Introduction to Neurospora biology and genetics,
a cytological perspective
Namboori B. Raju
Shear and Dodge (1927 J. Agr. Res., 34:1019-1042) discovered mating types (mat A and mat a) in Neurospora, and described the life histories of two eight-spored heterothallic species (N.crassa and N. sitophila) and one four-spored homothallic species (N. tetrasperma, later called pseudohomothallic). They named the genus Neurospora because of the nerve-like ornamentation (striations) on developing ascospore walls (Fig. 1).
Fig. 1. Ascospore ornamentation in N. crassa, hence the genus name Neurospora.
Dodge (1927 J. Agr. Res., 35:289-305) described the nuclear basis for heterothallism in the eight-spored species and for pseudohomothallism in the four-spored species. In N. tetrasperma, ascus development is programmed so that each of the four ascospores encloses two nuclei of opposite mating type; single-ascospore cultures are thus self-fertile (see also Raju 1992a Mycol. Res. 96:103-116; Raju and Perkins 1994 Dev. Genet. 15: 104-118). In the eight-spored N. crassa, the linearly ordered ascospore pairs reflect the underlying genetic events during meiosis, and provide the clearest visual demonstration that crossing over occurs at the four-strand stage during meiotic prophase. When a gene marker is close to the centromere (no crossing over between the gene and centromere), the two alleles are likely to segregate from one another at the first division of meiosis and result in a 4:4 ascospore pattern for the segregating alleles. In contrast, when a gene marker is far from the centromere (cross over likely) the two alleles often segregate at the second division of meiosis, and such asci show a 2:2:2:2 or 2:4:2 ascospore pattern for the segregating alleles (Fig. 2).
Dodge also publicized the virtues of N.
crassa for genetic studies, and it was Dodge (then at New York Botanical
Garden) who provided the Neurospora
strains to Stanford’s George Beadle and Edward Tatum for their 1941 landmark
studies, linking gene mutations with nutritional requirements -- later came to
be known as one-gene one-enzyme hypothesis for which they were awarded Nobel
Prize in 1958. With these humble beginnings, Neurospora became the model filamentous fungus for numerous subsequent
discoveries on vegetative incompatibility, recombination, gene conversion,
intragenic complementation, biological clocks, repeat-induced point mutations,
signal transduction, chromosome rearrangements, meiotic drive, population
biology, and post-transcriptional gene silencing (see Perkins 1992, Genetics
130: 687-701; Davis and Perkins 2002, Nature Reviews Genetics 3:397-403). For
general information on Neurospora
genetics and genes, see
Dodge (1927) initiated the early studies on ascus development, and Barbara McClintock (1945, Amer. J. Bot. 32: 671-678) showed that meiosis and chromosome behavior in Neurospora are very similar to that of higher plants and animals (see Singleton 1953 Amer. J. Bot 40: 124-144). McClintock, Singleton, and E.G. Barry (at Stanford) have skillfully used an aceto-orcein staining procedure for studying meiotic chromosome behavior. I use an iron-hematoxylin staining procedure (Raju and Newmeyer 1977 Exp. Mycol. 1: 152-165), which stains chromosomes, nucleolus, spindles, spindle pole bodies, and ascus apical pore very well. A ten-fold dilution of ferric acetate and hematoxylin solutions is also good for preparing unfixed rosettes of maturing asci for photography (Figs. 2-5). The DNA-specific acriflavin “staining” and fluorescence microscopy have also been used for detailed meiotic chromosome analysis (Raju 1986 Mycologia 78: 901-906).
We routinely grow the protoperithecial parent (female) on Petri plates containing synthetic crossing medium for 5 days (at 25 C) and fertilize with conidia (or mycelial fragments) from a strain of opposite mating type. Developing perithecia contain
Fig. 4. Wild type x Round spore. A rosette of maturing asci. All eight ascospores (R as well as R+) are round, because the Round-spore mutation is ascus dominant.
longitudinally and they are also well spaced at the second division. The third-division spindles are aligned across the ascus, but all eight nuclei line up in single file in the elongated narrow ascus prior to ascospore delimitation. Another mitosis occurs in the young ascospores and four or five additional mitoses occur in the mature black ascospores (Fig. 6). An illustrated account of meiosis and ascospore genesis is given in Raju (1980, Eur. J. Cell Biol. 23: 208-223). Many gene mutations result in defective
meiosis, abnormal ascus or ascospore morphology and size (reviewed in Raju 1992b Mycol. Res. 96: 241-262).
Turner and Perkins (1979, Genetics 93: 587-606) discovered Spore killers in Neurospora that behave like meiotic drive elements in Drosophila. When heterozygous (Killer x Sensitive), meiosis and ascospore delimitation are normal, but every ascus contains four large, black, viable ascospores and four small, white, dead ascospores; all viable ascospores carry the killer allele (Fig. 5). Thus spore killers distort genetic ratios of Sk-linked genes. Sensitive nuclei are rescued when they are enclosed together with one or more killer nuclei in the same giant ascospore (Raju 1979 Genetics 93: 607-623; 1994 Mycologia 86: 461-473; Raju and Perkins 1991 Genetics 129: 25-37). David Perkins has single handedly analyzed more than 350 chromosome rearrangements in N. crassa, and showed their usefulness for gene mapping and for studies on heterokaryon incompatibility.
Fig. 5. Sk-2K x Sk-2S. A rosette of maturing asci from a cross heterozygous for Spore killer-2. Most asci show four normal (larger) spores and four small aborted spores. The few asci not showing the 4:4 pattern are still immature. All the maturing ascospores are Sk-2K. The first-division segregation (4K:4S) in all asci indicates that there is no crossing over between Spore killer-2 and the centromere.
Fig. 6. Mature black ascospores contain up to 64 nuclei resulting from 4-5 mitoses.
Figs. 6-9. The nuclei are tagged by fusing the chromosomal protein gene histone H1 with the green fluorescent protein (GFP) gene from a jellyfish.
In the last three years, I have provided cytological support for novel (and more trendy) studies on meiotic gene silencing, and expression of GFP-tagged genes during ascus development. Shiu et al. (2001, Cell 107: 905-915) have shown that an unpaired
Fig. 7. hH1-GFP x hH1-GFP. A rosette of maturing asci at eight days after fertilization. All eight ascospores show glowing nuclei; the ascospores are binucleate at this stage. When homozygous, histone H1 is expressed throughout meiosis and ascospore maturation.
ectopically inserted gene, whose function is essential for meiosis, triggers post- transcriptional gene silencing when made heterozygous in a cross. The silencing occurs not only of the duplicated ectopic gene but also of any other paired or unpaired gene sequence(s) elsewhere that are homologous to that of the ectopic gene (see Kasbekar 2002 J. Biosciences, for a commentary). Ascus development in such heterozygous crosses is arrested at a characteristic stage reflecting the function of the inserted gene. Homozygous crosses carrying the same ectopic gene insert do not show silencing and develop normally. We have demonstrated this novel phenomenon using several cloned genes whose functions are known to be required for normal meiosis or ascus development. With the recent use of GFP-tagged histone H1 and b-tubulin genes, we are able to visualize the expression or silencing of these genes during ascus development in homozygous (Figs. 7) and heterozygous crosses (Figs. 8, 9), respectively. The meiotic silencing by unpaired DNA (MSUD) in heterozygous asci does not extend into autonomously developing ascospores. Thus, in a heterozygous cross of hH1-GFP x wild type, four of the eight ascospores in each ascus begin to show hH1-GFP expression (glowing nuclei) approximately12-24 hours after spore delimitation (Fig. 9). Apparently, the MSUD in heterozygous asci is a defense mechanism against invading foreign DNA.
Figs. 8. Wild type x hH1-GFP. Histone H1-GFP is completely silenced in the developing heterozygous asci until after ascospores are cut out. However, the silencing does not extend into autonomously developing ascospores. Four of the eight ascospores in each ascus show the glowing nuclei.