Developing Hindlimb Bud Research Articles

Pitx1 and Pitx2 are required for development of hindlimb buds.

Two closely related homeobox transcription factors, Pitx1 and Pitx2, have been implicated in patterning of lateral plate mesoderm derivatives: Pitx1 for specification of hindlimb identity and Pitx2 for determination of laterality. We show that, together, Pitx1 and Pitx2 are required for formation of hindlimb buds and, when present in limited doses, for development of proximal (femur) and anterior (tibia and digit 1) hindlimb structures. Although Pitx1 is expressed throughout developing hindlimb buds, Pitx2 is not expressed in limb bud mesenchyme itself, but is co-expressed with Pitx1 in the presumptive hindlimb field before bud growth. Thus, Pitx1 and Pitx2 genes are required for sustained hindlimb bud growth and formation of hindlimbs.

The mouse polydactylous mutation, luxate (lx), causes anterior shift of the anteroposterior border in the developing hindlimb bud.

Pattern formation along the anterior-posterior axis of the vertebrate limb is established upon activation of Sonic Hedgehog (SHH) in the zone of polarizing activity (ZPA). Since many mouse mutants with preaxial polydactyly show ectopic expression of Shh at the anterior margin of the limb buds, it has been thought to be a primary defect caused by these mutations. We show here that the mouse mutation luxate (lx) exhibits dose-dependent reduction in the size of the Fgf8 expression domain in the ectoderm from the initial stage of limb development. This aberration was independent of Fgf10 expression in the limb mesenchyme. Shh was induced in the mesenchyme underlying the posterior end of the Fgf8 expression domain, indicating an anterior shift of Shh expression in lx hindlimb buds. Prior to the ectopic induction of Shh, the expression domains of genes downstream from Shh, namely dHAND, Gli1, Ptc and Gre, which are normally expressed in posterior mesenchyme of limb buds, expanded anteriorly on the lx hindlimb buds. Conversely, the expression domains of anterior mesenchymal markers such as Gli3and Alx4 decreased in size. Thus, ectopic Shh is not a primary defect of the lx mutation. Rather, our results indicate that the lx mutation affects the positioning of the anteroposterior border in developing hindlimb buds.

Limb development: Farewell to arms

Forelimbs and hindlimbs are, clearly, quite different, and it has long been appreciated that their differences are assigned early in development; the genetic basis of these differences has been more mysterious, however. Recent work has now shown that the homeobox gene Pitx1 imparts identity to the developing hindlimb bud.

Behavior of cells in artificially made cell aggregates and tissue fragments after grafting to developing hind limb buds in Xenopus laevis.

During vertebrate limb development, the limb bud grows along the proximo-distal (P-D) direction, with the cells changing their adhesiveness. To know whether the position-related differences in cell adhesiveness are actually utilized by morphogenesis to constitute limb structures, we grafted cell aggregates made of dissociated cells derived from different positions and stages of developing hind limb buds into developing hind limb buds and observed the behavior of the cells. Cell aggregates made of dissociated mesenchymal cells from two different origins were implanted in different positions and stages of limb buds or grafted on limb stumps made by cutting. The two grafted cell populations in the aggregate always sorted out from each other, but their patterning of sorting-out was quite different according to the transplanted regions. In summary, cells in the aggregate that have closer positional identity to the transplanted site were always situated at the boundary between host and donor cells. The pattern of sorting-out seemed to be determined by the relative adhesiveness of surrounding cells to the constituent cells of the aggregates. We also transplanted fragments dissected out from different regions along the P-D axis into st. 50 limb buds. The descendants of grafted cells moved distally to the region corresponding to their positional identity and participated in the formation of more distal structures from that point. These results suggest that the difference in cell adhesiveness may probably play a role in arranging cells along the P-D axis of a developing limb bud.

Existence of gradient in cell adhesiveness along the developing Xenopus hind limb bud, shown by a cellular sorting-out experiment in vitro.

To examine the possibility of a difference in cell adhesiveness along the developing Xenopus hind limb bud axes, single mesenchymal cells from developing hind limb buds were cultured, allowing them to form an aggregate in a gyratory culture system. By observing the distribution of cells within aggregates, it was found that sorting-out occurred between cells from different positions and different stages. Cells derived from more distal positions tended to be situated interiorly in the aggregates. According to Steinberg's differential adhesion hypothesis, these results support the idea that there is a graded difference in cell adhesiveness along the proximo-distal axis of the developing limb, with adhesiveness increasing distally. Although similar sorting-out was observed between anterior and posterior cell populations, it could not be determined which cell populations were definitely more cohesive. These properties may be correlated with the experimentally demonstrated 'positional value', which should be different among cells located at different positions along the axes of the developing vertebrate limb bud.

Backfoot is a novel homeobox gene expressed in the mesenchyme of developing hind limb.

Homeobox genes play important roles in pattern formation during development. Here, we report the cloning and temporal and spatial expression patterns of a novel homeobox gene Backfoot (BFT for the human gene, and Bft for the mouse gene), whose expression reveals an early molecular distinction between forelimb and hind limb. BFT was identified as a sequence-specific DNA-binding protein. In addition to the homeodomain, it shares a carboxyl-terminal peptide motif with other paired-like homeodomain proteins. Northern hybridization analysis of RNAs from human tissues revealed that human BFT is highly expressed in adult skeletal muscle and bladder. During midgestation embryogenesis, mouse Bft is expressed in the developing hind limb buds, mandibular arches, and Rathke's pouch. The expression of Bft begins prior to the appearance of hind limb buds in mesenchyme but is never observed in forelimbs. At later stages of limb development, the expression is progressively restricted to perichondrial regions, most likely in tendons and ligaments. The timing and pattern of expression suggest that Bft plays multiple roles in hind limb patterning, branchial arch development, and pituitary development. Bft is likely identical to a mouse gene, Ptx1, that was recently isolated by Lamonerie et al. ([1996] Genes Dev. 10:1284-1295) and that has been suggested to play a role in pituitary development.

An immunocytochemical analysis of the expression of thyroid hormone receptor alpha and beta proteins during natural and thyroid hormone-induced metamorphosis in Xenopus.

Amphibian metamorphosis is characterized by the upregulation of thyroid hormone receptor (TR) mRNA in all tissues of tadpole during both the natural and thyroid hormone (TH)-induced development. The two TR genes, termed alpha and beta, are members of a large multigene family of nuclear receptors related to the cellular homolog of the oncogene c-erbA. The phenomenon of upregulation is more marked for the beta than the alpha isoform. To determine whether or not the auto-induction of the transcripts is paralleled by that of TR proteins, non-cross-reacting monoclonal antibodies were prepared against Xenopus laevis TR alpha and beta (xTR alpha, beta) in order to analyze immunocytochemically their expression and localization. Three tadpole tissues that exemplify three major consequences of gene re-programing during natural and TH-induced metamorphosis were studied: (i) Liver that undergoes extensive functional switching; (ii) small intestinal epithelium that exhibits substantial cell death prior to major structural and biochemical modifications; and (iii) hind limb-bud as an example of de novo morphogenesis. It was shown that xTR alpha protein is generally more abundant in these tissues, and its expression is developmentally and hormonally less regulated, than is xTR beta. The auto-induction of xTR beta was particularly intense at 5 days after administration of triiodo-thyronine (T3) to both pre-metamorphic (stage 52) tadpoles and at the onset of natural metamorphosis (stage 55). In the developing hind limb-bud at both stages the upregulation of TR beta is topologically restricted, being particularly intense in dense pockets of cells, presumably rich in chondrocytes. It was concluded that the distribution and expression of xTR alpha and beta proteins match partially, but not fully, those of their transcripts during natural and hormone-induced metamorphosis.

Expression and function of Pax 1 during development of the pectoral girdle.

Pax 1 is a member of the paired-box containing gene family. Expression has previously been observed in the developing sclerotomes and later in the anlagen of the intervertebral discs. Analysis of Pax 1-deficient undulated mice revealed an important role for this gene in the development of the axial skeleton, in which Pax 1 apparently functions as a mediator of notochordal signals during sclerotome differentiation. Here we demonstrate that Pax 1 is also transiently expressed in the developing limb buds. A comparative phenotypic analysis of different undulated alleles shows that this expression is of functional significance. In mice that are mutant for the Pax 1 gene severe developmental abnormalities are found in the pectoral girdle. These include fusions of skeletal elements which would normally remain separate, and failures in the differentiation of blastemas into cartilaginous structures. Although Pax 1 is also expressed in the developing hindlimb buds and Wolffian ridge, no malformations could be detected in the corresponding regions of Pax 1 mutant mice. These findings show that, in addition to its role in the developing vertebral column, Pax 1 has an important function in the development of parts of the appendicular skeleton.

Development of fast synaptic transmission in bullfrog sympathetic ganglia

Extracellular recordings were made of postsynaptic responses originating in sympathetic ganglia 9 and 10 of bullfrog tadpoles and adults. At stage III, when the length and diameter of the developing hindlimb bud are equal, preganglionic stimulation elicits postganglionic action potentials in spinal nerves 9 and 10 near the sciatic plexus. Although they fluctuate in amplitude, these responses follow short trains of repetitive stimuli at 20 Hz. Their mediation by nicotinic synapses was demonstrated by reversible blockade in low Ca 2+, high Mg 2+ Ringer and in nicotine. Parallel sympathetic B and C systems are clearly defined by stage III. They can be selectively activated by appropriate segmental stimulation of the sympathetic chain and are characterized by distinct conduction velocities which both lie in the C fiber range (< 1 m/s). Throughout subsequent tadpole stages, the conduction velocities of the developing B and C systems gradually double while the magnitudes of their compound action potentials grow exponentially by 100-fold. Conduction velocities reach adult values after completion of metamorphosis. These results provide physiological evidence that synapse formation in sympathetic ganglia supplying the hindlimbs begins by the earliest stages of limb bud development, is selective, and progresses over a protracted period of months, prior to myelination.

Evidence for regulation following amputation and tissue grafting in the developing mouse limb.

Procedures are now available to experimentally manipulate postimplantation mouse embryos in situ and allow development to continue into postnatal life (Muneoka, K., N. Wanek, and S.V. Bryant (1986) J. Exp. Zool., 239:289-293). We have investigated the ability of the well-formed hindlimb bud to regulate following two experimental operations: amputation and wedge grafts designed to confront anterior and posterior cells. After comparing the resultant limbs with the fate maps of the relevant stages, we conclude that the developing hindlimb bud at stage 7/8 (equivalent to stage 27/28 of the chick) is capable of partial regeneration of the peripheral digits following amputation and capable of supernumerary digit tip formation after grafting of a wedge of anterior tissue to a posterior position.

The effects of DDT, dieldrin and 2,4-D on amphibian spawn and tadpoles

Spawn of the common frog ( Rana temporaria) or tadpoles of the common frog, common toad ( Bufo bufo) or smooth newt ( Triturus vulgaris) were maintained for 24 or 48 h in amphibian saline to which pesticide had been added. After treatment they were transferred to fresh saline. Tadpoles that hatched from treated spawn and tadpoles that had been treated were observed for up to three weeks. DDT did not penetrate well-developed spawn and was only detected in hatching tadpoles after freshly laid spawn had been treated. These tadpoles became hyperactive after their external gills were lost. In the experiments in which frog and toad tadpoles were exposed to DDT, tadpoles were most susceptible either just before or just after developing hind limb buds and at these and later stages they became hyperactive when tissue concentrations reached 2–3 and 3–4 ppm respectively. During tail resorption small frogs, unlike small toads, were susceptible to tissue residues of DDT that had been acquired during larval development. At every stage of development, toads were more resistant to DDT than frogs, some toad tadpoles surviving despite tissue concentrations of >300 ppm. After treatment with DDT, DDe was often detected in newt tadpoles and in frog and toad tadpoles with hind limbs. Dieldrin caused lower mortality amongst tadpoles than DDT and had less effect on behaviour, although, like DDT, it produced distinct behavioural changes and morphological abnormalities. Only frog tadpoles were treated with 2,4-D. It had no visible effect and no tissue residues could be detected even after treatment in 50 ppm for 48 h.

Tetrazolium salt and electron-microscopic studies of cellular degeneration and necrosis in the interdigital areas of the developing chick limb.

ABSTRACT Cellular degeneration and necrosis were studied in the interdigital areas of developing hind limb buds of normal chick embryos by means of enzyme-specific tetrazolium salts and electron microscopy. Using succinic acid as a substrate and the tetrazolium salt, nitro blue tetrazolium, succinate dehydrogenase was specifically demonstrated via a colour reaction in which degenerating and necrotic cells—those with no enzyme activity—did not stain, while those with enzyme activity stained deeply blue-black.The interdigital cells exhibited near absent levels of succinate dehy-drogenase as early as stage 26–27, one to two days prior to when morphological evidence of degeneration and necrosis was present. It was postulated that the mechanism of cell death resulted from decreased activity and/or loss of strategic cellular enzymes such as succinate dehydrogenase, with a subsequent fall in the cellular adenosine triphosphate (ATP) level and a resultant compromise in vital cellular processes, eventually leading to cell death. The evidence indicated that ‘biochemical degeneration’ occurred prior to morphological changes in cells. The ultrastructural events of degenerating and necrotic cells were also studied. Most ‘dying’ cells observed had already been phagocytosed and were observedin various stages of degenera-tion. The degenerating cells exhibited both nuclear and cytoplasmic changes. There was evidence of active intracellular digestion within the phagocytes. Numerous lysosomes were observed within these cells and some appeared fused with the digestive vacuole’s membranes.The origin of the phagocytes was not determined. No definite information was obtained concerning the utility of cellular degeneration and necrosis in the interdigital areas of the hind limb buds. It appeared to be at least partially causal in separation of the digits.