Intravital imaging of mouse embryos.

Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution.

Neuropilin 1 signaling guides neural crest cells to coordinate pathway choice with cell specification.

Neural crest cells (NCCs) are highly motile embryonic stem cells that delaminate from the neuroectoderm early during vertebrate embryogenesis and differentiate at defined target sites into various essential cell types. To reach their targets, NCCs follow 1 of 3 sequential pathways that correlate with NCC fate. The firstborn NCCs travel ventrally alongside intersomitic blood vessels to form sympathetic neuronal progenitors near the dorsal aorta, while the lastborn NCCs migrate superficially beneath the epidermis to give rise to melanocytes. Yet, most NCCs enter the somites to form the intermediate wave that gives rise to sympathetic and sensory neurons. Here we show that the repulsive guidance cue SEMA3A and its receptor neuropilin 1 (NRP1) are essential to direct the intermediate wave NCC precursors of peripheral neurons from a default pathway alongside intersomitic blood vessels into the anterior sclerotome. Thus, loss of function for either gene caused excessive intersomitic NCC migration, and this led to ectopic neuronal differentiation along both the anteroposterior and dorsoventral axes of the trunk. The choice of migratory pathway did not affect the specification of NCCs, as they retained their commitment to differentiate into sympathetic or sensory neurons, even when they migrated on an ectopic dorsolateral path that is normally taken by melanocyte precursors. We conclude that NRP1 signaling coordinates pathway choice with NCC fate and therefore confines neuronal differentiation to appropriate locations.

Segmental neurovascular syndromes in children.

The concept of segmental vascular syndromes with different, seemingly unrelated, diseases is based on the embryology of the neural crest and the mesoderm migration of cells that share the same metameric origin. Migrating patterns of these cells link the brain, the cranial bones, and the face on the same side. A somatic mutation developing in the region of the neural crest or the adjacent cephalic mesoderm before migration can, therefore, be postulated to produce arterial or venous metameric syndromes, including PHACES, CAMS, Cobb syndrome, and Sturge-Weber syndrome. Although these diseases may be rare, their relationships among each other and their postulated linkage with the development of the neural crest and the cephalic mesoderm may shed light on the complex pathology and etiology of various cerebral vascular disorders.

Ets-1 expression is associated with cranial neural crest migration and vasculogenesis in the chick embryo.

The transcription factor Ets-1 is expressed in many different migratory cell types, suggesting that it may play an important role in regulating motility. To determine whether its expression in the neural crest is consistent with such a function, we have performed a detailed analysis of its expression during early chick embryogenesis. Our results show that this transcription factor is up-regulated in the cranial neural folds and dorsal neural tube approximately 4-6 h prior to commencement of neural crest migration. c-Ets-1 continues to be expressed by migrating cranial neural crest cells and subsequently by some neural crest-derived tissues. In addition to neural crest, we find expression of c-Ets-1 in endothelial cells of blood vessels, in somitic and intermediate mesoderm, in limb buds and in the heart.

Developing blood vessels and associated extracellular matrix as substrates for neural crest migration in Japanese quail, Coturnix coturnix japonica.

Japanese quail embryos were used to examine paths of neural crest cell (NC) migration in relationship to the embryonic vasculature. Immunolabeling for NC, angioblasts and the extracellular matrix (ECM) glycoproteins fibronectin (FN), laminin (LN) and tenascin (TN) revealed several instances where spatiotemporal patterns of NC migration coincide with the embryonic vascular pattern and its associated ECM. An in vitro model for angiogenesis was modified to include NC, and associations with "capillary-like" endothelial cell structures were demonstrated. A working hypothesis is that the embryonic vasculature may, in specific instances, be used as a substratum for directed NC migration and that these interactions are mediated primarily through the adhesive interactions of FN. Some members of the TN family of glycoproteins, through their relatively non-adhesive properties, may act to help guide neural crest cells to the FN-rich blood vessel surface.