The morphogenetic properties causing germ-layer spreading and stratification in amphibian gastrulation were called "tissue affinities" by Holtfreter. The differential adhesion hypothesis (DAH) attributes such liquid-like tissue rearrangements to forces generated by intercellular adhesions within and between the migrating cell populations. This theory predicts that, among the primary germ layers, the cohesiveness of deep ectoderm should be the greatest, that of deep mesoderm should be intermediate, and that of deep endoderm should be the least. Also, the cohesiveness of differentiating neural ectoderm should increase after induction, causing it to internalize and segregate from epidermis. The DAH also explains why the cohesiveness of "liquid" tissues, whose cells are free to rearrange, should be measurable as tissue surface tensions. Using a specially designed tissue surface tensiometer, we demonstrate that (i) aggregates of Rana pipiens deep germ layers do possess liquid-like surface tensions, (ii) their surface tension values lie in precisely the sequence necessary to account for germ-layer stratification in vitro and in vivo, and (iii) the surface tension of deep ectoderm just underlain by the archenteron roof is twice that of not-yet-underlain deep ectoderm. These measurements provide direct, quantitative evidence that the "tissue affinities" governing germ-layer flow during early stages of vertebrate morphogenesis are reflected in tissue surface tensions.

Using counterimmunoelectrophoresis, DNA may be detected at concentrations as low as 1.5 &ml In p h and 0.2 pg/ml in serum. Serum, however,was not suitable for this study because DNA was sporadically released into serum during the clotting process. Counterimmunoelectrophoresis is at least ten times more sensitive than simple immunodiffusion in the detection of DNA. DNA was found in plasma in a variety of illnesses which are delineated in this study. Tan et al, in 1966, using the method of gel diffusion, reported the detection of DNA in the sera of patients with systemic lupus erythematosus (SLE) and other diseases (1). In the same year, Barnett and Vaughan (2) suggested that normal human sera may contain small amounts of antigenic nuclear material, since rabbits immunized with whole human serum in adjuvant often developed antibodies to DNA. Barnett (3), in 1968, employed complement fixation to demonstrate the presence of DNA in sera and synovial fluids. The possibility that circulating DNA might induce pathogenetic antibodies (4) with a role in conditions such as lupus nephritis has been suggested (5). More recently, Hughes et a l , (6) diffusion in agarose gel, reported the detection of DNA in the sera and synovial fluids of patients with a variety of illnesses and suggested that the presence of DNA in blood may be a relatively nonspecific phenomenon. The level of DNA which Hughes was able to detect was approximately 10 rg/ml of serum. Using counterimmunoelectrophoresis (CIE), a technic previously described in this laboratory primarily for the determination of anti-DNA (7), we were able to detect DNA at concentrations as low as 1.5 rg/ml in plasma and 0.2 Pg/ml in serum. With the development of a rapid, reproducible and more sensitive screening technic in which multiple samples could be analyzed simultaneously with appropriate controls, we assayed a random hospital population as well as healthy subjects for circulating DNA. MATERIALS AND METHODS Blood was collected from the University of VirginiaClinical Laboratories in 7-ml tubes (100 x 13 mm) containing 9 mg of sodium EDTA. These blood specimens were obtained on a random basis, promptly refrigerated and processed within 18 hours of venapuncture. Plasma was separated by centrifugation at 2000 rpm for 15 minutes and was then heated at 56" C for 1 hour to destroy complement activity. (Heating at 56" C for 1 hour instead of 30 minutes increased our detection sensitivity from 3.0 to 1.5 pg/ml, whereas we were able to detect no less than 20 rg DNA/ml in fresh plasma.) The heated plasma was again centrifuged at 3000 rpm for 20 minutes in order to remove a flocculent 52

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Emerging chick limb-buds at first grow only in length, not width. The growth parameters of limb mesoderm — cell shapes, distributions, division patterns and cleavage orientations — are incompatible with representations of this tissue as an elongating solid composed of proliferating but immobile cells. We observe that samples of both early limb mesoderm and also surrounding flank mesoderm round up like liquid droplets in organ culture. Therefore, liquid-like tissue rearrangments, including cell shuffling movements and neighbor exchanges, may occur in limb and flank mesoderm during in vivo limb budding. If so, differences in limb-flank surface tension properties would have to be present to keep these two fluid cell populations segregated into distinct tissues and properly positioned underneath limb and flank ectoderm. Previous studies have shown that tissue surface tensions are reflected in the spreading behavior of fused pairs of cell aggregates. To determine whether or not they possess differing surface tension properties, we pair excised pieces of early leg-bud, wing-bud or intervening flank mesoderm with pieces of 5¾-day heart or liver in hanging drop cultures. For more rapid determinations of relative liquid-tissue cohesiveness than can be obtained in conventional, long-term experiments, aggregate pairs are fixed shortly after fusion. Since partial-envelopment configurations depend upon relative aggregate sizes as well as their tissue surface tensions, new procedures are used to deduce relative aggregate cohesiveness from cross-sections of these briefly fused aggregate pairs. The envelopment tendencies of aggregates fixed 6–9 h after fusion are similar to those fixed 15–19 h after fusion: heart tends to surround leg; heart and wing surround each other with similar frequencies, but flank tends to surround heart. Also, liver tends to surround leg and wing, but flank tends to surround liver. When the effects of relative aggregate size are taken into account, these non-random, tissue-specific patterns of aggregate envelopment indicate that the relative cohesiveness of these tissues falls into the sequence: leg > heart ∼ wing > liver > flank. The in vitro behavior of early limb-bud and neighboring flank mesoderm in these studies suggests that they are not simply mechanically identical portions of a single liquid tissue. We have previously proposed that early limb-bud mesoderm may act like a non-dispersing, cohesive liquid droplet which is embedded within a less cohesive fluid layer of flank tissue (and which is molded distally into paddle-shaped conformations by solid-like limb ectoderm and/or subjacent extracellular matrix). This proposal is not only compatible with the growth parameters of limb-bud mesoderm in vivo, but is also consistent with our observation that flank mesoderm surrounds tissues which surround limb mesoderm in these aggregate-fusion-experiments. Our model suggests that differences in the surface tension properties of limb vs. flank mesoderm may combine with differential cell proliferation, and possibly with active limb ectoderm expansion, to generate initial proximodistal limb outgrowth.