G.S. Sohal

VENTRALLY EMIGRATING NEURAL TUBE CELLS CONTRIBUTE TO THE FORMATION OF MECKEL'S AND QUADRATE CARTILAGE

A population of multipotential neuroepithelial cells originating in the ventral portion of the hindbrain neural tube has been shown recently to emigrate at the site of attachment of the trigeminal nerve. These ventrally emigrating neural tube cells populate the mesenchyme of the first pharyngeal (branchial) arch. Because the Meckels and the quadrate cartilage develop from this mesenchyme, we sought to determine whether these ventrally emigrating neural tube cells contributed to their development. The ventral neural tube cells were tagged with a replication‐deficient retroviral vector containing the LacZ gene. This method permanently labels the descendents of the neural tube cells; thus, they can be subsequently tracked during development. The viral concentrate was microinjected into the lumen of the rostral hindbrain of chick embryos, after the emigration of neural crest is finished, on embryonic day 2 (stage 14). In control embryos, the virus was placed on top of the neural tube. Embryos were killed on days 3, 4, and 7 and processed for the detection of LacZ‐positive cells. By day 7, the Meckels and the quadrate cartilage can be easily recognized. LacZ‐positive cells were seen in both cartilages. They were located in perichondrium and in the cartilage. Immunostaining with the neural crest cell marker HNK‐1 indicated that the LacZ‐positive cells were HNK‐1 negative. The HNK‐1‐positive neural crest–derived cells were located in the cartilage but not in the perichondrium. These results indicate that the chondrocytes in the Meckels and the quadrate cartilage differentiate from two sources of cells; the ventrally emigrating neural tube cells and the neural crest. The developmental significance of differentiation of cartilage from the ventral neural tube cells and of the heterogeneous origin of chondrocytes in morphogenesis remains to be established. Dev Dyn 1999;216:37–44.

DiI labeling and homeobox gene Islet-1 expression reveal the contribution of ventral neural tube cells to the formation of the avian trigeminal ganglion

Cells of the neural tube are thought to be committed to form only the central nervous system, whereas the peripheral nervous system is believed to be derived from neural crest cells and from placodes, which are specialized regions of the surface ectoderm. Neural crest cells arise early from the dorsal part of the neural tube. The possibility that after emigration of the neural crest cells, another population of cells arising from the ventral part of the neural tube also emigrates via a different route was examined. Here we report that, after labeling cells of the ventral neural tube in the rostral hindbrain of E3 duck embryos with DiI, they were later found in the trigeminal ganglion of the fifth cranial nerve. A trail of labeled cells could be traced from the ventral part of the neural tube to the peripheral ganglion. Further, expression of the homeobox gene Islet‐1 in cells of the neural tube and the ganglion also indicated that some ventral neural tube cells may normally emigrate to the trigeminal ganglion. It is concluded that not all neural tube cells are committed to form the central nervous system; the ventral part of the neural tube also provides cells for the formation of the trigeminal ganglion. These results raise the possibility that the ventral neural tube may serve as an additional source of cells for the formation of various other components of the peripheral nervous system.

A second source of precursor cells for the developing enteric nervous system and interstitial cells of Cajal.

The enteric nervous system is believed to be derived solely from the neural crest cells. This is partly based on the belief that the neural crest cells are the sole neural tube‐derived cells colonizing the gastrointestinal tract. However, recent studies have shown that after the emigration of neural crest cells an additional population of cells emigrates from the cranial neural tube. These cells originate in the ventral part of the hindbrain, emigrate through the site of attachment of the cranial nerves, and colonize a variety of developing structures including the gastrointestinal tract. This cell population has been named the ventrally emigrating neural tube (VENT) cells. We followed the fate of these cells in the gastrointestinal tract. Ventral hindbrain neural tube cells of chick embryos were tagged with replication‐deficient retroviral vectors containing the LacZ gene, after the emigration of neural crest from this region. In control embryos, the viral concentrate was dropped on the dorsal part of the neural tube. Embryos were sacrificed from embryonic days 3–12 and processed for the detection of LacZ positive ventrally emigrating neural tube cells. These cells colonized only the foregut, specifically the duodenum and stomach. Immunostaining with the neural crest cell marker HNK‐1 showed that they were HNK‐1 negative, indicating that they were not derived from neural crest. Cells were detected in three locations: (1) the myenteric and submucosal plexus of the enteric nervous system; (2) circular smooth muscle cell layer; and (3) mucosal lining of the lumen. A variety of specific markers were used to identify their fate. Some ventrally emigrating neural tube cells differentiated into neurons and glial cells, indicating that the enteric nervous system in the foregut develops from an additional source of precursor cells. It was also found that some of these cells differentiated into interstitial cells of Cajal, which mediate impulses between the enteric nervous system and smooth muscle cells, whereas others differentiated into epithelium. Altogether, these results indicate that the ventrally emigrating neural tube cells are multipotential. More importantly, they reveal a novel source of precursor cells for the neurons and glial cells of the enteric nervous system. The developmental and functional significance of the heterogeneous origin of the cell types remains to be established.

Ventrally emigrating neural tube cells differentiate into vascular smooth muscle cells

A multipotential cell population originating in the ventral part of the hindbrain neural tube, the ventrally emigrating neural tube cells (VENT cells), has recently been shown to migrate into the craniofacial mesenchyme. Because vascular smooth muscle cells develop from this mesenchyme, we sought to determine if the VENT cells contributed to their differentiation. VENT cells were tagged with replication-deficient retroviral vector with LacZ by microinjection into the lumen of the rostral hindbrain of chick embryos on day 2. Embryos were processed for the detection of LacZ positive cells on day 7. LacZ-positive cells were present in the wall of craniofacial arteries and veins. Immunostaining with the smooth muscle alpha-actin confirmed the labeled cells to be smooth muscle cells. It is concluded that some vascular smooth muscle cells differentiate from neural tube cells. the developmental and functional significance of which remains to be established.

Emigration of neuroepithelial cells from the hindbrain neural tube in the chick embryo

It is generally believed that after the emigration of neural crest, the neuroepithelial cells of the neural tube are committed to differentiate only as neurons and supporting cells of the central nervous system. Neural crest cells arise from the dorsal portion of the developing neural tube and contribute to the formation of the peripheral nervous system and a variety of non‐neural structures. In contrast to this view we have recently shown, by focal application of the vital dye Dil in duck embryos, that an additional population of cells emigrates from the neural tube. By using an entirely different technique we confirm and extend these observations in the chick embryo. Replication‐deficient retroviral vector LZ12 containing the gene LacZ was utilized to label the neural tube cells. The viral concentrate was microinjected into the lumen of the rostral hind‐brain neural tube, considerably after the completion of emigration of neural crest cells. The labeled cells were monitored in whole mounts and histological sections. Initially, the labeled cells were restricted to the neuroepithelium of the hindbrain neural tube. Subsequently, they were seen in the neural tube and in the ganglion of the fifth cranial nerve (trigeminal ganglion). Later, they migrated beyond the trigeminal ganglion, i.e., into the mesenchyme of the first pharyngeal arch. Immunostaining with the neural crest cell marker, HNK‐1, indicated that the emigrated neuroepithelial cells were HNK‐1 negative. It is concluded that in the chick embryo some neuroepithelial cells emigrate at the site of attachment of the trigeminal nerve, migrate into the ganglion and then into the mesenchyme of the first arch. This cell population differs antigenically from the neural crest cells.

Ventral neural tube cells differentiate into hepatocytes in the chick embryo

Abstract. A population of ventral neural tube cells has recently been shown to migrate out of the hind brain neural tube via the vagus nerve and contribute to the developing gastrointestinal tract. Since liver is also innervated by the vagus nerve, we sought to determine if these cells also migrate into the liver. Ventral neural tube cells in the caudal hindbrain of chick embryos were tagged with a replication-deficient retroviral vector containing the LacZ gene on embryonic day 2. Embryos were processed for detection of labeled cells on embryonic day 5 and 11. Labeled cells were seen in the liver on both days and identified as hepatocytes. Previously, it was believed that all hepatocytes develop from the gut endoderm. Results of the present study show an additional source for the formation of liver cells.

Ventrally emigrating neural tube cells contribute to the normal development of heart and great vessels

We investigated the contributions of a recently described population of neural tube cells, which participates in the development of a variety of tissues, to the development of the heart and great vessels. These cells, termed as the ventrally emigrating neural tube (VENT) cells, originate in the ventral part of the hindbrain neural tube, emigrate at the site of attachment of the cranial nerves, and populate their respective target tissues. VENT cells of the caudal hindbrain neural tube at the level of the vagus nerve, which were previously reported to migrate into the heart, were tagged with replication-deficient retroviruses containing the LacZ gene in chick embryos, after the emigration of neural crest from this region. In older embryos, VENT cells were detected in a variety of locations including the ventricles, atria, their septa, aorticopulmonary septum, and great vessels of the heart. Immunostaining with a specific marker revealed that VENT cells differentiated into smooth muscle cells of great vessels. Differentiation of VENT cells into cardiac muscle cells was reported previously. Extirpation of the VENT cells prior to their departure from the neural tube resulted in some common cardiovascular malformations: thin-walled ventricles and atria, ventricular and atrial septal defects, persistent truncus arteriosus, and stenosis of the great vessels. These results suggest that a novel population of neural tube cells also contributes to the normal development of the heart and great vessels. Thus, the heart and great vessels develop from three sources of cells: mesoderm, neural crest, and VENT cells.

Development of postsynaptic-like specializations of the neuromuscular synapse in the absence of motor nerve.

It was previously reported that the acetylcholine receptor clusters and acetylcholinesterase appear on embryonic superior oblique muscle cells developing in vivo without motor nerve contacts. The objective of this study was to examine whether some other components of neuromuscular junction also form on muscle cells developing in vivo in the absence of motor neurons. In the present study, postsynaptic specializations such as junctional folds, postsynaptic density and basal lamina were studied in normal and aneural muscles. The superior oblique muscle of duck embryos was made aneural by permanent destruction of trochlear motor neurons by cauterizing midbrain on embryonic day 7; 3 days before the motor neurons normally project their axons into the muscle. Normal and aneural muscles from embryonic days 10 to 25 were processed for electron microscopy. The results indicate that morphological specializations such as junction‐like folds, postsynaptic‐like density, and basal lamina also develop in the absence of motor neuron contacts. Whether the differentiation of specialized synaptic basal lamina is dependent on the presence of motor neurons was examined by utilizing a monoclonal antibody against heparan sulfate proteoglycan. Immunohistochemical studies indicate that specialized synaptic basal lamina differentiates in the absence of motor neurons. Thus, the mechanism of development of postsynaptic components of neuromuscular junction in this muscle is not dependent on motor neuron contacts. These results also suggest that the postsynaptic cell plays a more active role in synapse formation than previously realized. The results are discussed in relation to the control of synapse numbers by the postsynaptic cell.

Synapse formation on quail trochlear neurons transplanted in duck embryos before naturally occurring motor neuron death

About half of the trochlear motor neurons in duck and quail embryos die during normal development. In a previous study the role of target muscle in controlling the number of surviving motor neurons was investigated by reducing the number of neurons innervating the muscle. This was accomplished by removing the midbrain of the duck embryo and grafting in its place the midbrain of the quail embryo before motor neuron death begins. It was observed that the number of surviving trochlear motor neurons in the quail‐duck chimera embryos was not significantly different from that of the normal quail. The present investigation was undertaken to determine whether trochlear motor neurons in the chimera embryos received afferent synapses. Brains of duck, quail and chimera embryos on days 16 and 20 were processed for electron microscopical observations. Synapses formed on motor neurons of the chimera embryos. Surprisingly, synapses on motor neurons of quail differed from those of duck, both qualitatively and quantitatively. Synapses on the motor neurons of the chimera embryos developed in a fashion similar to that for the duck motor neurons. Our failure to rescue trochlear motor neurons in the chimera embryos suggests that the developing motor neurons may respond to a larger target muscle only if they received a normal complement of afferent synaptic input.

Synapse formation on trochlear motor neurons under conditions of increased and decreased cell death during development

There is a normally occurring death of about half of the trochlear motor neurons during development. Early removal of the target muscle results in death of almost all neurons whereas neuromuscular blockade prevents neuron death. The present investigation was undertaken to determine whether the number of central afferent synapses on motor neurons is altered under conditions which either accentuate cell loss or rescue neurons. The sole peripheral target of innervation of the trochlear motor neurons, the superior oblique muscle, was extirpated in duck embryos before the motor axon out‐growth begins. The neuromuscular blockade was achieved by application of paralyzing dosages of alpha bungarotoxin on to the vascularized chorioallantoic membrane. This treatment began prior to the onset of cell death and embryos were treated daily throughout the period of cell death. Brains were processed for electron microscopy and quantitative observations were made on synapses at the onset, during the period of, and at the end of cell death. It was found that there was no significant difference in the number of synapses on neurons following target removal, following neuromuscular blockade, and those developing normally. This observation indicates that the number of central afferent synapses on cell soma is not altered under conditions which either decrease or increase neuron survival. These results suggest that the synapse number per se may not be directly involved in the process of naturally occurring cell death. The results also suggest that the number of synapses on trochlear motor neurons is independent of interactions with the target.