Muscle-derived factors that support survival and promote fiber outgrowth from embryonic chick spinal motor neurons in culture

The purposes of the experiments reported is to provide an unambiguous demonstration that embryonic skeletal muscle contains factors that act directly on embryonic spinal motor neurons both to support their survival and to stimulate the outgrowth of neurites. Cells of lumbar and brachial ventral spinal cords from 6-day-old chick embryos were separated by centrifugation in a two-step metrizamide gradient, and a motor neuron enriched fraction was obtained. Motor neurons were identified by retrogradely labeling with rhodamine isothiocyanate, and were enriched fourfold in the motor neuron fraction relative to unfractionated cells. In culture, the isolated motor neurons died within 3-4 days unless they were supplemented with embryonic chick skeletal muscle extract. Two functionally distinct entities separable by ammonium sulfate precipitation were responsible for the effects of muscle extracts on motor neurons. The 0-25% ammonium sulfate precipitate contained molecules that alone had no effect on neuronal survival but when bound to polyornithine-coated culture substrata, stimulated neurite outgrowth and potentiated the survival activity present in muscle. Most of this activity was due to a laminin-like molecule being immunoprecipitated with antisera against laminin, and immunoblotting demonstrated the presence of both the A and B chains of laminin. A long-term survival activity resided in the 25-70% ammonium sulfate fraction, and its apparent total and specific activities were strongly dependent on the culture substrate. In contrast to the motor neurons, the cells from the other metrizamide fraction (including neuronal cells) could be kept in culture for a prolonged time without addition of exogenous factor(s).     

Mouse Prickle1 and Prickle2 are expressed in postmitotic neurons and promote neurite outgrowth

The Drosophila planar cell polarity (PCP) gene prickle has been previously indicated as one of the regulators of gastrulation in the early embryonic stage. However, the functional role of prickle in the brain in particular is not known. We first indicated that mouse Prickle1 and Prickle2 are continually expressed in the brain throughout the embryonic stages and are observed to be specifically expressed in the postmitotic neurons. Furthermore, Prickle1 or Prickle2 depletion effectively decreases the neurite outgrowth levels of mouse neuroblastoma Neuro2a cells. These results indicate that mouse Prickle1 and Prickle2 possibly regulate positive neurite formation during brain development.     

Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins.

The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: the N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expression of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals.     

Evidence that the two sucrose synthetase genes in maize are related

Summary Evidence is presented that the sucrose synthetase coding sequence at the Shrunken locus is distantly related to the sequence encoding a second, minor sucrose synthetase present in maize endosperm. Three doubly mutant sh bz strains lacking at least part of the Sh coding sequence produce an antigenically cross-reactive protein having the same electrophoretic mobility as the Sh-encoded, 92-kD sucrose synthetase monomer, but differing in primary structure. An mRNA is present in endosperm of mutants with deletions at the Sh locus that is weakly homologous to the Sh coding sequence and encodes a 92-kD protein precipitable with antiserum to sucrose synthetase. We conclude that the genes encoding the two different proteins are related.     

Two vertebrate homeobox genes related to the Drosophila empty spiracles gene are expressed in the embryonic cerebral cortex.

We cloned two homeobox genes, Emx1 and Emx2, related to empty spiracles, a gene expressed in very anterior body regions during early Drosophila embryogenesis, and studied their expression in mouse embryos. Emx1 expression is detectable from day 9.5 of gestation whereas Emx2 appears to be already expressed in 8.5 day embryos. Both genes are expressed in the presumptive cerebral cortex and olfactory bulbs. Emx1 is expressed exclusively there, whereas Emx2 is also expressed in some neuroectodermal areas in embryonic head including olfactory placodes in earlier stages and olfactory epithelia later in development.     

Proteoglycan-Lb, a small dermatan sulfate proteoglycan expressed in embryonic chick epiphyseal cartilage, is structurally related to osteoinductive factor.

We have isolated cDNA clones encoding the core protein of PG-Lb, proteoglycan which has been shown to be preferentially expressed in the zone of flattened chondrocytes of the developing chick limb cartilage (Shinomura, T., Kimata, K., Oike, Y., Yano, S., and Suzuki, S. (1984) Dev. Biol. 103, 211-220). The deduced amino acid sequence from the cDNA analysis indicates the presence of consensus leucine-rich repeats which are present in other small proteoglycans, decorin, biglycan, and fibromodulin. However, the homology analysis revealed that chick PG-Lb showed a higher homology (about 50% in the region containing leucine-rich repeats) to human osteoinductive factor, OIF, rather than to the other small proteoglycans. Furthermore, 6 cysteine residues are detected in both PG-Lb and OIF with invariant relative positions. Therefore, such an evolutionarily conserved structure in the PG-Lb core protein might be involved in some important biological functions of this molecule. In close relation to the structural similarity to OIF, the unique expression of PG-Lb in the ossifying area of cartilage suggested the possible participation of this proteoglycan in osteogenic processes.     

Identification and preliminary characterization of two human digitalis-like substances that are structurally related to digoxin and ouabain.

In order to characterize the structure of endogenous digitalis-like immunoreactive factor (DLIF), we utilized peritoneal dialysis fluid from patients with chronic renal failure as a source of endogenous digitalis-like immunoreactive factor (DLIF), and subjected it to one-step ion exchange chromatography, followed by one step reverse HPLC. Crude dialysis fluid contained 0.09 ng/ml of DLIF, and using Amberlite XAD-2 chromatography we extracted 110 ng of DLIF from 800 ml of dialysis fluid. By applying this partially purified DLIF to our HPLC system, we discerned three peaks of DLIF activity, with retention times of 34, 58 and 63 minutes. The first peak overlapped the elution profile of ouabain, and the third peak co-eluted precisely with digoxin. The second DLIF peak was not in proximity to any of the digitalis-like markers employed. Thus, our results indicate that DLIF isolated from peritoneal dialysis fluid exists in three distinct forms, one of which resembles ouabain, and one which is identical to digoxin.     

Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense.

The coexistence of two different PII, proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding PII) adjacent to glnA. Strains were grown under conditions of nitrogen limitation or nitrogen excess. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by [32P]phosphate or [3H]uracil labeling or by cross-reaction with an anti-A. brasilense PII-antiserum. After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains. The second PII protein, named Pz, was uridylylated under conditions of nitrogen limitation. The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of PII. Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation. No glnZ mutant is yet available, but it is clear that PII and Pz are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters. The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A. brasilense.     

Mesenchyme of embryonic reproductive ducts directs process outgrowth of Retzius neurons in the medicinal leech.

In the two segments of the medicinal leech (Hirudo medicinalis) that contain the male (segment 5) and the female (segment 6) reproductive ducts, the paired Retzius (Rz) neurons are distinguished by several unique properties. For example, the muscles and glands of the body wall are the primary peripheral targets of Rz neurons in standard segments [Rz(X)], whereas the muscles and glands of the reproductive ducts are the primary peripheral targets of Rz neurons in the two reproductive segments [Rz(5,6)]. In this paper, we show that organogenesis and differentiation, which generate an epithelial tube surrounded by mesenchymal cells, occur in the embryonic reproductive ducts at approximately the time when Rz processes first contact these structures. The growth cones leading one branch of the posterior axon of Rz(5,6) contact the duct mesenchymal cells. Following initiation of this contact, these posterior growth cones enlarge and send out numerous filopodia. Secondarily, growth cones leading the anterior axon of each Rz(5,6) also modify their shapes and trajectories. When embryonic reproductive ducts were transplanted into posterior (nonreproductive) segments, the branch of the posterior Rz axon near the ectopic reproductive tissue produced enlarged growth cones and extended several secondary branches into the mesenchyme of the ectopic tissue. This result suggests that the reproductive mesenchyme is attractive to, and can modify the growth of, all Rz neurons. The behavior of Rz(5,6) growth cones suggests that the reproductive mesenchyme cells provide guidance cues that control the location in which Rz axons elaborate their peripheral arborization and form synapses, and that the mesenchyme may also stimulate the production of a densely branched arbor.     

Tech: a RhoA GEF selectively expressed in hippocampal and cortical neurons

Recent studies implicating the Rho family of small G proteins in the regulation of neuronal morphology have focused attention on identifying key components of Rho signaling pathways in neurons. To this end, we have conducted studies aimed at defining the localization and function of Tech, a Rho guanine nucleotide exchange factor (GEF) family member that is highly enriched in brain. We have found that Tech is selectively expressed in cortical and hippocampal neurons with prominent Tech immunostaining apparent in the cell bodies and dendrites of these cells. In vitro studies with prototypical members of the major Rho subfamilies, RhoA, Rac1 and Cdc42, indicate that Tech binds selectively to and activates RhoA. To assess whether Tech may be involved in the regulation of neuronal morphology, we examined the effects of Tech constructs on the morphology of cortical neurons grown in primary culture. We found that a constitutively active Tech construct, Tech 245DeltaC, decreases the number of dendritic processes present on these neurons. This reduction appears to be mediated by activation of RhoA as it is blocked by insertion of a point mutation into the DH domain of Tech which blocks its ability to activate RhoA or coexpression of a dominant negative RhoA construct. As Tech protein levels increase during post-natal development and remain at peak levels into adulthood, these results indicate that Tech regulates RhoA signaling pathways in developing and mature forebrain neurons.     

Identification of two novel genes specifically expressed in the D-group neurons of the terrestrial snail CNS

A search for genes specifically expressed in the giant interneurons of parietal ganglia of the snailHelix lucorum yielded, among others, two genes named HDS1 and HDS2. According to data obtained by Northern hybridization and whole-mountin situ hybridization, both genes are neurospecific and expressed almost exclusively in the peptidergic D-group neurons (Sakharov, 1974) located in the right parietal ganglion.In situ hybridization of the HDS1 and HDS2 probes with CNS of several related species of the Helicoidea superfamily identified in all cases similarly located homologous groups of neurons. Sequencing of the near full-length cDNA copies of the HDS1 and HDS2 genes revealed open reading frames 107 and 102 amino acids long for HDS1 and HDS2, respectively. Both putative proteins contain a hydrophobic leader peptide and putative recognition sites for furin-like and PC-like endopeptidases. Predicted amino acid sequences of the HDS1 and HDS2 proteins were found to be moderately homologous to each other, as well as to the LYCP preprohormone expressed by the light yellow cells of the freshwater snailLymnaea stagnalis. These results confirm an earlier hypothesis that the D-group of theHelix family and the light yellow cells ofLymnaea stagnalis represent homologous neuronal groups. Our data suggest that the HDS1 and HDS2 genes encode precursors of secreted molecules, most likely neuropeptides or neurohormones.     

Vaccinia virus encodes two proteins that are structurally related to members of the plasma serine protease inhibitor superfamily.

Nucleotide sequencing adjacent to the right inverted terminal repetition of the vaccinia virus genome revealed two genes encoding polypeptides that are structurally related to members of the plasma serine protease inhibitor superfamily (SPI). Inclusion in the superfamily is based on extensive amino acid sequence similarities as well as a consensus sequence adjacent to the active-site region near the carboxyl ends of the proteins. The genes designated SPI-1 and SPI-2 are located 10,000 and 17,000 base pairs from the right end of the genome, respectively. The predicted SPI-1 polypeptide is 11 amino acids longer than that of SPI-2, and the deduced masses are 40,471 and 38,125 daltons, respectively. Similarities between SPI-1 and SPI-2 are indicated by the percentage of identical amino acids (44%) and corresponding hydrophobicity plots. The maximum amino acid sequence diversity occurs precisely in the putative active-site region, suggesting that SPI-1 and SPI-2 may inhibit different proteases. SPI-2 is homologous to a previously described cowpox virus gene (D. J. Pickup, B. S. Ink, W. Hu, C. A. Ray, and W. K. Joklik, Proc. Natl. Acad. Sci. USA 83:7698-7702, 1986). Evidence for a cowpox virus homolog of SPI-1 was obtained by DNA hybridization. Thus, the presence of two genes that belong to the plasma serine protease inhibitor superfamily may be characteristic of orthopoxviruses.     

Functional analyses of a variety of chimeric dioxygenases constructed from two biphenyl dioxygenases that are similar structurally but different functionally.

The biphenyl dioxygenases (BP Dox) of strains Pseudomonas pseudoalcaligenes KF707 and Pseudomonas cepacia LB400 exhibit a distinct difference in substrate ranges of polychlorinated biphenyls (PCB) despite nearly identical amino acid sequences. The range of congeners oxidized by LB400 BP Dox is much wider than that oxidized by KF707 BP Dox. The PCB degradation abilities of these BP Dox were highly dependent on the recognition of the chlorinated rings and the sites of oxygen activation. The KF707 BP Dox recognized primarily the 4'-chlorinated ring (97%) of 2,5,4'-trichlorobiphenyl and introduced molecular oxygen at the 2',3' position. The LB400 BP Dox recognized primarily the 2,5-dichlorinated ring (95%) of the same compound and introduced O2 at the 3,4 position. It was confirmed that the BphA1 subunit (iron-sulfur protein of terminal dioxygenase encoded by bphA1) plays a crucial role in determining the substrate selectivity. We constructed a variety of chimeric bphA1 genes by exchanging four common restriction fragments between the KF707 bphA1 and the LB400 bphA1. Observation of Escherichia coli cells expressing various chimeric BP Dox revealed that a relatively small number of amino acids in the carboxy-terminal half (among 20 different amino acids in total) are involved in the recognition of the chlorinated ring and the sites of dioxygenation and thereby are responsible for the degradation of PCB. The site-directed mutagenesis of Thr-376 (KF707) to Asn-376 (LB400) in KF707 BP Dox resulted in the expansion of the range of biodegradable PCB congeners.     

The 70-kd mammalian heat shock proteins are structurally and functionally related to the uncoating protein that releases clathrin triskelia from coated vesicles.

It is shown that in immunological, structural and functional terms the uncoating protein, which catalyses ATP-dependent dissociation of clathrin triskelia from clathrin-coated vesicles is intimately related to two major stress proteins of mammalian cells. These proteins of hitherto unknown functions have polypeptide mol. wts. of 73 kd and 72 kd, respectively. They are normal cell constituents which are synthesized in increased abundance under adverse environmental circumstances, such as non-physiological temperatures or treatment with amino acid analogues.     

F-spondin: a gene expressed at high levels in the floor plate encodes a secreted protein that promotes neural cell adhesion and neurite extension.

The floor plate is a cell group implicated in the control of neural cell pattern and axonal growth in the developing vertebrate nervous system. To identify molecules that might mediate the functions of the floor plate, we have used subtractive hybridization techniques to isolate floor plate-enriched cDNA clones. One such clone encodes a novel secreted protein, F-spondin, which is expressed at high levels in the floor plate. The C-terminal half of the protein contains six repeats identified previously in thrombospondin and other proteins implicated in cell adhesion. F-spondin is expressed in the floor plate at the time that axons first extend and at lower levels in the peripheral nerve. Recombinant F-spondin promotes the attachment of spinal cord and sensory neuron cells and the outgrowth of neurites in vitro. F-spondin may contribute to the growth and guidance of axons in both the spinal cord and the PNS.