Sonic hedgehog promotes the survival of specific CNS neuron populations and protects these cells from toxic insult In vitro.

Sonic hedgehog (Shh), an axis-determining secreted protein, is expressed during early vertebrate embryogenesis in the notochord and ventral neural tube. In this site it plays a role in the phenotypic specification of ventral neurons along the length of the CNS. For example, Shh induces the differentiation of motor neurons in the spinal cord and dopaminergic neurons in the midbrain. Shh expression, however, persists beyond this induction period, and we have asked whether the protein shows novel activities beyond phenotype specification. Using cultures derived from embryonic day 14.5 (E14. 5) rat ventral mesencephalon, we show that Shh is also trophic for dopaminergic neurons. Interestingly, Shh not only promotes dopaminergic neuron survival, but also promotes the survival of midbrain GABA-immunoreactive (GABA-ir) neurons. In cultures derived from the E15-16 striatum, Shh promotes the survival of GABA-ir interneurons to the exclusion of any other cell type. Cultures derived from E15-16 ventral spinal cord reveal that Shh is again trophic for interneurons, many of which are GABA-ir and some of which express the Lim-1/2 nuclear marker, but it does not appear to support motorneuron survival. Shh does not support the survival of sympathetic or dorsal root ganglion neurons. Finally, using the midbrain cultures, we show that in the presence of MPP+, a highly specific neurotoxin, Shh prevents dopaminergic neuron death that normally would have occurred. Thus Shh may have therapeutic value as a protective agent in neurodegenerative disease.

Sonic hedgehog: making the gradient.

The amino-terminal peptide of Sonic hedgehog is a cell-tethered molecule, which nevertheless seems to provide a developmental signal that acts at a distance and has different effects depending on its concentration. Recent structural data suggest that zinc-dependent proteolysis may somehow be involved in Sonic hedgehog's function.

Combinatorial signaling by Sonic hedgehog and Wnt family members induces myogenic bHLH gene expression in the somite.

We have demonstrated previously that a combination of signals from the neural tube and the floor plate/notochord complex synergistically induce the expression of myogenic bHLH genes and myogenic differentiation markers in unspecified somites. In this study we demonstrate that Sonic hedgehog (Shh), which is expressed in the floor plate/notochord, and a subset of Wnt family members (Wnt-1, Wnt-3, and Wnt-4), which are expressed in dorsal regions of the neural tube, mimic the muscle inducing activity of these tissues. In combination, Shh and either Wnt-1 or Wnt-3 are sufficient to induce myogenesis in somitic tissue in vitro. Therefore, we propose that myotome formation in vivo may be directed by the combinatorial activity of Shh secreted by ventral midline tissues (floor plate and notochord) and Wnt ligands secreted by the dorsal neural tube.

Induction of dopaminergic neuron phenotype in the midbrain by Sonic hedgehog protein.

Loss of substantia nigra dopaminergic neurons, which develop from the ventral region of the midbrain, is associated with Parkinson's disease. During embryogenesis, induction of these and other ventral neurons is influenced by interactions with the induction of mesoderm of the notochord and the floor plate, which lies at the ventral midline of the developing CNS. Sonic hedgehog encodes a secreted peptide, which is expressed in notochord and floor plate cells and can induce appropriate ventral cell types in the basal forebrain and spinal cord. Here we demonstrate that Sonic hedgehog is sufficient to induce dopaminergic and other neuronal phenotypes in chick mesencephalic explants in vitro. We find that Sonic hedgehog is a general ventralizing signal in the CNS, the specific response being determined by the receiving cells. These results suggest that Sonic hedgehog may have utility in the induction of clinically important cell types.

Distribution of Sonic hedgehog peptides in the developing chick and mouse embryo.

Sonic hedgehog (Shh) encodes a signal that is implicated in both short- and long-range interactions that pattern the vertebrate central nervous system (CNS), somite and limb. Studies in vitro indicate that Shh protein undergoes an internal cleavage to generate two secreted peptides. We have investigated the distribution of Shh peptides with respect to these patterning events using peptide-specific antibodies. Immunostaining of chick and mouse embryos indicates that Shh peptides are expressed in the notochord, floor plate and posterior mesenchyme of the limb at the appropriate times for their postulated patterning functions. The amino peptide that is implicated in intercellular signaling is secreted but remains tightly associated with expressing cells. The distribution of peptides in the ventral CNS is polarized with the highest levels of protein accumulating towards the luminal surface. Interestingly, Shh expression extends beyond the floor plate, into ventrolateral regions from which some motor neuron precursors are emerging. In the limb bud, peptides are restricted to a small region of posterior-distal mesenchyme in close association with the apical ectodermal ridge; a region that extends 50-75 microns along the anterior-posterior axis. Temporal expression of Shh peptides is consistent with induction of sclerotome in somites and floor plate and motor neurons in the CNS, as well as the regulation of anterior-posterior polarity in the limb. However, we can find no direct evidence for long-range diffusion of the 19 x 10(3) Mr peptide which is thought to mediate both short- and long-range cell interactions. Thus, either long-range signaling is mediated indirectly by the activation of other signals, or alternatively the low levels of diffusing peptide are undetectable using available techniques.

Somite differentiation. Sonic signals somites.

Sonic hedgehog, a secreted signalling molecule known to play a role in the patterning of the central nervous system and the limb in vertebrates, also controls differentiation of the somites.

Requirement of 19K form of Sonic hedgehog for induction of distinct ventral cell types in CNS explants.

The identity and patterning of ventral cell types in the vertebrate central nervous system depends on cell interactions. For example, induction of a specialized population of ventral midline cells, the floor plate, appears to require contact-mediated signalling by the underlying notochord, whereas diffusible signals from the notochord and floor plate can induce ventrolaterally positioned motor neurons. Sonic hedgehog (Shh), a vertebrate hedgehog-family member, is processed to generate two peptides (M(r) 19K and 26/27K) which are secreted by both of these organizing centres. Moreover, experiments in a variety of vertebrate embryos, and in neural explants in vitro, indicate that Shh can mediate floor-plate induction. Here we have applied recombinant Shh peptides to neural explants in serum-free conditions. High concentrations of Shh bound to a matrix induce floor plate and motor neurons, and addition of Shh to the medium leads to dose-dependent induction of motor neurons. All inducing activity resides in a highly conserved amino-terminal peptide (M(r) 19K). Moreover, antibodies that specifically recognize this peptide block induction of motor neurons by the notochord. We propose that Shh acts as a morphogen to induce distinct ventral cell types in the vertebrate central nervous system.

Proteolytic processing yields two secreted forms of sonic hedgehog.

Sonic hedgehog (Shh) is expressed in tissues with known signalling capacities, such as the notochord, the floor plate of the central nervous system, and the zone of polarizing activity in the limb. Several lines of evidence indicate that Shh is involved in floor plate induction, somite patterning, and regulation of anterior-posterior polarity in the vertebrate limb. In this report, we investigate the biochemical behavior of Shh in a variety of expression systems and embryonic tissues. Expression of mouse Shh in Xenopus oocytes, COS cells, and baculovirus-infected insect cells demonstrates that in addition to signal peptide cleavage and N-linked glycosylation, chicken and mouse Shh proteins undergo additional proteolytic processing to yield two peptides with molecular masses of approximately 19 kDa (amino terminus) and 27 kDa (carboxy terminus), both of which are secreted. In transfected COS cells, we show that the 19-kDa peptide does not accumulate significantly in the medium unless heparin or suramin is added, suggesting that this peptide associates with the cell surface or extracellular matrix. This retention appears to depend on sequences in the carboxy-terminal part of the peptide. Finally, detection of the 19-kDa product in a variety of mouse and chicken embryonic tissues demonstrates that the proteolytic processing observed in cell culture is a normal aspect of Shh processing in embryonic development. These results raise the possibility that amino- and carboxyl-terminal regions of Shh may have distinct functions in regulating cell-cell interactions in the vertebrate embryo.

UHF-1, a factor required for maximal transcription of early and late sea urchin histone H4 genes: analysis of promoter-binding sites.

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: [sequence: see text]. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.

In vitro analysis of germ cell genotoxicity in testis explant cultures: spermatid micronucleus assays.

Explant cultures of testes from the frog Xenopus laevis have been employed to evaluate the application of testis culture to the routine screening of potential germ cell genotoxicants. Testis explants were incubated with varied concentrations of 3 model mutagens (9,10-dimethyl-1,2-benzanthracene, cyclophosphamide, adriamycin) and solvent controls. Round spermatids were isolated from testes cultured 2-30 days after exposure to each mutagen. The spermatids were then stained with Hoechst 33258 and spermatid micronuclei were scored with a fluorescence microscope. Acute exposure of testes to each mutagen resulted in a dose-dependent increase in spermatid micronuclei that was stage specific and proportional to the length of the exposure period. The assay sensitively detected clastogenic effects by 10(-7) M adriamycin (4-h exposure period) and 10(-6) M cyclophosphamide and dimethylbenzanthracene (24-h exposure). The results demonstrate the feasibility of in vitro approaches to the routine screening and investigation of genotoxicity in the premeiotic S through meiotic division stages of vertebrate spermatogenesis.

In vitro maintenance of spermatogenesis in Xenopus laevis testis explants cultured in serum-free media.

Spermatogenesis has been maintained for extended periods in Xenopus laevis testis explants cultured in serum-free media supplemented with bovine serum albumin, insulin, transferrin, follicle-stimulating hormone, dihydrotestosterone, testosterone, retinol, ascorbate, and tocopherol. The organization of the testis fragments was maintained for 28 days, and all stages of development were present throughout the culture period. 3H-Thymidine-labeled secondary (Type B) spermatogonia developed in 28 days into spermatids at the acrosomal vesicle stage whereas labeled zygotene spermatocytes became mature spermatids in 28 days. Spermatogonial proliferation also continued in vitro for 28 days. Germ cell differentiation was not dependent upon exogenous testosterone, ascorbate, or tocopherol since 3H-labeled spermatogonia became mature spermatids in testes cultured 35 days in media lacking these supplements. Autoradiography demonstrated that 55% of the luminal sperm present in explants cultured 10 days had differentiated in vitro. Sperm from testes cultured 10-35 days were similar to sperm from freshly dissected testes with regard to motility and fecundity, and eggs fertilized with sperm from explant cultures developed normally into swimming tadpoles. The results demonstrate the feasibility of maintaining vertebrate spermatogenesis in culture and suggest that in vitro analysis of Xenopus spermatogenesis using defined media may provide important insights into the evolution of regulatory mechanisms in spermatogenesis.

Changes in DNA topology during spermatogenesis.

DNA topology in histone- and protamine-depleted nuclei (nucleoids) from somatic cells, sperm, and spermatogenic cells was studied to determine if the superhelical configuration of DNA looped domains is altered during spermatogenesis. The expansion and contraction of nucleoid DNA was measured with a fluorescence microscope following exposure of nucleoids to different concentrations of ethidium bromide (EB). Nucleoids from Xenopus laevis erythrocytes, primary spermatocytes, and round spermatids, and from Rana catesbeiana sperm all exhibited a biphasic change (condensed-relaxed-condensed) in size as a function of exposure to increasing concentrations (0.5-100 micrograms/ml) of EB, indicating that they contain negatively supercoiled DNA. In contrast, DNA in sperm nucleoids from Xenopus laevis and Bufo fowleri was relaxed and expanded at low (0.5-6 micrograms/ml) EB concentrations, but became gradually condensed as the EB concentration was increased (6-100 micrograms/ml). Nucleoids prepared from all cell types retained the general shape of the nucleus regardless of the superhelical configuration of the nucleoid DNA. Sperm nucleoid DNA condensed by 100 micrograms/ml EB was relaxed by exposure to UV light, DNase I, proteinase K, or 4 M urea, but not by RNase A or 10 mM dithiothreitol. These results demonstrate that the DNA in sperm nucleoids is constrained in domains of supercoiling by nonbasic nuclear proteins. Negatively supercoiled DNA is present in nucleoids from cells with a full complement of histones, including Rana sperm, but not in nucleoids from Xenopus and Bufo sperm in which histones are replaced by "intermediate-type" protamines. Histone replacement in these species, therefore, is accompanied by unfolding of nucleosomal DNA and active removal of the negative supercoils. Results presented also suggest an important role for the nonbasic nuclear proteins of sperm in the morphogenesis of the nucleus and the arrangement of DNA.