Introduction Ceratopteris richardii(C-Fern) is a model organism in the study of plant biology due to its rapid life cycle and

the ease at which it grows in vitro. Like all plants C-Fern undergoes a process known as the alternation of generations, in which it spends part of its life as a diploid sporophyte which can grow several meters tall and another part of its life as a haploid gametophyte, both of which exist as nutritionally independent organisms. The sporophytes produce haploid spores via meiosis in their sporangia, which then divide by mitosis to form a gametophyte. Germination of a spore can take as little as 3 days at 28C, attaining sexual maturity about 8 days later. As adults the C-Fern gametophytes are only 2mm in diameter and consists of a primitive leaf and root structure known as the prothallus and rhizoid, respectively, along with vegetative and reproductive cells. These reproductive organs(known as gametangia) are composed of the female archegonium and the male antheridium. The female archegonium contains only a single egg cell at the bottom of a column with approximately four rows of cells surrounding central "neck" cells. On the other hand the male antheridium produces up to 16 sperm cells, but is much smaller than the archegonium. In the presence of sufficient water the cells of both the archegonium and the antheridium swell up and burst, with the archegoniums central "neck" cells depositing their contents at the tops of the column and the antheridium releasing its motile spermatozoids, which follow chemical messengers known as chemotaxis toward the egg resulting in fertilization. After several days the embryo becomes visible as the diploid sporophyte divides rapidly and with a few weeks the haploid gametophyte dies off. Eventually the sporophyte will produce spores of its own, restarting the alternation of generations. Gametophytes contain both male and female sexual organs but instead of having male and female genders they can instead be either male or hermaphroditic(which contains both reproductive organs). Sexual differentiation from the default hermaphrodite form into the male form is induced by antheridiogen(ACe), a pheromone produced by crowded hermaphrodites. Ace causes latent germinating spores to grow only antheridium, which results in a much smaller male gametophyte. The absence of a

female specific gender is due to the fact that more females would not be advantageous for a population of immobile organisms. Since plants are immobile they must grow toward their nutrients in the soil and since female sex organs have the energy demanding task of nurturing an embryo until it reaches autonomy, too many hungry females could result in population wide malnourishment. Males however are only needed for their sperm and usually die shortly after their antheridium bursts. The previous laboratory exercise examined the effects of population density on the development of two strains of C-Fern, a wild-type(WT) strain and a her1 strain. The results of the experiment revealed that mature WT strains consistently show the presence of males at varying population densities, in which the identities of the males were confirmed by hydration with 1-2 drops of water causing them to swell up and burst, releasing spermatozoids. However the her1 strains showed few if any males, indicating some kind of inability for the her1 hermaphrodites to differentiate into males and was suspected to be the result of a faulty ACe ligand/receptor signaling system.To get a more precise picture of how the her1 mutation prevents sexual differentiation, an experiment was set up in which the filtrate of WT and her1 strains containing ACe would be applied to cultures containing germinating WT or her1 spores. It was hypothesized that if the mutation occurred in the gene that codes for the ACe pheromone, then the her1 filtrate should cause little to no differentiation in WT cultures. However if the mutation occurred in the gene responsible for the receptor to ACe, then the her1 culture should not respond to the functional ACe present in WT filtrates. Procedure In the previous lab each student was assigned a task to collect several plates of a specific strain and then apply filtrates from either WT or her1 gametophytes(Wernick, 2012). Student #15 was assigned three WT plates with population densities of 1:8(with 1:1 being equal to 300 gametophytes), in which two of the plates received a WT derived filtrate at a concentration of 11%, while the remaining plate was treated with a 3.7% WT filtrate. After a week of germination, the plates assigned to student #15 were collected and examined to

determine the percentage of males each plate contained. The results of all the participants in the class were compiled with all the other participating lab sections for analysis. Results Figure 1. Male Proportions of Wild-Type/Her1 Strains at Varying Population Densities

Figure 2. Linear Regression Analysis of Wild-Type/Her1 Population Density Data

Figure 3. Male Population Proportions of Wild-Type/ Strains After Filtrate Treatments

Discussion Analysis of the her1 genes role in the sexual differentiation of C-Fern gametophytes can be broken down into two sections: correlation and causation. The correlation between population density and the amount of sexual differentiation is shown in Figure 1 and Figure 2. Figure 1 shows the average proportion of males(%) when the WT/Her1 strains were cultured at population densities of 1:2, 1:4, 1:8, 1:16 and 1:32(with a 1:1 equaling 300 gametophytes). As can be seen in population densities 1:32, 1:16, 1:8 and 1:2 the proportion of male WT/Her1 gametophytes does indeed go up as the population density. The WT strain shows only small deviations with the average male proportion being 57.6%, but the her1 strain shows a significant deviation from the predicted results at density 1:4. At a population density of 1:4 the her1 strain actually shows an average male proportion of 28.1%, which is significantly larger than all the other her1 population densities. The experimental hypothesis predicted that as the population density increases, the percent males for both WT/Her1 gametophytes should increase, except that the her1 strains should show no males, or at the very least an insignificant number of them. Figure 2 shows a coefficient of determination(R2) of 0.0959, indicating a slight but still existent positive correlation between population densities and male sexual differentiation in her1 strains, whereas

the WT strains R2 is only 0.0302, significantly less than predicted These unexpected results directly conflict with what the experimental hypothesis would have predicted. Not only does the her1 strain have males present, but the correlation between the population density and the amount of male gametophytes is actually greater in the her1 strain than in the WT strain. This correlation is only slight and can easily be explained by considering that R2 describes how much variation in y can be attributed to x, or how the variations in the proportions of male can be explained by looking at the population densities. An R2 of 0.0959 indicates that 90.41% percent of the variation in the her1 male populations cannot be explained by any changes in the population densities, and thus the population density is not a good predictor of the proportion of males in the her1 strains. One possible reason for these contradictory results is that of experimental error, in which a lab section might have mistaken undifferentiated hermaphrodites for males and miscounted the actual male populations. Another possible reason can be seen from the fact that only the 1:4 her1 population density shows a significant number of males, whereas in all the other densities the occurrence of males is rare. The vast majority of male sightings in 1:4 population density slides might be the result of mistakes during sample preparation in which WT spores were mistakenly used in the preparation of the 1:4 her1 plates. This would be a sound explanation for the unusually high male population in the 1:4 slides, but even this explanation is based on the assumption that the inexperienced experimenters can correctly and consistently identify hermaphrodites from male gametophytes. Future laboratory exercises can avoid this mistake by preparing not only the slides with more care but also in validating the experimenters ability to tell a hermaphroditic gametophyte from a male gametophyte. Figure 3 displays the average proportion of males in the WT/her1 control plates and in the WT/her1 plates that were treated with either a WT or a her1 filtrate. The WT plates showed consistently higher rates of sexual differentiation than her1 plates, which were predicted to have very little to none. It is important to consider two factors when analyzing Figure 3: 1) the filtrate treatment was not the only source of ACe, as the hermaphroditic gametophytes did not have their secretions inhibited and 2) the

original hypotheses considered only a fault in the receptor/ligand complex. It is important to consider these as the results show a somewhat intermediate reality between the two possible sites of malfunction. For example, if the hypothesis that assumes a faulty ACe receptor is indeed true then the administration of filtrate from any source would only be effective at catalyzing the differentiation of WT gametophytes as the ACe would be unable to bind to the germinating her1 spores who would produce few if any males. However if the her1 mutation instead produced a malfunctioning ACe ligand (as opposed to its receptor) then her1 filtrates would be ineffective at differentiating both the WT and her1 spores, resulting in few if any WT/Her1 males. Figure 3 shows that neither are completely true, and instead an intermediate explanation is likely. The her1 control group showed <2% males developed in the absence of filtrate, in which addition of WT filtrate to the her1 treatment plates produced a significant increase in male gametophytes over the control group(the smallest increase was 8%; the average increase 14%). Clearly the functioning WT ACe is successfully binding to the her1 receptor, indicating that the presence of a mutated, dysfunctional ACe receptor is unlikely. If the her1 receptor is indeed the same as the WT receptor then it is hypothesized that the her1 filtrate should have little to no effect on the WT spores, with any minor sexual differentiation being attributed to the ACe secreted from the WT hermaphrodites. Figure 3 contradicts this prediction as well as the her1 filtrate treatment resulted in a male population of 35%, the same as the WT control group. This is strong support for the hypothesis that the her1 mutation has no affect on the pheromone itself as a mutated pheromone should produce fewer if any males. The WT treatment group that received WT filtrates showed an average male proportion of 40% which is slightly higher than the WT control or WTHer1 treatment group. If the WT filtrates and the her1 filtrates can elicit a response in either strain then both hypotheses cannot 100% correct, but they aren't 100% false either. When ACe binds to its receptor the signal transduction pathway recruits many enzymes, each of which are coded by genes that are themselves susceptible to mutation, especially if ACe undergoes modifications in the gametophyte(61 Eberle,J.R.

1996). A study by Professor Jo Ann Banks of Perdue University used the mutagen ethyl methanesulfonate(EMS) to induce mutations in WT and her1 spores, which were then allowed to germinate. The resulting gametophytes were then crossed in a variety of ways, in which Banks noted that "the her phenotype did not segregate as a single Mendelian trait but was heritable through at least two generations of back-crossing by wild-type sperm." By crossing mutated her strains with mutated WT strains Banks analyzed how the mutations passed from parental to filial generations in relation to the her mutation and how these mutant ACe products performed when applied to WT gametophytes. Banks concluded that:

"If the secreted form of ACE is converted or modified to an active form by the gametophyte in order to be effective in directing male development, her mutations may also affect this processing. Although many independent her mutations have been identified thus far, we do not know how many different gene loci are represented by these mutations. There are likely to be several her loci given that mutation of any of the genes involved in the ACE signal transduction pathway(s) will yield a her phenotype."(63 Banks,J.A. 1994)

While the conclusion doesn't exactly confirm either experimental hypothesis, it does acknowledge that the her mutation is an interplay of multiple genes that probably affect the ACe signal transduction pathway in some way. Both hypotheses focused on the possibility of the mutation involving ACe as a ligand and the ACe receptor, but neither considered the signal transduction pathway after binding or the ability of the C-Fern gametophyte to modify ACe after secretion.

References

Banks JA. 1994. Sex-determining genes in the homosporous fern ceratopteris. Development 120(7):1949-58. Wernick, Naomi. 2012. Experimental Biology II Course #81.118 Spring Laboratory Manual. Laboratory 1, Part 2: SimBio Virtual Labs: EvoBeaker, pp 19-35. University of Massachusetts Lowell, MA Eberle JR and Banks JA. 1996. Genetic interactions among sex-determining genes in the fern ceratopteris richardii. Genetics 142(3):973-85.

The Effects of Her1 Mutants on the Sexual Differentiation of Ceratopteris Richardii Gametophytes

Zachary Thorpe Experimental Biology (811) TA Emmanuelle Paiva 04/10/2012