalytical technology, has expanded the molecular toolbox of protein detection and characterization (proteomics). This chapter overviews the characteristics, advantages, and challenges of this biological model and technology.

Six1 proteins with human branchio-oto-renal mutations differentially affect cranial gene expression and otic development.

Single-nucleotide mutations in human result in amino acid substitutions in either the protein-protein interaction domain or the homeodomain, and cause ∼4% of branchio-otic (BOS) and branchio-oto-renal (BOR) cases. The phenotypic variation between patients with the same mutation, even within affected members of the same family, make it difficult to functionally distinguish between the different mutations. We made four of the BOS/BOR substitutions in the Six1 protein (V17E, R110W, W122R, Y129C), which is 100% identical to human in both the protein-protein interaction domain and the homeodomain, and expressed them in embryos to determine whether they cause differential changes in early craniofacial gene expression, otic gene expression or otic morphology.

Proteomic Characterization of the Neural Ectoderm Fated Cell Clones in the Xenopus laevis Embryo by High-Resolution Mass Spectrometry.

The molecular program by which embryonic ectoderm is induced to form neural tissue is essential to understanding normal and impaired development of the central nervous system. Xenopus has been a powerful vertebrate model in which to elucidate this process. However, abundant vitellogenin (yolk) proteins in cells of the early Xenopus embryo interfere with protein detection by high-resolution mass spectrometry (HRMS), the technology of choice for identifying these gene products.