John Sechrist

Noelin-1 is a secreted glycoprotein involved in generation of the neural crest

The vertebrate neural crest arises at the border of the neural plate during early stages of nervous system development; however, little is known about the molecular mechanisms underlying neural crest formation. Here we identify a secreted protein, Noelin-1, which has the ability to prolong neural crest production. Noelin-1 messenger RNA is expressed in a graded pattern in the closing neural tube. It subsequently becomes restricted to the dorsal neural folds and migrating neural crest. Over expression of Noelin-1 using recombinant retroviruses causes an excess of neural crest emigration and extends the time that the neural tube is competent to generate as well as regenerate neural crest cells. These results support an important role for Noelin-1 in regulating the production of neural crest cells by the neural tube.

Birth and differentiation of reticular neurons in the chick hindbrain: Ontogeny of the first neuronal population

To understand better early neuronal birth and differentiation in higher vertebrates, we have examined the time when presumptive neurons in the chick embryo first withdraw from the cell cycle and express neurofilaments. Hindbrain reticular neurons arise prior to the definitive streak stage of gastrulation and represent the first neuronal population to be born. The birth of hindbrain reticular neurons and spinal interneurons occurs in a rostrocaudal sequence that closely parallels regression of Hensens node. More rostral brain stem neurons are born shortly after those in the hindbrain. Neurofilament expression in reticular neuroblasts first occurs by the 7-somite stage, followed by axon outgrowth by the 15-somite stage. When neural plate morphogenesis is inhibited, neurofilament expression occurs on schedule in neurons that are undergoing or have completed terminal mitosis. Our results suggest that the inductive cues governing the birth and initial differentiation of reticular neurons are imparted by gastrulation and early neurulation.

Relationship between spatially restricted Krox-20 gene expression in branchial neural crest and segmentation in the chick embryo hindbrain.

Previous studies have suggested that the rostrocaudal patterning of branchial arches in the vertebrate embryo derives from a coordinate segmental specification of gene expression in rhombomeres (r) and neural crest. However, expression of the Krox‐20 gene is restricted to neural crest cells migrating to the third branchial arch, apparently from r5, whereas this rhombomere contributes cells to both the second and third arches. We examined in the chick embryo how this spatially restricted expression is established. Expression occurs in precursors in both r5 and r6, and we show by cell labelling that both rhombomeres contribute to Krox‐20‐expressing neural crest, emigration occurring first from r6 and later caudally from r5. Krox‐20 transcripts are not detected in some precursors in rostral r5, presaging the lack of expression in cells migrating rostrally from this rhombomere. After transposition of r6 to the position of r4 or r5, many Krox‐20‐expressing cells migrate rostral to the otic vesicle, whereas when r5 is transplanted to the position of r4, only a small number of migrating cells express Krox‐20. These results indicate that, in the chick, Krox‐20 expression in branchial neural crest does not correlate with rhombomeric segmentation, and that there may be intrinsic differences in regulation between the r5 and r6 Krox‐20‐expressing populations.

Cell division and differentiation in avian embryos: techniques for study of early neurogenesis and myogenesis.

Publisher Summary This chapter discusses the techniques for the study of early neurogenesis and myogenesis of the cell division and differentiation in avian embryos. Cell division and differentiation have long been recognized as two fundamental processes in the development of multicellular organisms. Early cytologists provided morphological descriptions of mitotic activity and changes in cytoplasmic organelles based on light microscopic observations of developing embryos. The cell cycle consists of a mitotic phase in which the chromosomes condense and separate as the nucleus divides and an interphase during which there are growth in the size, replication of DNA (S or synthetic phase), and other elaborate preparations for the next division. The regulatory mechanisms that control the number of cell divisions in any given tissue are not only crucial for producing a normal multicellular organism but also may contribute significantly to species differences. During and after the gastrulation stage of development, a few nerve cell precursors begin to exit the cell cycle (to enter a long Go phase) followed later by exiting skeletal muscle precursors. One characteristic that many neuroblasts and skeletal myoblasts have in common is that their terminally differentiated state is postmitotic.

Combined Vital Dye Labelling and Catecholamine Histofluorescence of Transplanted Ciliary Ganglion Cells

We have utilized the carbocyanine dye, DiI, to label suspensions of dissociated ciliary ganglion cells removed from 6 to 12 day old quail embryos. Some of the cells were injected into the trunk somites of 2.5 - 3 day old chick embryos along pathways where neural crest cells migrate to form sensory and sympathetic ganglia, aortic plexuses and the adrenal medulla; the remainder of the cells were cultured to check their viability and the persistence of the DiI label. Embryos were incubated for 1 – 8 days post-injection, fixed in 4% paraformaldehyde/0.25% glutaraldehyde and processed for cryostat sectioning. DiI-labelled cells were readily identifiable in culture and in sections of embryos at all stages examined. Several cell types were identified, based on their morphology and soma size. These included cells with large cell bodies and bright DiI-labelling that appeared to be neurons and smaller, more weakly labelled cells that appeared non-neuronal. The latter presumably had divided several times, accounting for their reduced levels of dye. Many of the DiI-labelled cells were found in and around neural crest-derived sympathetic ganglia, aortic plexuses and adrenomedullary cords, but were rarely observed in dorsal root ganglia. The aldehyde fixative (Faglu mixture) used in this study reacts with catecholamines to form a bright reaction product in adrenergic cells including those in the sympathetic ganglia and the adrenal medulla. The catecholamine biproduct and the DiI in the same cell can easily be viewed with different fluorescent filter sets. A variable number of the DiI-labelled cells in these adrenergic sites contained catecholamines. Cells derived from younger 6 day ciliary ganglion dissociates exhibited detectable catecholamine neurotransmitters earlier and more frequently than those derived from 8 day embryos. The presence of cells exhibiting both bright DiI and catecholamine fluorescence is consistent with previous indications that post-mitotic ciliary ganglion neurons can undergo phenotypic conversion from cholinergic to adrenergic when transplanted to the trunk environment.

Age-Dependent Neurotransmitter Plasticity of Ciliary Ganglion Neurons

We have examined neurotransmitter plasticity in postmitotic cholinergic neurons isolated from 6.5- to 11-day-old embryonic quail ciliary ganglia. Purified neurons were labeled with DiI, transplanted into the trunk of young chick embryos, and assayed for catecholamine content and [3H]thymidine uptake 4 to 5 days later. For ciliary neurons derived from 6.5- to 8-day-old embryos, as many as 25% (average of 9% overall) expressed catecholamines in the host sympathetic ganglia, migratory stream, aortic plexuses, and adrenal medulla. In contrast, neurons from >8-day-old ganglia did not acquire or produce detectable catecholamines, indicating a limited time period over which phenotypic conversion can occur in vivo. As a control, ciliary neurons were also injected into the head mesenchyme of young embryos; no catecholamine expression was observed. Interestingly, after transplantation some DiI-labeled postmitotic ciliary neurons took up [3H]thymidine with or without phenotypic change. These results suggest that phenotypic plasticity in ciliary neurons is age-dependent, is location-dependent, and may involve resumption of DNA replication, a characteristic feature of some differentiating adrenergic sympathetic neurons. Apoptosis of a few proliferating transplanted cells may be induced independently or in association with transmitter change.

Early regulative ability of the neuroepithelium to form cardiac neural crest.

The cardiac neural crest (arising from the level of hindbrain rhombomeres 6-8) contributes to the septation of the cardiac outflow tract and the formation of aortic arches. Removal of this population after neural tube closure results in severe septation defects in the chick, reminiscent of human birth defects. Because neural crest cells from other axial levels have regenerative capacity, we asked whether the cardiac neural crest might also regenerate at early stages in a manner that declines with time. Accordingly, we find that ablation of presumptive cardiac crest at stage 7, as the neural folds elevate, results in reformation of migrating cardiac neural crest by stage 13. Fate mapping reveals that the new population derives largely from the neuroepithelium ventral and rostral to the ablation. The stage of ablation dictates the competence of residual tissue to regulate and regenerate, as this capacity is lost by stage 9, consistent with previous reports. These findings suggest that there is a temporal window during which the presumptive cardiac neural crest has the capacity to regulate and regenerate, but this regenerative ability is lost earlier than in other neural crest populations.