INTRODUCTION

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In mycelial fungi, hyphae extend by a highly polarized process of cell extension known as tip growth. As the tip extends, periodic branches are formed at or near the apex of the tip. These branches also extend in a polarized manner as new tips. The two processes of branching and tip growth permit the organism to colonize and efficiently utilize a substrate, and they are rarely found in organisms other than fungi, leading to their being termed hallmarks of the fungal kingdom (8).

Attempts to understand tip growth and branching have employed various approaches. Cytological analysis has identified several key substances involved in the process, most notably actin and calcium (6, 8, 11, 12, 13). Ultrastructural studies have demonstrated the importance of tip-growth vesicles (1, 2, 3, 19, 22, 23). Genetic analysis of induced and naturally occurring mutants has identified over 100 loci that encode products that can affect tip growth and branching in Neurospora crassa (17, 20). Generally, the phenotypic outcome of mutation in these genes is either unsupplementably slow or aberrant growth or increased branch density (hyperbranching). Decreased branch density (hypobranching) is almost never observed as a mutant phenotype. Tip extension proceeds via the polarized exocytosis of tip-growth vesicles (1, 2, 3, 9, 19, 22, 23). Vesicle deposition appears to be orchestrated by the Spitzenkörper, a loose collection of vesicles near the hyphal apex (7, 10).

Branching is thought to be an extension of the basic tip growth mechanism. In N. crassa, Zalokar (27), studying regional variations in protein and RNA production, showed that material for tip extension can come from regions of the mycelium at least 12 mm from the colony margin. Katz et al. (14) proposed that precursors from such distant regions are transported to growing tips and that accumulation of one or more of these precursors could be the trigger for branch initiation. Specifically, branching would be initiated when the rate of supply of vesicles outpaces the rate of their incorporation at the growing tip. The vesicular basis of hyphal growth and branching was incorporated into a model by Trinci (22). A key element of this model was the hyphal growth unit, defined as the ratio of total hyphal length to the total number of tips. This growth unit represents the mean length of hyphae that contribute to the extension of an individual tip. The initiation of a new branch has been proposed to be controlled by changes in the cytoplasmic volume, so that branching occurs when a critical value of the mean hyphal growth unit is attained. In this way, the protoplasm considerably distant from the growing tip could have a contributing role in branch initiation. In a further elaboration of this model, Prosser and Trinci (19) proposed that the concentrations of vesicles and nuclei regulate the increase in hyphal length and the occurrence of branches and septa.

Watters et al. (25) showed that in N. crassa the distribution of branch intervals is independent of tip extension rate, as controlled by temperature. Although rapid cooling disturbs this distribution, the normal default distribution of branch intervals was soon restored at the new temperature. Thus, the statistical distribution of branch-to-branch intervals along a hypha seems to constitute a homeostatic set point. Prompted by this observation, our objectives in this study were to develop and test a model of lateral branch initiation that explains the apparent independence of branch interval and temperature yet permits a dramatic response to changes of temperature. This model extends previous work (1, 2, 3, 19, 22, 23) by including the kinetics of growth. In the proposed model, supply and deposition of tip extension factors henceforth assumed to be tip-growth vesicles, in accordance with previously published models of tip growth and branching (1, 2, 3, 19, 22, 23), are proportional to metabolic rate, resulting in a fixed set point for branch interval that is essentially temperature compensated.

The model. Any comprehensive model for tip growth and branching must incorporate the main phenomenology associated with these processes. Tip extension occurs via apical exocytosis of tip-growth vesicles manufactured subapically and transported to the tip (1, 2, 9, 19, 22). Thus, the tip concentration of vesicles and any other tip extension factors depends on the balance between the rates of supply (synthesis and transport) and consumption (either deposition or destruction). Branching, which is triggered by the rate of accumulation at the tip, is proportional to the excess of vesicle production over tip deposition. Branching has previously been shown to be at least partially controlled by factors at or proximal to the previous branch point (26).

	MATERIALS AND METHODS

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Strains and media. The standard N. crassa Oak Ridge wild-type 74-OR81-1a (FGSC #988 Fungal Genetics Stock Center, Microbiology Department, University of Kansas Medical Center, Kansas City, Kans.) was used in all experiments unless otherwise noted. Media and culturing procedures were those described in the work of Davis and deSerres (5). Colonial mutants used were col-4 (allele 70007c), col-8 (allele R2356), and col-16 (allele R2539).

Photomicroscopy. Cultures were photographed on TMX400 film (Eastman Kodak Company, Rochester, N.Y.) with a Zeiss Axioskop microscope (Carl Zeiss, Inc., Thornwood, N.Y.) that was fitted with a 35-mm camera. Negatives were printed to a constant magnification, and growth and branching were measured to the nearest 10 µm. Branch segments were measured following 15 to 25 mm of growth on the plate, in order to allow the colony to reach steady-state growth conditions. A single branch interval is the distance between branch points along a hypha.

Statistical analysis. Changes in branching were analyzed by comparing distributions of branch interval lengths. In most cases, this distribution was skewed toward shorter intervals with an extended tail representing occasional long branch intervals. In form, this distribution matched the gamma-distributed growth observed for several fungal species by Kotov and Reshetnikov (15). Because of this skew, we chose the median as a descriptor of the distribution. The data were graphed and analyzed statistically using the programs Cricket Graph III (Computer Associates Int. Inc., Islandia, N.Y.) and Statworks (Cricket Software, Philadelphia, Pa.) on a Macintosh SE/30 personal computer. The significance of the difference between pairs of distributions of branch intervals was estimated using the nonparametric Mann-Whitney test, which was chosen for its suitability for non-normal distributions.

	RESULTS

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Temperature shifts. The distribution of branch intervals measured at 10° (Fig. 1) matched the default distribution (median = 160 µm) (25). In a transition phase (lasting approximately 2 h) following the shift to 25°C, the distribution of branch lengths was shortened (median = 70 µm) (Fig. 1). Following the transition period, branching recovered, returning to the default distribution characteristic of steady-state growth. Statistical comparison of the transition- and recovery-phase branch distributions to those observed before the shift showed that the length of branch intervals in the transition phase was significantly different (P = 7.4 × 10⁷), while that in the recovery phase was not (P = 0.88).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Hyphal branching associated with a temperature downshift. The distribution of branch intervals is shown for continuous growth at 10°C () as well as during the transition phase following a shift to 25°C (----). Sample sizes were 50 (10°C) and 108 (25°C) branch intervals. The distributions have been smoothed (5-point binomial) and normalized. The transition distribution was shown to be different from the initial (10°C) distribution by using the Mann-Whitney test (P = 7.4 × 107). Following this transition, branching returned to the default distribution (P = 0.88).

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View larger version (18K): [in this window] [in a new window]   FIG. 2.   Branching associated with a temperature upshift. The distribution of branch intervals is shown for continuous growth at 25°C () as well as for the first postshift branch interval (----). Sample sizes were 148 and 45 branch intervals, respectively. The transition distribution was shown to be different from the initial (25°C) distribution by using the Mann-Whitney test (P < 107). Following this transition, branching returned to the default distribution (P = 0.34).

Hyphal damage. We also measured branch length intervals in growing hyphal tips that had been isolated from their colony (Fig. 3), as well as the resulting tipless hyphae (the segments still attached to the colony). In this experiment, all the tips of a colony were severed by cuts approximately 5 mm from the periphery of the colony. Following minor loss of cytoplasm on both sides of the cut, septa in both the isolated tip and the tipless hypha are plugged (24). Within 5 min following isolation of a tip from its colony, most tips produce a single dichotomous branch. After this initial response, the following two branch intervals are, on average, longer than usual (P = 4 × 10⁶) (Fig. 3). As with the temperature shift experiments, branching returns to the default distribution following a transition phase (P = 0.70).

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Response of the tip to proximal hyphal damage. The distribution of branch intervals near the tip was measured prior to the time of damage () and compared to the distribution of lengths of branch intervals formed fully postdamage on the same hyphae (----). Sample sizes were 93 and 54 branch intervals, respectively. The transition distribution was shown to be different from the initial (predamage) distribution by using the Mann-Whitney test (P = 4 × 106). Following this transition, branching returned to the default distribution (P = 0.70).

View larger version (164K): [in this window] [in a new window]   FIG. 4.   Branching in tipless hyphae. The bulk of the colony lies to the right, and the point of damage is to the left (out of frame). Visible immediately to the left of the hyperbranched region is a collapsed section of hypha. Bar, 100 µm.

Colonial mutants. We subjected several hyperbranching mutants (col-4, col-8, and col-16 mutants) to growth at various constant temperatures (Fig. 5). In contrast to the result in wild-type N. crassa, the distribution of branch intervals in these mutants was dependent on tip extension rate. The most obvious shift in branch distribution was in the col-8 mutant (Fig. 5B), in which incubation at reduced temperature resulted in longer branch intervals. The other two hyperbranching strains (Fig. 5A and C) had more modest, but statistically significant, shifts toward longer branches when grown at reduced temperatures.

View larger version (16K): [in this window] [in a new window]   FIG. 5.   Relationship between branch interval distribution and incubation temperature among colonial mutants. Branch interval distributions for three colonial mutants col-4 (A), col-8 (B), and col-16 (C) are shown following incubation at three temperatures: 7°C (), 25°C (----), and 30°C (-·-·-). Sample sizes range from 155 to 195, except for col-16 at 30°C (sample size of 72). For all three mutant strains, the distribution of branch intervals at 7°C was shown to be different than that at 30°C via a Mann-Whitney test (P < 107 in each case).

	DISCUSSION

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We have considered a model for the control of branching in which a branch is initiated when the concentration of tip-growth vesicles reaches a threshold value. The model requires that the rates of supply and consumption of vesicles be proportional to the metabolic rate and that the deposition rate determine the tip growth rate. These assumptions ensure that the accumulation of vesicles that trigger a branch occurs at consistent intervals along the hypha during growth, regardless of the growth rate.

The model was tested first by observing the effects of temperature shifts that produce proportional shifts in tip extension rate. We found that after raising the temperature, the branch intervals decreased, suggesting that the rate at which vesicles are supplied exceeds the rate of consumption. Lowering the temperature results in a temporary increase in the lengths of branch intervals, implying that the supply of vesicles lags behind their consumption at the tip.

The response to temperature shifts may impact our understanding of the growth of fungi in the field. While N. crassa is not generally found in climates which would be expected to suffer such severe environmental shifts, the same could not be said for any number of plant pathogenic fungi found in more temperate climates, where typical day-night temperature cycles could easily reach the ranges shown to trigger a response.

The model also predicts that branching at the tip should enter into a transitional hypobranching phase following physical separation from the colony, as was observed. In contrast, proximal to the point of damage, hyperbranching is induced; we interpret this to mean that blocked septa restrict the flow of tip-growth vesicles destined for the tips, causing a buildup of vesicles and triggering branching. This result mirrors that observed by Trinci and Collinge (24). In that study, the tips of spco-9 mutants were damaged by osmotic shock in order to observe the plugging of septa during repair. The present results show that the results of Trinci and Collinge are not a peculiarity of spco-9 mutants.

The observation of hyperbranching proximal to the site of damage demonstrates that branches can potentially form at any point along the hyphae. Only the dynamics of tip extension cause branching to be normally confined to regions near the apex. The spacing of initiation points within the hyperbranched region is not explained by the proposed model, as the model was designed to address a growing tip. The observation of significant branching proximal to the point of damage argues against models in which branching is absolutely dependent on the division of some resource or structure at the tip itself (such as the Spitzenkörper).

The normally rare dichotomous branch form was induced both by temperature downshifts and in severed tips. The existence of mutations (pk, col-15) and environmental treatments that increase the frequency of dichotomous branches argues that the processes leading to the formation of lateral and dichotomous branches are distinct. Specifically, dichotomous branch points are not simply an occasional random variation on the normally lateral branch form but are triggered by a distinct set of circumstances. Both of the conditions associated with the induction of dichotomous branches in this study are those that produce longer intervals between lateral branches during their transition phases. This finding leads to the seemingly contradictory conclusion that dichotomous branch points may be induced by conditions similar to those that lead to longer intervals between lateral branches. Stated simply, dichotomous branch points may represent a failure to form a lateral branch. This observation may explain the lack of mutations that result in decreased branching. Namely, mutations that could result in longer branch point intervals instead cause closely spaced dichotomous branching and thus are not scored as "loose branch" mutations.

The proposed model also explains the observation that the distribution of branch intervals is dependent on the tip extension rate for colonial mutants, in contrast to the result for wild-type strains. The observation of rate dependency for all three strains tested argues that the effect is general and not the result of a cryptic temperature-sensitive mutation. Our interpretation of the result is that growth at lower temperatures reduces the rate of supply of vesicles to a level that the tips of the colonial mutant are better equipped to handle. Thus, the rate of accumulation slows and branching returns to a more normal distribution. This interpretation carries the implicit assumption that the primary defect in these mutants is in their ability to incorporate tip-growth vesicles at the tip and not a reduction in the supply of such vesicles. The observation of generalized temperature-dependent branching among colonial mutants is a novel observation that bears further examination in a wider spectrum of morphological mutants. This observation calls into question the nature of other colonial mutants previously identified as being temperature sensitive. They may represent cases of the above response and not mutations in temperature-sensitive proteins.

In conclusion, we have developed a model in which branching is triggered by a critical buildup of a colony-produced tip-extension-associated factor (probably tip-growth vesicles). This buildup results from the difference in the rates of supply and consumption of these vesicles. The model explains how branching can be independent of tip extension rate under steady-state conditions while responding dramatically to changing conditions. The model also is consistent with the results of tip isolation experiments and explains the lack of mutations resulting in longer branch intervals as well as the observed temperature and extension-rate dependence of branching in colonial mutants.