A sub-population of the neural crest is known to play a crucial role in development of the cardiac outflow tract. Studies in avians have mapped the complete migratory pathways taken by `cardiac' neural crest cells en route from the neural tube to the developing heart. A cardiac neural crest lineage is also known to exist in mammals, although detailed information on its axial level of origin and migratory pattern are lacking. We used focal cell labelling and orthotopic grafting, followed by whole embryo culture, to determine the spatio-temporal migratory pattern of cardiac neural crest in mouse embryos. Axial levels between the post-otic hindbrain and somite 4 contributed neural crest cells to the heart, with the neural tube opposite somite 2 being the most prolific source. Emigration of cardiac neural crest from the neural tube began at the 7-somite stage, with cells migrating in pathways dorsolateral to the somite, medial to the somite, and between somites. Subsequently, cardiac neural crest cells migrated through the peri-aortic mesenchyme, lateral to the pharynx, through pharyngeal arches 3, 4 and 6, and into the aortic sac. Colonisation of the outflow tract mesenchyme was detected at the 32-somite stage. Embryos homozygous for the Sp2H mutation show delayed onset of cardiac neural crest emigration, although the pathways of subsequent migration resembled wild type. The number of neural crest cells along the cardiac migratory pathway was significantly reduced in Sp2H/Sp2H embryos. To resolve current controversy over the cell autonomy of the splotchcardiac neural crest defect, we performed reciprocal grafts of premigratory neural crest between wild type and splotch embryos. Sp2H/Sp2H cells migrated normally in the +/+environment, and +/+ cells migrated normally in the Sp2H/Sp2H environment. In contrast, retarded migration along the cardiac route occurred when either Sp2H/+ or Sp2H/Sp2H neural crest cells were grafted into the Sp2H/Sp2Henvironment. We conclude that the retardation of cardiac neural crest migration in splotch mutant embryos requires the genetic defect in both neural crest cells and their migratory environment.
During early embryonic development, cell migration is one of the most important morphogenetic processes. Neural crest cells arise from the dorsal part of the neural tube and migrate along different pathways to numerous locations where they differentiate into a variety of tissues. In the mouse, studies of neural crest cell migration have been difficult partly because of the absence of specific markers which can label neural crest cells throughout their migration from their origin to the site of differentiation. Nevertheless, the use of different experimental strategies involving extrinsic, intrinsic or genetic cell markers has already led to a good understanding of this migration. In our studies, extrinsic markers such as wheat germ agglutinin-gold conjugates and DiI and genetic markers including Hoxb2-lacZ and green fluorescent protein have been employed in tracing migrating neural crest cells. The labelling procedures and the strength and weaknesses of the tracing methods are reviewed herein.
Background: Neural crest cells are a special group of embryonic cells which are able to emigrate from the dorsal neural tube (the developing central nervous system) to the mesenchyme and form different derivatives in embryos. In contrast to the situation in the avian embryo, where the use of quail-chick chimeras has provided much information on neural crest migration, the early migratory pathways of hindbrain neural crest cells are not well understood in mammals. Aims: In the present study, an exogenous dye, wheat germ agglutinin-gold conjugate (WGA-Au), was used to label the early migrating hindbrain neural crest cells to track their migration at three axial levels, namely (1) pre-otic, (2) post-otic and pre-somitic, and (3) somitic (somites 1–4) levels, in mouse embryos cultured in vitro. Results: It was found that neural crest cells had already started their emigration from the dorsal part of the neural tube at the 9-somite stage and migrated into the mesenchyme subjacent to the neural tube. When the labelled embryos were cultured to more advanced stages, neural crest cells migrated to more ventral regions. At the 13-somite stage, most neural crest cells at the pre-somitic hindbrain migrated to the mesenchyme lateral to the dorsal aorta, while by the 17-somite stage, labelled cells reached the mesenchyme adjacent to the pharynx, which is located more ventrally than the dorsal aorta. At the 21-somite stage, more labelled cells were found in the mesenchyme lateral and ventral to the pharynx. At the somitic level, neural crest cells migrated along three distinct routes, namely the dorsolateral (between the surface epithelium and the somite), medial (between the somite and the neural tube) and intersomitic (between two consecutive somites) pathways. Conclusions: Our results indicate that whole embryo culture is able to support the normal development of mouse embryos at early organogenesis in vitro for 26 h, and that WGA-Au is an ideal short-term cell marker for tracking neural crest migration in the mouse. We also show that the migratory patterns of hindbrain neural crest cells in the mouse largely resemble those observed in the chick.
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