Rhombomere rotation reveals that multiple mechanisms contribute to the segmental pattern of hindbrain neural crest migration.

Hindbrain neural crest cells adjacent to rhombomeres 2 (r2), r4 and r6 migrate in a segmental pattern, toward the first, second and third branchial arches, respectively. Although all rhombomeres generate neural crest cells, those arising from r3 and r5 deviate rostrally and caudally (J. Sechrist, G. Serbedzija, T. Scherson, S. Fraser and M. Bronner-Fraser (1993) Development 118, 691-703). We have altered the rostrocaudal positions of the cranial neural tube, adjacent ectoderm/mesoderm or presumptive otic vesicle to examine tissue influences on this segmental migratory pattern. After neural tube rotation, labeled neural crest cells follow pathways generally appropriate for their new position after grafting. For example, when r3 and r4 were transposed, labeled r3 cells migrated laterally to the second branchial arch whereas labeled r4 cells primarily deviated caudally toward the second arch, with some cells moving rostrally toward the first. In contrast to r4 neural crest cells, transposed r3 cells leave the neural tube surface in a polarized manner, near the r3/4 border. Surprisingly, some labeled neural crest cells moved directionally toward small ectopic otic vesicles that often formed in the ectoderm adjacent to grafted r4. Similarly, they moved toward grafted or displaced otic vesicles. In contrast, surgical manipulation of the mesoderm adjacent to r3 and r4 had no apparent effects. Our results offer evidence that neural crest cells migrate directionally toward the otic vesicle, either by selective attraction or pathway-derived cues.

Origins of neural crest cell diversity.

The neural crest is a population of migratory cells, arising from the ectoderm, that invades many sites within the embryo and differentiate into a variety of diverse cell types. Pigment cells, most cells of the peripheral nervous system, adrenal medullary cells, and some cranial cartilage are derived from the neural crest. Despite a wealth of knowledge concerning their pathways of migration and vast array of derivatives, little is known about the formation of neural crest cells or their acquisition of positional identity. This review focuses on the origin of neural crest cells from the ectoderm and the generation of differences in neural crest cell fates along the rostrocaudal axis. In addition, we consider the role of temporal restriction in the developmental potential of premigratory neural crest cells. While evidence for the existence of multipotent stem cells is strong, some experiments also suggest that there may be heterogeneity among neural crest cell precursors, perhaps due to differences in origin, that might explain commitment events occurring early in neural crest development.

Regulative capacity of the cranial neural tube to form neural crest.

In avian embryos, cranial neural crest cells emigrate from the dorsal midline of the neural tube shortly after neural tube closure. Previous lineage analyses suggest that the neural crest is not a pre-segregated population of cells within the neural tube; instead, a single progenitor in the dorsal neural tube can contribute to neurons in both the central and the peripheral nervous systems (Bronner-Fraser and Fraser, 1989 Neuron 3, 755-766). To explore the relationship between the 'premigratory' neural crest cells and the balance of the cells in the neural tube in the midbrain and hindbrain region, we have challenged the fate of these populations by ablating the neural crest either alone or in combination with the adjoining ventral portions of the neural tube. Focal injections of the vital dye, DiI, into the neural tissue bordering the ablated region demonstrate that cells at the same axial level, in the lateral and ventral neural tube, regulate to reconstitute a population of neural crest cells. These cells emigrate from the neural tube, migrate along normal pathways according to their axial level of origin and appear to give rise to a normal range of derivatives. This regulation following ablation suggests that neural tube cells normally destined to form CNS derivatives can adjust their prospective fates to form PNS and other neural crest derivatives until 4.5-6 hours after the time of normal onset of emigration from the neural tube.

Segmental migration of the hindbrain neural crest does not arise from its segmental generation.

The proposed pathways of chick cranial neural crest migration and their relationship to the rhombomeres of the hindbrain have been somewhat controversial, with differing results emerging from grafting and DiI-labelling analyses. To resolve this discrepancy, we have examined cranial neural crest migratory pathways using the combination of neurofilament immunocytochemistry, which recognizes early hindbrain neural crest cells, and labelling with the vital dye, DiI. Neurofilament-positive cells with the appearance of premigratory and early-migrating neural crest cells were noted at all axial levels of the hindbrain. At slightly later stages, neural crest cell migration in this region appeared segmented, with no neural crest cells obvious in the mesenchyme lateral to rhombomere 3 (r3) and between the neural tube and the otic vesicle lateral to r5. Focal injections of DiI at the levels of r3 and r5 demonstrated that both of these rhombomeres generated neural crest cells. The segmental distribution of neural crest cells resulted from the DiI-labelled cells that originated in r3 and r5 deviating rostrally or caudally and failing to enter the adjacent preotic mesoderm or otic vesicle region. The observation that neural crest cells originating from r3 and r5 avoided specific neighboring domains raises the intriguing possibility that, as in the trunk, extrinsic factors play a major role in the axial patterning of the cranial neural crest and the neural crest-derived peripheral nervous system.

Phosphorylation of myosin-II regulatory light chain by cyclin-p34cdc2: a mechanism for the timing of cytokinesis.

To understand how cytokinesis is regulated during mitosis, we tested cyclin-p34cdc2 for myosin-II kinase activity, and investigated the mitotic-specific phosphorylation of myosin-II in lysates of Xenopus eggs. Purified cyclin-p34cdc2 phosphorylated the regulatory light chain of cytoplasmic and smooth muscle myosin-II in vitro on serine-1 or serine-2 and threonine-9, sites known to inhibit the actin-activated myosin ATPase activity of smooth muscle and nonmuscle myosin (Nishikawa, M., J. R. Sellers, R. S. Adelstein, and H. Hidaka. 1984. J. Biol. Chem. 259:8808-8814; Bengur, A. R., A. E. Robinson, E. Appella, and J. R. Sellers. 1987. J. Biol. Chem. 262:7613-7617; Ikebe, M., and S. Reardon. 1990. Biochemistry. 29:2713-2720). Serine-1 or -2 of the regulatory light chain of Xenopus cytoplasmic myosin-II was also phosphorylated in Xenopus egg lysates stabilized in metaphase, but not in interphase. Inhibition of myosin-II by cyclin-p34cdc2 during prophase and metaphase could delay cytokinesis until chromosome segregation is initiated and thus determine the timing of cytokinesis relative to earlier events in mitosis.

Separate domains of KAR1 mediate distinct functions in mitosis and nuclear fusion.

The yeast KAR1 gene is essential for mitotic growth and important for nuclear fusion. Mutations in KAR1 prevent duplication of the spindle pole body (SPB), and affect functions associated with both the nuclear and cytoplasmic microtubules. The localization of hybrid Kar1-lacZ proteins, described elsewhere (Vallen, E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press), suggest that the protein is associated with the SPB. In this paper, we report a deletion analysis demonstrating that the mitotic and karyogamy functions of KAR1 are separate and independent, residing in discrete functional domains. One region, here shown to be essential for mitosis, coincided with a part of the protein that is both necessary and sufficient to target Karl-lacZ hybrid proteins to the SPB (Vallen, E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press). Complementation testing demonstrated that deletions in this interval did not affect nuclear fusion. A second region, required only for karyogamy, was necessary for the localization of a Kar3-lacZ hybrid protein to the SPB. These data suggest a model for the roles of Kar1p and Kar3p, a kinesin-like protein, in nuclear fusion. Finally, a third region of KAR1 was found to be important for both mitosis and karyogamy. This domain included the hydrophobic carboxy terminus and is sufficient to target a lacZ-Kar1 hybrid protein to the nuclear envelope (Vallen E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press). Altogether, the essential mitotic regions of KAR1 comprised 20% of the coding sequence. We propose a model for Kar1p in which the protein is composed of several protein-binding domains tethered to the nuclear envelope via its hydrophobic tail.

Asymmetric mitotic segregation of the yeast spindle pole body.

The yeast KAR1 gene is required for spindle pole body (SPB) duplication and nuclear fusion. We determine here that KAR1-beta-galactosidase hybrid proteins localize to the outer face of the SPB. Remarkably, after SPB duplication, the hybrid protein was found associated with only one of the two SPBs, usually the one that enters the bud. Using an ndc1 mutant, which forms a defective SPB at the nonpermissive temperature, we found that the hybrid was exclusively associated with the "new" SPB. Two regions of KAR1 contribute to its localization; an internal 70 residue region was necessary and sufficient to localize hybrids to the SPB, and the hydrophobic carboxyl terminus localized proteins to the nuclear envelope. The localization domains correspond to two functional domains required for SPB duplication. We suggest that KAR1 is anchored to the nuclear envelope and interacts with at least one other SPB component during the cell cycle.

Monoclonal antibodies specific for thiophosphorylated proteins recognize Xenopus MPF.

Maturation promoting factor, (MPF), is a crucial regulatory component of the eukaryotic cell cycle. Though it is ubiquitous, MPF has been difficult to purify to homogeneity, and little is known about its physical properties or composition. In an attempt to further characterize and purify this protein, we have isolated five monoclonal antibodies that immunoadsorb MPF activity, and inhibit the activity in solution. However, all the antibodies recognize many proteins in partially purified MPF. We have shown that antibody binding is dependent on previous exposure of the preparation to ATP gamma S. This suggests that the antibodies specifically recognize thiophosphoproteins, although not all thiophosphorylated proteins in MPF are immunoprecipitated. Using one antibody, MPF was partially purified by immunoadsorption chromatography. These experiments provide the first evidence that MPF from Xenopus is a phosphoprotein that becomes thiophosphorylated upon addition of ATP gamma S.

The events of the midblastula transition in Xenopus are regulated by changes in the cell cycle.

The midblastula transition (MBT) in Xenopus can be initiated prematurely by blocking the fundamental cell-cycle oscillator with cycloheximide, in which case motility and transcription are quickly initiated. Using various inhibitors of specific events of the cell cycle that do not inhibit the autonomous oscillator, we have shown that transcription is activated when DNA synthesis is interrupted and motility is activated when cell cleavage is inhibited. Furthermore, very low levels of transcription are found to occur before the MBT. These results demonstrate that the pre-MBT egg is fully competent for transcription and motility and suggest that different features of the rapid early cell cycle normally suppress these events.

Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells.

Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h.

Regulation of mRNA levels for microtubule proteins during nerve regeneration.

The molecular regulation of tubulin synthesis was investigated in the regenerating goldfish retina. Previous in vivo studies pointed to an increase in tubulin synthesis in the retina during regeneration of the injured goldfish optic nerve. Using labeled cDNA probes, we showed that this increase occurs as a result of enhanced tubulin mRNA levels. Analysis of labeled in vivo products revealed enhanced beta 2-tubulin synthesis accompanied by an increase in the level of the low-Mr microtubule-associated proteins identified as TAU factors. The results are discussed with respect to the possible involvement of these proteins in the process of nerve regeneration.

Mapping of distinct structural domains on microtubule-associated protein 2 by monoclonal antibodies.

Monoclonal antibodies against microtubule-associated protein 2 (MAP2) were prepared and their specificity was verified by visualization of the antigens using the antibody overlay technique and by radioimmunoassay. MAP2 was cleaved by alpha-chymotrypsin to generate a series of high-molecular-mass fragments ranging between 270 and 140 kDa. The precursor-product relationship of these fragments was suggested from the rate of their appearance and from the analysis of the tryptic peptide map of each fragment. A group of monoclonal antibodies was found to react predominantly with the intact 270-kDa MAP2 molecule and a fragment having a mass of 240 kDa and to some extent with a 215-kDa fragment. Another group of monoclonal antibodies reacted with an antigenic determinant which was located on the 270-kDa molecule as well as on fragments as small as 140 kDa. None of the two groups of monoclonal antibodies reacted with the microtubule-binding domain of MAP2. These results suggest that one group of antibodies reacts with sites located at or dependent upon a terminal 60-kDa domain(s) distal to the microtubule-binding site of MAP2. The second group of antibodies, which can still bind to smaller proteolytic products, appear to be associated with the central region of the MAP2 molecule. Indirect immunofluorescence experiments with the antibody preparations indicated that at least some of the antigenic determinants are exposed when MAP2 is associated with microtubules in the cell body and neurite outgrowths of differentiated rat brain neuroblastoma B104 cells.

Modulation of mRNA for microtubule-associated proteins during brain development.

The heterogeneity of tau microtubule-associated proteins from rat brain is developmentally determined. Newborn rat brain contains two tau polypeptides (tau 0) with somewhat different molecular weights than the five tau components associated with microtubules from 12-day-old brain (tau 12). tau 0 and tau 12 are immunologically related and crossreact with antibodies against tau 12 proteins. Enrichment of the tau mRNA was achieved by prior hybridization of unfractionated poly(A)-containing mRNA to cDNA preparations containing tubulin and actin sequences. The remaining unhybridized mRNA was further fractionated by electrophoresis on methylmercury hydroxide agarose gels. Experiments involving cell-free translation of mRNA indicated that the major differences in the composition of tau proteins from newborn and developing brain are controlled at the mRNA level. The mRNA from newborn rat brain directed the synthesis of five tau proteins, two of which are specific for newborn brain, whereas the other three forms are characteristic of the developing brain. Thus, the appearance in newborn brain of mRNA species specific for three tau 12 forms precedes the phase of the synthesis of these proteins in the cell. By contrast, mRNA from 12-day brain directed the synthesis of four tau proteins specific for the developing brain, one of which is not synthesized by mRNA from newborn brain. None of the newborn tau 0 forms were synthesized with mRNA isolated from 12-day brain.

ADP-ribosylation of microtubule proteins as catalyzed by cholera toxin.

Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8-3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5'-AMP, respectively labeled with the corresponding isotope. Two-dimensional separation by isoelectric focusing and SDS-PAGE of labeled and native tubulin revealed that labeling occurs at least in four different isotubulins. The isoelectric point of the labeled isotubulins was slightly lower than that of native purified tubulin. This shift in mobility is probably due to additional negative charges involved with the incorporation of ADP-ribosyl residues into the tubulin subunits. SDS-PAGE of peptides derived from [32P]ADP-ribosylated alpha and beta tubulin subunits by Staphylococcus aureus protease cleavage showed a peptide pattern identical with that of native tubulin. Microtubule-associated proteins (MAP1 and MAP2) of high molecular weight were also shown to undergo ADP-ribosylation. Incubation of permeated rat neuroblastoma cells in the presence of [32P]NAD and cholera toxin results in the labeling of only a few cell proteins of which tubulin is one of the major substrates.