4.2.1 Making reaggregates from different regions of leg mesenchyme
Reaggregates were made essentially as section 2.2.2. The only d ifferen ce was that in addition to using the w hole leg, experim ents were done where only specific regions of the leg were excised (Figure 4.1). For anterior third reaggregates, a cut at the level of the inter-somite border between somites 27 and 28 was made at right angles to the main body axis. A further cut was made to separate the anterior mesenchyme from the trunk and the roughly triangular piece of tissue removed to sterile PBS.
26
32
Anterior
third
Posterior
third
Whole
leg
Figure 4.1
Schematic diagram o f the leg at around stage 20, showing the regions o f mesenchyme taken to make each type o f reaggregate. The dotted lines indicate the levels o f incision made with reference to the level o f the somites. The square bracket indicates the mesenchyme used for whole leg reaggregates.
F or p o sterio r third reaggregates, the sam e procedure was follow ed with a cut at the 30/31 inter-som ite boundary. The thirds o f limb bud thus collected were trypsinised for 10 minutes and the subsequent procedure was as normal.
4.2.2 Assigning digit identity
The criterion for identifying the digits of the norm al foot is phalanx num ber (Figure 4.2). Discounting digit I, which was alm ost never seen in reaggregates, the other three digits each consist o f a long m etatarsal, topped by a differing num ber of phalanges. Digit I I has three phalanges, digit I I I has four an d digit IV has five.
The same criteria were used for identifying the digits in reaggregates, however, a number of problems were encountered. Therefore, reaggregate digits could only be assigned an identity if the follow ing criteria were met. The digit should have: an elongated m etatarsal, to identify the basal phalanx; a terminal, claw -shaped phalanx, to ensure the digit was not distally tru n c a te d ; and the p h alan g es in the d ig it sh o u ld be proximodistally distinct from each other.
Som etim es, a jo in t sw elling developed betw een two phalanges, but no joint space was visible. It was im possible to determ ine whether this was indicative of two fused phalanges, or a single phalanx with abnormal morphology. As the aim of this w ork was to determ ine if anterior m esenchym e gave rise to anterior-type digits rather than posterior, and vice versa, these digits could be assigned a rough identity based on the number of other phalanges in the digit, as long as the other conditions for identity were met. Thus, if there were two other phalanges in the digit it was scored as I I /I I I , whereas if there were three other phalanges it was scored as III/IV .
C artilage growths not m eeting the above criteria, but show ing some evidence of proxim odistal segm entation, were scored as U (unidentifiable). Cartilage nodules, or spikes, not showing any evidence of segmentation were scored as O.
Figure 4.2
Normal chick hindlimb at stage 37, stained with Alcian Green to visualise cartilage. Roman numerals indicate digit number, mts, metatarsals; fe, femur; f, fibula; t, tibia. Proximal is up, anterior is right. Scale bar = 2000 |im.
/
fe
mts
4.2.3 Morphometric analysis of digit identity
To further investigate w hether reaggregate digits could be properly assigned an 'identity' corresponding to those of normal digits, a m orphom etric assessment of selected aspects o f digit morphology was performed. It can readily be seen that the digits o f the normal chick foot, at ten days of incubation, do not vary enorm ously in length or width, yet each contains a different number of phalanges. Each of these phalanges has a characteristic shape that differs between the digits, such that digit Ils phalanges are relatively long and thin com pared to the short, thick phalanges of digit IV (Figure 4.2). The ratio of length:width is therefore greater in the phalanges of digit I I than IV . The idea, then, was to determine if the ratios were com parable between normal and reaggregate digits.
The lengths and widths o f phalanges in the toes was measured, and the ratios worked out for each phalanx in each digit. Length was m easured as the m axim um proxim odistal length of the phalanx. W idth was m easured perpendicular to length, at the thinnest part of the phalanx (generally midway along the length). The same was then done for the phalanges in reaggregate digits.
Some problem s were encountered in determ ining how exactly to com pare the ratios found betw een norm als and reaggregates. W ith each digit having a different num ber o f phalanges, it was not clear which phalanx to compare with which (Figure 4.3). The protocol adopted was as follows.
Reaggregates were harvested at 6.5 days after grafting, a total of 10 days incubation of their mesenchyme. As reaggregates could not be accurately staged, control eggs were windowed at 3.5 days and further incubated to a total 10 days and then harvested also. As each egg does not age at precisely the same speed in the incubator, the aim was to get a spread of ages of normals to compensate for the expected spread of ages o f the reaggregates. One slight problem was that there may have been a developmental retardation of reaggregates compared to normals. Therefore the absolute values of the ratios obtained were not com pared between reaggregate and normal. Instead, the trends