Characterization of concentration gradients of a morphogenetically active retinoid in the chick limb bud

It has long been suggested that the generation of biological patterns depends in part on gradients of diffusible substances. In an attempt to bridge the gap between this largely theoretical concept and experimental embryology, we have examined the physiology of diffusion gradients in an actual embryonic field. In particular, we have generated in the chick wing bud concentration gradients of the morphogenetically active retinoid TTNPB, (E)-4-[2-(5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-2-naphthalenyl)-1-prope nyl] benzoic acid, a synthetic vitamin A compound. Upon local application of TTNPB the normal 234 digit pattern is duplicated in a way that correlates with the geometry of the underlying TTNPB gradient; low doses of TTNPB lead to a shallow gradient and an additional digit 2, whereas higher doses result in a steep, far-reaching gradient and patterns with additional digits 3 and 4. The experimentally measured TTNPB distribution along the anteroposterior axis, can be modeled by a local source and a dispersed sink. This model correctly predicts the site of specification of digit 2, and provides an empirical estimate of the diffusion coefficient (D) of retinoids in embryonic limb tissue. The numerical value of approximately 10(-7) cm2s-1 for D suggests that retinoids are not freely diffusible in the limb rudiment, but interact with the previously identified cellular retinoic acid binding protein. In addition, D affords an estimate of the time required to establish a diffusion gradient as 3 to 4 h. This time span is in a range compatible with the time scale of pattern specification in developing vertebrate limbs. Our studies support the view that diffusion of morphogenetic substances is a plausible mechanism of pattern formation in secondary embryonic fields.

[1]  Gregor Eichele,et al.  Identification and spatial distribution of retinoids in the developing chick limb bud , 1987, Nature.

[2]  B. Alberts,et al.  Microcontrolled release of biologically active compounds in chick embryos: beads of 200-microns diameter for the local release of retinoids. , 1984, Analytical biochemistry.

[3]  L. Honig Positional signal transmission in the developing chick limb , 1981, Nature.

[4]  B. Alberts,et al.  Studies on the mechanism of retinoid-induced pattern duplications in the early chick limb bud: temporal and spatial aspects , 1985, The Journal of cell biology.

[5]  C. Tickle,et al.  A quantitative analysis of the effect of all-trans-retinoic acid on the pattern of chick wing development. , 1985, Developmental biology.

[6]  M. Maden,et al.  Retinoic acid-binding protein in the chick limb bud: identification at developmental stages and binding affinities of various retinoids. , 1986, Journal of embryology and experimental morphology.

[7]  D. Summerbell The effect of local application of retinoic acid to the anterior margin of the developing chick limb. , 1983, Journal of embryology and experimental morphology.

[8]  M. Sporn,et al.  Structure-activity relationships of a new series of retinoidal benzoic acid derivatives as measured by induction of differentiation of murine F9 teratocarcinoma cells and human HL-60 promyelocytic leukemia cells. , 1983, Cancer research.

[9]  V. Hamburger Morphogenetic and axial self-differentiation of transplanted limb primordia of 2-day chick embryos† , 1938 .

[10]  C. Kimmel,et al.  Identification of the cellular retinoic acid binding protein (cRABP) within the embryonic mouse (CD-1) limb bud. , 1985, Teratology.

[11]  J. Westwater,et al.  The Mathematics of Diffusion. , 1957 .

[12]  L. Wolpert,et al.  Local application of retinoic acid to the limb bond mimics the action of the polarizing region , 1982, Nature.

[13]  A. Mastro,et al.  Diffusion of a small molecule in the cytoplasm of mammalian cells. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[14]  P. Loeliger,et al.  AROTINOIDS, A NEW CLASS OF HIGHLY ACTIVE RETINOIDS , 1980 .

[15]  B. Geiger,et al.  Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery , 1982, Cell.

[16]  L. Wolpert Positional information and the spatial pattern of cellular differentiation. , 1969, Journal of theoretical biology.

[17]  D. Ong Cellular retinoid-binding proteins. , 1987, Archives of dermatology.

[18]  L. Honig,et al.  The Control of Pattern Across the Antero-Posterior Axis of the Chick Limb Bud by a Unique Signalling Region , 1982 .

[19]  S. Bryant,et al.  Views of limb development and regeneration , 1986 .

[20]  A. Caplan,et al.  The establishment of vascular-derived microenvironments in the developing chick wing. , 1983, Developmental biology.

[21]  F. Crick Diffusion in Embryogenesis , 1970, Nature.

[22]  H. Meinhardt Models of biological pattern formation , 1982 .

[23]  J. C. Jaeger,et al.  Conduction of Heat in Solids , 1952 .

[24]  K. Paigen,et al.  A simple, rapid, and sensitive DNA assay procedure. , 1980, Analytical biochemistry.

[25]  G. Eichele Retinoids Induce Duplications in Developing Vertebrate LimbsRetinoic acid is a good candidate for the signaling compound thought to be involved in limb pattern formation , 1986 .

[26]  M. Shaw,et al.  Diffusion coefficient measurement by the "stop-flow" method in a 5% collagen gel. , 1981, Biophysical journal.

[27]  L. Rosenhead Conduction of Heat in Solids , 1947, Nature.