Stripe Pattern Formation in the Alligator Experiments

As has been reiterated many times in this book in regard to spatial pattern formation we simply do not know what the actual mechanism is in the developmental process. Although we study several possible pattern generating mechanisms such as reaction diffusion, chemotaxis diffusion and mechanical systems the experimental evidence for a specific mechanism is still lacking. One of the major drawbacks is that in many situations we do not know when in development the pattern generating mechanism is operative; we only observe the results. In the case of the striping on the zebra discussed in Chapter 3 we deduced that the mechanism was probably operative at a specific time in development determined by counting the stripes and measuring their size and number relative to the size of the embryo at that time. However, firm experimental verification was lacking due to the paucity of data on developing zebra embryos. Because of the relative ease of embryonic manipulation and reliability of growth data obtainable with alligator embryos Murray et al. (1990) decided to study the stripe patterning on the alligator to try and determine the time of initiation of the patterning mechanism and to quantify the effect of size on the stripe pigmentation pattern. It is in part their work that we describe. We then use the results to show how the theory (Murray 1989) suggested specific experiments (Deeming and Ferguson 1989a) to resolve a recurring question in development, namely the role of genetics in pattern determination, as it applies to the stripe pattern on alligators. An interesting result is that we show that genetics does not play a role in the detailed stripe formation as had been often stated.

Experimental Results of the Effect of Incubation Temperature on the Number of Stripes on A. Mississippiensis

Hatchling alligators are dark brown/black (a result of melanin production) with a series of white stripes down their dorsal side from the nape of the neck to tail tip as typically shown in Figure 4.1. Individual hatchlings exhibit variation in both the intensity of the dark regions of the body and in the number of stripes along the body and tail (Deeming and Ferguson 1989a). Less regular 'shadow stripes' often exist on the body sides; these approximately interdigitate with the principal stripe patterning as seen in Figure 4.1; we discuss these shadow stripes later. We know from Chapter 4 that incubation temperature determines sex in alligators with females from eggs incubated at lower temperatures and males at higher temperatures. Females at 30°C are generally paler and have fewer white stripes than males, incubated from eggs incubated at 33°C (Deeming and Ferguson 1990). During development the pattern is first apparent at stage 23 of development which corresponds approximately to 41-45 days of incubation; see the detailed facts in the seminal review by Ferguson (1985). The gestation period is around 65 to 70 days.

In alligators an incubation temperature of 33°C gives rapid differentiation and growth of the embryo compared to 30° C. For any given day of incubation, or stage

Figure 4.1. Typical stripe pattern on an alligator hatchling. There are generally 8 white stripes from the nape of the head to the rump with 12 from the rump to tail tip. Note the shadow stripes on the trunk towards the ventral side and their position relative to the main stripes. This embryo is at stage 25 which is around 51-60 days of gestation. Ruler is in millimetres. (Photograph courtesy of Professor M.W.J. Ferguson)

Figure 4.1. Typical stripe pattern on an alligator hatchling. There are generally 8 white stripes from the nape of the head to the rump with 12 from the rump to tail tip. Note the shadow stripes on the trunk towards the ventral side and their position relative to the main stripes. This embryo is at stage 25 which is around 51-60 days of gestation. Ruler is in millimetres. (Photograph courtesy of Professor M.W.J. Ferguson)

of development, prior to hatching, embryos at the higher temperature are heavier (Ferguson 1985; Deeming and Ferguson 1989b). As pointed out in Chapter 4, Volume I the optimal temperature for both females and males is in the region of 32°C.

In the first three sections we examine the link between embryo size and the pigmentation pattern exhibited by individual hatchlings and address the important question of when in gestation the actual pattern generation mechanism is operative. If we are ever to discover a real biological pattern formation mechanism from experiment it is clearly essential to know when (and of course where) during development to look for it: it is too late after we see the pattern. Although there is essentially no mathematics in this part of the chapter we use mathematical modelling concepts which result in real verifiable (and already verified) biological implications.

Murray et al. (1990) counted the number of white stripes along the dorsal (top) side of alligator hatchlings, from the nape of the neck to the tip of the tail; there are no stripes on the head. The number of stripes on the body (nape to rump) and on the tail (rump to tail tip) was recorded together with the colour of the tail tip. The total length of the animal, the nape-rump length and rump-tail tip length were also measured to the nearest 0.1 mm at various times during development. Hatchlings from two incubation temperatures, 30°C and 33°C (which resulted respectively in 100% female and 100% male hatchlings) were examined (these were identical animals to those examined by Deeming and Ferguson 1989a).

To investigate the effects of sex on pigmentation pattern (specifically the number of stripes), hatchlings from eggs of a pulsed 'shift twice' experiment (Deeming and Ferguson 1988) were analysed. In these 'shift twice' experiments eggs were incubated at 33°C, except for days 7 to 14 when they were incubated at 30°C. This incubation treat ment produced 23 male and 5 female hatchlings despite the male-inducing temperature of 33°C for the rest of the incubation (Deeming and Ferguson 1988). The fact that there were some females is that sometime during this time period and with this temperature the sex was determined to be female.

In a second experiment various measurements of embryos, at 30°C and 33°C, were taken from days 10 to 50 of incubation. These included total length of the animal, nape-rump length and rump-tail tip length (Deeming and Ferguson 1989b). The embryos were assigned a stage of development (Ferguson 1985). Regression estimates were calculated for embryo growth at the two temperatures. Morphometric measurements were also taken for a third group of embryos incubated for 32, 36, 40, 44, 48 and 52 days. These embryos were also assigned a stage of development and were examined using a dissecting microscope for the earliest macroscopic indication of pigmentation pattern.

Samples of skin containing both black and white stripes were removed from the tails of alligator embryos at stage 28, that is, well after the stripe pattern was evident, and methods for demonstrating neural and melanistic cells were applied to them.

Temperature clearly affected the pigmentation pattern of hatchling alligators (see Table 1 in Murray et al. 1990). There was a higher number of stripes on animals incubated at 33°C compared to those incubated at 30°C. Those animals with a white tip to their tail had, on average, one more stripe than those with a black tip at both temperatures. Generally there are 8 stripes on the body and 12 on the tail. There was no significant effect of temperature upon hatchling length nor was there any direct relationship between hatchling size and the number of stripes. The number of stripes was not sex linked: male hatchlings from eggs incubated at 33°C (30°C between 7 to 14 days) had a mean number of stripes of 19.96 (+/ — 1.15) whereas females from the same treatment had 20.00 (+/ — 0.71) stripes.

Regression estimates for the relationship between time and the length of the tail and the nape-rump length for embryos incubated at 30°C and 33°C are shown in the following section in Figures 4.3 and 4.4 respectively. For ease of comparison the growth at the two temperatures is shown in Figure 4.5. Embryos at the higher temperature grew more rapidly. The time at which a ratio of nape-rump to rump-tail tip equalled 8/12, as predicted from the growth curve data, was influenced by temperature: at 30°C this occurred at 46.5 days and at 33°C at 36.5 days. These times of incubation are similar to those recorded for stage 23 (Ferguson 1985). Total length of the embryos was also related to temperature (Figure 4.6(a)). A regression analysis showed that despite being longer at any given time, embryos at 33°C were equivalent to embryos at 30°C at any given stage (Figure 4.6(b)).

The time at which pigmentation was apparent in embryos occurred much earlier at 33°C (day 36 compared with day 44 at 30°C). The pattern was also more apparent on the body of the embryo before that on the tail.

Melanocytes (melanin producing cells) were found to be present in the basal layer of the epidermis of the alligator embryos although the distribution of both cells and melanin was different in white and black stripes. In black stripes melanocytes were abundant and there was a high concentration of melanin. In white stripes melanocytes were present but rare and although they produced some melanin it was limited to the cells and their immediate environment. There was a very sharp demarcation between the black and white stripe regions of the skin, that is, those with and without melanin; see Murray et al. (1990) for experimental details and data.

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