By then, winged mice were already on the verge of becoming the stuff of legend. Even so, more than one of Mr. Tamura’s friends had told him that they had seen a winged mouse faintly glowing like a flame on the riverbank at night. One of those friends had even witnessed a winged mouse shedding tears. In the region, sightings of a glowing or weeping winged mouse were considered to be bad omens that, ironically, portended good luck for the particular individual who witnessed the occurrence.
What Dr. Akedera picked up from this was the bit about “faintly glowing like a flame on the riverbank at night.” To be spotted in a thicket at night, he conjectured, winged mice would have to be emitting light.
Part of the plan for the second day was canceled and Dr. Sakakibara was called upon once again. The objective was to interview those who had captured two winged mice at the same time (unlike with Ponta and Ai, who had been caught separately). Most of the recorded captors had already passed away or moved from the area, but they finally found one person, a retired police officer, and obtained his testimony.
This was Takanori Abe (formerly of the Fukagawa PD).
Located halfway up Kamuikotan’s mountains, Indian Peak affords vistas of the entire gorge. At the time, Officer Abe was using that location to check for speeders. The tunnel had yet to be completed, and the only road ran along the river. When Officer Abe saw lights that were moving much faster than the others, he radioed in so that a patrol car standing by on the roadside could head out to apprehend the violator. Originally, the peak had no name. Some readers may associate “Indian” with the famous Indian Watermill in Chitose, also in Hokkaido, but there is no connection whatsoever. The name was inspired by Westerns, where North American Indians fall upon stagecoaches on cue from a lookout.
Dr. Akedera interviewed Mr. Abe in the lobby of a hotel. What follows is a summary of the former police officer’s testimony.
Mr. Abe was serving at Indian Peak together with a sergeant from around 10 p.m. According to the records, “Visibility [was] poor due to [a] light fog.” They could not adequately carry out their surveillance from inside their patrol car, and since the sergeant was a veteran of the force, Mr. Abe volunteered to assume a post outside of the vehicle. A spot slightly down from the top offered a vantage point unhindered by any foliage. Mr. Abe left the patrol car every thirty minutes, his radio transceiver in hand, to conduct his lookout from that location. The standard practice was to let drivers be when it was raining, foggy, or conditions were otherwise unfavorable to speeding. A couple of fatal accidents had occurred just the day before, however, and lookout duty was to extend late into the night.
The monitoring shifts ran a half-hour each starting from 22:00 and 23:00, and then came the period in question, the thirty minutes from midnight on. Seated on a large cold slab of rock and absently following the flow of headlights, Mr. Abe noticed a car moving very fast and reached for his transceiver. It was then that he noticed, at the edge of the rock, two small unfamiliar creatures that resembled tailless mice.
Although at first he assumed that they were dead since they crouched stock still, the warmth and slight stirring he felt when he took them in his hands revived legends of the winged mice for the young Officer Abe. He brought back the pair to his cruiser right away.
That was the extent of his story. He had left the force for another line of work quite some time ago, and his memory was riddled with vague patches. Perhaps for fear of leading him, Dr. Akedera did not immediately ask whether or not the mice had been glowing.
After confirming some trivial details, he did, however, ask if there was anything more about the appearance of the mice that Mr. Abe could recall. No doubt having been in an excited state at the time, Mr. Abe only retained inaccurate recollections. His own interest was centered on the fact of capture itself, and the circumstantial details he managed to provide seemed quite suspect. Any emission of light had been too feeble to leave an impression on him, to say the least.
Dr. Akedera then confirmed with Mr. Abe that there were no artificial sources of light such as street lamps on Indian Peak. Establishing that the only illumination by which Mr. Abe could have spotted the mice, then, came from the stars, the headlights of the patrol car, the faint light that shone from down by the river, or from the flashlight he carried, Dr. Akedera considered the likelihood of each of those sources in turn.
Under foggy conditions, starlight could not have been of any help. It was also hard to imagine that the headlights would have been left on. As for light from the road below, even if there were no fog, it hardly seemed to count as illumination. That ruled out everything but directly shining his flashlight on the mice. But would someone who wanted to capture a small animal bathe it in a beam of light?
At one point, Mr. Abe admitted that the area around the mice was bright, but he also seemed to contradict himself by saying that it was not as if the mice themselves had been glowing. The end result was a witness account dredged from deep down in Mr. Abe’s uncertain memories: “It was as if light leaking out from somewhere happened to fall on the two mice,” even though “Thinking about it more calmly now, there was no such light source,” and yet, “The area around the winged mice was definitely faintly illuminated overall.”
With the assistance of a private nature conservancy group in Fukagawa City, the Mutsumi Club, the rest of that afternoon was spent reviewing old literature and calling citizens mainly in regard to the issue of winged mice emitting light. The club members mobilized en masse and two days were spent on the investigation, but there were no new developments on either additional captures or the emission phenomenon.
Allow me to expand upon the activities of the Mutsumi Club. Its members, requested by Dr. Akedera to look into past reports of winged mice, kept up their investigations even after his return to Tokyo following the species’ all-but-certain extinction. Their detailed research, which aimed to uncover cases not included in Table 1, resulted in a final report a year later. In the process, club executive officer Seiji Iwasawa and others encountered a highly interesting fact as regards to why it had been so difficult to gather information on the capture of winged mice. In accordance with Dr. Akedera’s request for information on the captors at the time of capture, the final report that Mr. Iwasawa sent included several photos taken when or around when the individuals had caught the mice. Despite the various seasons and hours, many of the photos were travel souvenirs of families visiting Kamuikotan (i.e., at the time of capture), and when Dr. Akedera received these, he immediately noticed that every photo had children in it. There had been a basis for Dr. Akedera’s conjecture; the reader is invited to revisit the passage on Mr. Tamura, whose memories of winged mice belonged to his early years and were of his childhood friends sharing witness accounts with him. When the elementary school teacher found Ponta, she was leading children on a field trip, and when the reverend found Ai, the winged mouse was spotted by children playing on the shrine’s premises.
As a result of the renewed investigation ushered by Dr. Akedera, it became evident, as he had predicted, that children had been responsible for the animals’ initial sighting or actual capture in the majority of cases. On the other hand, the imprecision of early memories and many of the individuals moving away upon maturity were imagined to be the reason why so few cases had been reported. It was extremely rare for adults like Officer Abe to find winged mice, a fact that did not contradict the difficulties that Dr. Akedera faced in locating suitable interview subjects.
Currently, the Mutsumi Club alone conducts organized searches of winged mice. They employ a method based on the unique working hypothesis that Dr. Akedera tacitly proposed before he passed away: frequent searches on a small scale with children in tow. Putting aside the validity of Dr. Akedera’s conjecture, it is certainly an interesting approach to take, and if it yields results, the validity issue will have been settled after a manner. In addition, subsequent investigations by the Mutsumi Club determined that the families appearing in the photos were relatively close kin,
but this may simply have been a coincidence.
So much for our digression. At the suggestion of the Mutsumi Club, experimentation on the two mice in captivity was considered, but the city council and committee members’ consent proved nearly impossible to obtain on experiments outside the established protocols for species preservation. That avenue had to be abandoned.
Immediately afterwards, major set-up work at the Species Preservation Center got underway. Under Dr. Sakakibara’s ministrations, a compact clean bench, thermostatically controlled tanks, five incubators, an electronic scale, an autoclave, compressed gas, a phase-contrast stereomicroscope, disposable plastic products and more were brought into the pathology lab. In addition, five refrigerators, requisitioned from the town hall and other public facilities with the assistance of the Mutsumi Club, were placed in the adjacent room, and in them went the reagents and serum kits that had been delivered that afternoon.
In truth, this was not the first time an attempt had been made to culture cells from the winged mouse. The first researcher, Dr. Ishikawa, had already tried (using a mouse caught at Indian Peak that subsequently died). He had asked an acquaintance of his, the clinical lab technician Mitsuo Miura, to perform a primary culture of epidermal fibroblasts, but subsequent cultures had failed and it was left at that. Since the center had lacked the apparatuses for culturing cells, Mr. Miura had packed a clean box and a simple incubator into the back of a van –– sitting with them to guard against shaking –– while his wife Tsutako had driven them from a lab in Sapporo to Fukagawa.
In spite of such efforts, Dr. Ishikawa made no mention of it during his academic presentation.
Dr. Akedera had conducted a phone interview with Mr. Miura prior to departing for Hokkaido, but why the attempt failed remained unclear. Even after a detailed examination, no problems came to light on the basic points, such as the time between death and culturing, or the procedure itself.
Either the fetal bovine serum had not been suitable for the cells, or the conditions of the culturing medium had been problematic, or alternatively, the two factors together had been an infelicitous combination, Dr. Akedera concluded in the end. Cells can die off when a culture is contaminated with a resistant strain of bacteria, and perhaps such a possibility merited adequate consideration given how they had gone about it. De facto countermeasures, however, were already in place.
The work began by heating and deactivating as many as fifty types of serum and dissolving the glutamine. All sorts of combinations of the serums with thirty types of culturing medium, thirteen types of antibiotics and antifungal agents, growth factors, transferrin, insulin, etc., were created and subdivided in the clean bench. The effort lasted until after midnight, but ultimately nearly three hundred culturing media were stored in the refrigerators.
It may be that major discoveries owe to coincidence and luck regardless of the epoch. Good fortune smiled on Dr. Akedera, too, as if a strange destiny were guiding him.
Somehow, the content of a phone interview that had been conducted with Dr. Ishikawa’s wife was brought to Dr. Akedera’s attention only that night, via Dr. Sakakibara.
“That day, I had a phone call from the Mutsumi Club asking about the winged mouse. It might have been because the gas cylinder was being changed out at the time, but … the question concerned shining, so I replied that I had no idea what was inside the cylinder. I knew it had to be something important, but my husband never discussed his work with me, and I also assumed that Dr. Sakakibara would know, so I was the one who was surprised. […] Even after my husband disappeared, someone came once a month from Asahikawa to swap out the gas. It wasn’t such a large expense …” (Mrs. Sumiko Ishikawa).
Mrs. Ishikawa had simply misunderstood the intent of the phone interview, but preserved winged mouse tissue became available to the world as a result. Dr. Ishikawa had stored the remainder of the culture he had requested from Mr. Miura in liquid nitrogen.
Dr. Sakakibara and Mr. Tamura left immediately for the Ishikawa residence to speak to Mrs. Ishikawa. She consented on the spot since it would allow her husband’s work to be continued, and the tank was brought to the center for a culturing attempt. Dr. Ishikawa appeared to have been a very methodical person; thin slices of tissue had been deposited by organ in upwards of thirty storage tubes, and a list described the content of each.
Yet work did not begin straight away. Normally, when frozen tissue is brought back to room temperature, much of it is dead. More accurately, upon freezing, the water content within the cells crystallizes and causes damage. With only so much tissue, it might be better, ran the long discussion, for them to let it all slumber in liquid nitrogen until regeneration technologies were somewhat more advanced, just as Dr. Ishikawa seemed to have willed.
Dr. Akedera’s position was that they should culture all of the tissues and re-preserve them under better conditions, whereas Dr. Sakakibara emphasized the dangers of wasting the material. Messrs. Tamura and Miura were also in favor of storing the tissues, so there was something of a standoff. In the end, it became evident that epidermal tissue had been parceled among the various storage tubes, and an agreement was made to defrost one. Hence both Dr. Akedera and Dr. Sakakibara managed to stick to their guns: analysis and preservation, respectively.
Of all tissues, the epidermis includes cells that are the easiest to culture, with the highest success rates. Rapidly defrosted in a 37°C incubator, the tissue was cut into small pieces in a Petri dish, with some reserved for extracting nucleic acid and the rest placed in deactivated serum. After twenty-fours had passed, the material was transferred to a medium so that culturing could continue. According to the records, Dr. Akedera performed all of this work himself and alone with the utmost care.
Cells seeping beyond the tissue fragment were observed ten hours into the culturing process, and comparable growth obtained in all the bases after twenty-four more hours. At this point tissue fragments were retrieved from the Petri dish to be re-frozen.
Meanwhile, one gram of tissue fragment was placed in liquid nitrogen and destroyed, and the nucleic acids extracted using the MAD method8 –– which is known to damage genomic material (DNA) the least – and stored at −70°C in a state of ethanol precipitate.
Seventy-two hours into the process, overgrown cells were carefully detached from the Petri dish and replanted using EDTA isolation (later, it was imagined that this step was to blame for the failed culturing). A portion of the detached cells went into serum mixed with DMSO and stored in liquid nitrogen.
The experiment was proceeding smoothly, and Dr. Akedera seemed to have achieved his initial goal of preservation at the cellular and genetic level. Rapid changes began to occur ninety-six hours into culturing, however.
“Although they weren’t confluent [covering the surface without gaps], on the morning of the fourth day, the cells in the nearly thirty Petri dishes had floated up all at once. I ran to tell Dr. Akedera, and then things went a little haywire,” recalled Mr. Miura, who was serving as an assistant at that point. “The culturing work was being done with great care with varying concentrations of medium for the different Petri dishes, so it was very strange that all of the cells should suddenly die at once. Since it couldn’t have been bacterial contamination, we considered incubator malfunction or a defective gas cylinder but found nothing wrong with them either.”
The batch of frozen cells was defrosted once again and in a hurry to be cultured, but all of the cells floated up and failed to regenerate.
“Ordinarily, mouse cells multiply with extreme rapidity and become immortal through replanting. With chicken, we see more dead cells as the generations go by, but not until the fiftieth or so, after about six months of replanting half the culture each time” (Mr. Miura).
The conclusion that he and Drs. Sakakibara and Akedera finally came to: “They are either similar to highly segmented liver cells, which do not yield successive generations, or else are extremely ‘finicky’ cells that require extraordinarily specific conditions for
culturing.”
Dr. Akedera also inscribed in his log a personal hypothesis: “The cells meet death within a brief time span.”
The next day, an attempt was made to isolate genetic material, but the men realized that they could not confirm the results due to phoresis. This was believed to be caused by the breakdown of the genetic material by DNAse, but there was uncertainty as to why the enzyme had activated in conditions where it normally would not (the MAD method may have been tricky for some reason).
Professor Yoji Ogawa of the Asahikawa College of Science was quickly called in for a consultation, but “Why an enzyme strong enough to break down an organism’s own genetic material should be necessary is beyond me” (Prof. Ogawa).
As we can see from that statement, the mystery only deepened, and they were back to square one.
At the same time, they had shown that at least one generation of cells could be cultured from material frozen in liquid nitrogen, and if he so wished Dr. Akedera could claim that the sample within the liquid nitrogen met the objective of preservation. In other words, he now had the option of excusing himself and entrusting future researchers with the modicum of materials and data that had been obtained.
Retreat, however, was not part of Dr. Akedera’s vocabulary as is clear from the following passage in his journal.
“If an individual organism’s struggles have the preservation of the species as their purpose, then upon species extinction, that individual’s death loses meaning. If this is natural selection, then what is the energy called evolution trying to smother and what is it deeming fit to let live? Might not the principle of natural selection close the circle by selecting against all living things in the end? […] Will the truth guide us to preordained harmony or to chaos? Two winged mice await extinction in their separate cages. I need to figure out what, at this moment, I am able to do about that.”
Biogenesis Page 3