Trinkaus pursued his doctoral studies in biology at John Hopkins University, under the supervision of Benjamin H. Willier. Willier, adopted an approach similar to his own mentor, Frank R. Lillie at the University of Chicago, which consisted of assigning graduate students general research problems and then leaving them on their own; a pedagogical method that Trinkaus later used on his doctoral and post-doctoral students. Consistent with that method, Willier assigned Trinkaus to study the effect of estrogen on the development of the sexually dimorphic pigmentation patterns of Brown Leghorn chickens. During his first year at John Hopkins, Trinkaus worked on defining the problem more clearly, refining particular research questions, and learning to be skilled at “handling the material,” which included techniques in tissue culture and surgical techniques, such as avian orchiectomy. He performed the latter technique because, without testosterone, capons are able to regenerate feather germs and feathers quickly after its mature feathers are plucked, and Trinkaus needed to examine many feathers and feather germs to better understand their developmental process and to design experiments that would answer his research questions.
After a few false starts in the lab, Trinkaus finally designed an elegant transplantation experiment that would lead to insights into the development of pigmentation patterns in Brown Leghorns and the role of estrogen in this process. Trinkaus wanted to test whether melanoblast cells, the precursor cells to melanocytes, or pigmented skin cells, responded to the hormone, estradiol, in the same way in different cellular environments, or whether the specific environment in the breast feather germ played a role in the melanoblasts’ ability to respond to the hormone. When melanoblast cells responded to the hormone, they synthesized red melanin granules instead of black granules. Because of prior work by Mary Rawles on cell migration, Trinkaus knew that melanoblast cells retain their ability to migrate at different stages of development, and therefore he felt confident that his transplantation experiment would yield clear results. As he recalled in his autobiography, “The procedure was obvious: Transplant adult Brown Leghorn breast basal barb ridges containing responsive melanoblasts to the wing bud of a White Leghorn embryo and see if they migrate in the growing wing bud and enter the forming juvenile wing feather germs. Then determine whether they can express their ability to respond to estradiol in their new environment of immature barb ridge epidermis” (Trinkaus 2003, 81). After the experimental intervention, the transplanted melanoblasts did not respond to estradiol in their new environment, demonstrating that “melanoblasts with an incipient genetic capacity to respond to estradiol must be associated with mature, tract specific, competent epidermis to express it” (Trinkaus 2003, 82). In 1948, this unambiguous result formed the basis of Trinkaus’s dissertation and was published in the Journal of Experimental Zoology.