The collaborators at Yale and Caltech demonstrate with three different technologies -- immunostaining of proteins, in situ hybridization and multiplex RT-PCR of mRNAs -- that formation of neural crest cells in chick embryos is independent of both mesoderm and neural tissues. They also identify, Pax7, as an early marker of neural crest formation and prove that its function is required in the earliest stages of development.
Early chick embryo with Pax-7 (red) designating neural crest progenitor cells. Credit: Martín García-Castro
The neural crest is a population of stem cells that migrate extensively during development and give rise to many derivatives, including most of the bone and cartilage of the head skeleton, pigment cells of the skin, and cells of the peripheral nervous system.
In humans, cleft palate, heart valve malformations and various tumors are among the common malformations associated with disruption of neural crest development.
Chick embryos have well-characterized stages and are a valuable model for examining vertebrate development. While it was known that the ability to form neural crest cells declines after "stage 10," the researchers were seeking the earliest conditions surrounding formation of these important stem cells.
"Understanding the origin of neural crest cells -- where, when and how they arise -- is a critical step if we are to manipulate them for therapeutic purposes," said Martín García-Castro, assistant professor of molecular, cellular and developmental biology at Yale and principal investigator on the study. "Implications of these basic questions of biology and development reach far beyond these chicken and eggs."
Based on work from the 1940's before molecular tools were available, the neural crest was thought to form by interactions between neural and non-neural cell layers. "We show in this work that neural crest stem cell precursors are designated very early in development -- as early as the gastrula stages -- and in an independent fashion from those other tissues," said Martín García-Castro.
The researchers grew grafts of cells from "stage 3" chick embryos, before the neural plate formed, in non-inducing cultures. Surprisingly, restricted regions of the embryo generated both migrating neural crest cells and their derivative cell types, without any interaction with neural or mesodermal tissues.
"Our results are contrary to current text-book models and suggest that different modes of neural crest induction operate during development," said Martín García-Castro. "Interestingly, the one we have uncovered is related to the early, cranial neural crest cells, the only ones in higher vertebrates that retain bone and cartilage forming potential."
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