![Columbia University Fly Room with bananas.](javascript:dispOrigImg("43057/flylabAPS_FULL.jpg", "Columbia University Fly Room with bananas.", "Y", "Figure 1", "1222280464725-4114524462_1", 'Y', "Copyright American Philosophical Society.", '350', '256', "http://cdm.amphilsoc.org/cdm4/item_viewer.php?CISOROOT=/genetics&CISOPTR=14&CISOBOX=1&REC=11"); "Columbia University Fly Room with bananas.")
One day in 1910, American geneticist Thomas Hunt Morgan peered through a hand lens at a male fruit fly, and he noticed it didn't look right. Instead of having the normally brilliant red eyes of wild-type Drosophila melanogaster, this fly had white eyes. Morgan was particularly interested in how traits were inherited and distributed in developing organisms, and he wondered what caused this fly's eyes to deviate from the norm. Morgan's fly lab (Figure 1) at Columbia University was already in the habit of breeding Drosophila so that the researchers there could observe the transmission of genetic traits through successive generations, so Morgan chose to do a simple breeding analysis to find out more about white eyes. Little did Morgan know that, with this white-eyed fly, he was about to confirm the chromosome theory. In doing so, Morgan would also be the first person to definitively link the inheritance of a specific trait with a particular chromosome.
Morgan Detects an Unusual Pattern of Inheritance
Morgan's early days of scientific training had taught him that, in order to find an answer, he must design an experiment that asked the right question. Thus, he first performed a test cross between the white-eyed male fly and several purebred, red-eyed females to see whether white eyes might also occur in the next generation. The members of the resulting F1 generation had all red eyes, but Morgan suspected that the white-eye trait was still present yet unexpressed in this hybrid generation, like a recessive trait would be. To test this idea, Morgan then crossed males and females from the F1 generation to probe for a pattern of white eye reoccurrence. Upon doing so, he observed a 3:1 ratio of red eyes to white eyes in the F2 generation. This result is very similar to those reported for breeding experiments for recessive traits, as first shown by Mendel. Strangely, however, all of Morgan's white-eyed F2 flies were male, just like their grandfather—there were no white-eyed females at all! Correlation of a nonsexual trait with male or female identity had never been observed before. Why, Morgan puzzled, would this particular trait be limited to only males?
Table 1 provides a brief summary of Morgan's observed results, as well as the expected outcomes for a recessive trait that shows a normal Mendelian pattern of inheritance. In the Mendelian example, the 3:1 ratio of red eyes to white eyes would be shared equally among males (♂) and females (♀). Morgan's data, however, looked very different.
Table 1: Expected Mendelian Ratios versus Morgan's Actual Results
| Cross | Outcome | |
| Expected Phenotypes | Observed Phenotypes | |
| P1 Red ♀ × P1 White ♂ | F1 = All Red | F1 = All Red* |
| F1 Red ♀ × F1 Red ♂ |
75% Red ♀ and ♂ 25% White ♀ and ♂ |
50% Red ♀ 25% Red ♂ 25% White ♂ |
*Morgan did observe 3 white-eyed males in the F1 generation. His original paper suggested that these white-eyed males were evidence of "further sporting."
Morgan Explores Possible Explanations for This Pattern
Morgan was curious as to why female flies never had white eyes, and he considered several possible reasons for this phenomenon. One potential explanation was that white-eyed females never hatched, or that they died early in development. In other words, this hypothesis predicted that white eyes were lethal in female flies—therefore, among the progeny of a test cross of heterozygous (F1) red-eyed females to white-eyed males, there should be no white-eyed females. Morgan conducted this very cross to see whether the results matched his predictions. Surprisingly, this cross yielded a 1:1:1:1 ratio of red-eyed females to white-eyed females to red-eyed males to white-eyed males. Based on these results, Morgan arrived at three important conclusions:
- The appearance of white eyes in females shows that this trait is not lethal in females.
- All possible combinations of white eyes and sex are possible.
- The white-eye trait can be carried over to females when F1 females are crossed with white-eyed males.
So, why would white eyes show a bias toward males in the original F1 x F1 cross? Morgan knew of recent work by Nettie Stevens and E. B. Wilson that demonstrated that sex determination was related to the inheritance of an "accessory chromosome," more recently known as the X chromosome. He further recognized that the inheritance of the sex determination chromosomes in Drosophila seemed to follow closely with the inheritance of the white-eye phenotype. But what was the exact relationship between eye color and sex?
Principles of Sex Determination
In order to understand Morgan's experiments aimed at answering this question, it is first helpful to review the pattern of sex chromosome inheritance in fruit flies. Recall that when a female fly (denoted XX) is crossed with a male fly (denoted XY), their offspring will be 50% female and 50% male (Table 2). Furthermore, note that males have only one X chromosome, which means that the male phenotype is not reflective of a dominant or recessive trait, but rather, it is merely reflective of the only sex chromosome that the male fly carries. Geneticists refer to the state of the male genotype (with only one X chromosome) as hemizygous.
Table 2: Sex Chromosome Inheritance in Fruit Flies
| Male Gametes | |||
| X | Y | ||
| Female Gametes | X | XX | XY |
| X | XX | XY | |
If eye color is inherited along with the X chromosome, then it can be denoted as a linked trait by tagging the X chromosome with a symbol, as follows:
- X+ = Red-eye trait (wild type)
- Xw = White-eye trait
These tagged sex chromosome symbols can now be used to visualize Morgan's test crosses.
Morgan's Test Crosses
In his initial test cross aimed at exploring the precise relationship between eye color and sex, Morgan bred white-eyed males (XwY) with wild-type red-eyed females (X+X+). This cross yielded only red-eyed offspring, as summarized in Table 3.
Table 3: Morgan's First Test Cross
| Male Gametes | |||
| Xw | Y | ||
| Female Gametes | X+ | X+Xw | X+Y |
|
X+
|
X+Xw |
X+Y | |
Next, Morgan decided to cross two flies from the F1 generation—specifically, a red-eyed female (X+Xw) and a red-eyed male (X+Y)—to test for a recessive pattern of inheritance. This cross is depicted in Table 4.
Table 4: Morgan's Second Test Cross
| Male Gametes | |||
| X+ | Y | ||
| Female Gametes | X+ | X+X+ | X+Y |
| Xw | X+Xw | XwY | |
As shown in the table, the offspring of this cross exhibited a 3:1 ratio of red eyes to white eyes, which indicated that white eyes were recessive. Moreover, all of the white-eyed F2 offspring were male.
Next, as previously discussed, Morgan conducted a third cross to determine whether white eyes were lethal in female flies. Here, he bred red-eyed females (X+Xw) with white-eyed males (XwY), as summarized in Table 5.
Table 5: Morgan's Third Test Cross
| Male Gametes | |||
| Xw | Y | ||
| Female Gametes | X+ | X+Xw | X+Y |
| Xw | XwXw |
XwY | |
This third cross revealed that white eyes were in fact not lethal in females, because it produced a 1:1:1:1 ratio of red-eyed females to white-eyed females to red-eyed males to white-eyed males.
Finally, Morgan opted to conduct a fourth cross to determine whether the white-eye trait followed the inheritance of the X chromosome from maternal gametes to male offspring. This reciprocal F1 cross was the most crucial part of this series of experiments, because Morgan could make some very concrete predictions if the trait was indeed sex-linked. Specifically, because the white-eyed trait appeared to be recessive, Morgan could predict that a white-eyed female would probably be homozygous recessive. Moreover, because males inherit their only X chromosome from their mother, the use of a white-eyed mother would mean that an X-linked white-eyed trait would be the only trait male flies could inherit from a homozygous mother. Thus, Morgan could predict that all male offspring resulting from a cross between a white-eyed female and a red-eyed male would be white eyed. Likewise, because female offspring inherit the only X chromosome that exists in the paternal gametes, all female offspring of this particular cross would carry the red-eye trait, and this trait would mask the recessive white-eye trait they inherited via the maternal gametes.
To test these predictions, Morgan crossed a white-eyed female with a red-eyed male, as depicted in Table 6.
Table 6: Morgan's Fourth Test Cross
| Male Gametes | |||
| X+ | Y | ||
| Female Gametes | Xw | X+Xw | XwY |
| Xw | X+Xw |
XwY | |
Because this cross yielded all white-eyed males and all red-eyed females, Morgan could indeed conclude that the white-eye trait followed a sex-linked pattern of inheritance.
The Context of Morgan's Discovery
Morgan's conclusion—that the white-eye trait followed patterns of sex chromosome inheritance—was at once very specific and very grand. A few years prior to these test crosses, Mendelian ideas of inheritance had been enthusiastically discussed by many researchers in the context of new findings about chromosomes. Indeed, after observing meiotic reductive divisions and correlating them to chromosome counts in male and female offspring, cytologists Walter Sutton, Nettie Stevens, and E. B. Wilson had all promoted the idea that sex was determined via chromosome-based inheritance. Morgan, however, had long resisted the idea that genes resided on chromosomes, because he did not approve of scientific data acquired by passive observation. Furthermore, Morgan was not convinced that traits couldn't morph into new forms in an organism based on the blending of parental contributions, an idea leftover from pre-Mendelian scientists. Morgan was sure that Wilson and the other researchers who promoted the chromosome theory of inheritance were looking for an easy answer as to how independent assortment occurred in gamete formation, because he believed they ignored counterevidence in the face of excited conviction. In fact, he thought that the concept of genes was at best an invention intended to link the mysterious paths of chromosomes and discontinuous inheritance patterns. Morgan formalized his derision in a well-known publication (Morgan, 1909), wherein he called for a more experimental approach to the understanding of inherited factors and insisted that germ plasm should not be cast aside as a putative carrier of inherited traits.
Interestingly, within a year of this public criticism of chromosome theory, Morgan set out to test the idea of inherited chromosomal factors using Drosophila. Because Morgan was particularly interested in experiments designed to test hypotheses, he turned to the fly system to maximize data acquisition over short periods of time. Soon after launching these experiments, Morgan saw his white-eyed fly peering back at him through his hand lens. Then, many crosses later, Morgan became convinced by his own empirical evidence that traits could in fact be passed on in the same manner predicted by the inheritance of sex chromosomes. Morgan never looked back, and he developed a huge following of accomplished students over the next few decades. Indeed, for his work with Drosophila, Morgan was awarded the Nobel Prize in 1933.
References and Recommended Reading
Benson, K. R. T. H. Morgan's resistance to the chromosome theory. Nature Reviews Genetics 2, 469–474 (2001) doi:10.1038/35076532 (link to article)
Morgan, T. H. What are "factors" in Mendelian explanations? American Breeders Association Reports 5, 365–368 (1909) (link to article)
———. Sex-limited inheritance in Drosophila. Science 32, 120–122 (1910) (link to article)
