Regeneration of HoxD expression domains during pattern regulation in chick wing buds.

The expression domains of genes located at the 5' end of the HoxD (formerly Hox-4) complex appear to correlate with pattern along both the proximal-distal (PrDi) and the anterior-posterior (AP) axes of the developing limb bud, and it has been suggested that the HoxD gene products are involved in the specification of positional information during limb development. The apical ectodermal ridge is required for limb outgrowth and is thought to influence mesodermal cells at the distal end of the limb bud in a region within which patterning events occur. In this paper, we examine the expression of 5' HoxD genes during PrDi pattern regulation in chick wing buds. In limbs undergoing pattern regulation, we demonstrate that the domains of HoxD11 and HoxD13 gene expression are "regenerated" within 24 hr of removal of the distal mesenchyme. In contrast, in limbs which will not form distal structures, HoxD13 expression becomes reduced.

Genetic analysis of developmental mechanisms in hydra. XXI. Enhancement of regeneration in a regeneration-deficient mutant strain by the elimination of the interstitial cell lineage.

The interstitial cell lineage, including interstitial stem cells, nerve cells, and nematocytes, was eliminated from a regeneration-deficient mutant strain (reg-16) of Hydra magnipapillata. The resultant interstitial cell lineage-free (or "epithelial") reg-16 animals showed a marked enhancement in the ability to regenerate head structures. The epithelial reg-16 polyps regenerated nearly the same number of tentacles as was originally present within 8 days after head removal, while interstitial cell lineage-containing (or "complete") reg-16 polyps restored less than one-third of their original tentacle number under the same conditions. Lateral tissue transplantation was used to examine the head activation and inhibition potentials. The gradients of the two potentials along the body axis of intact epithelial 105 (a wild-type strain) and intact epithelial reg-16 polyps were nearly identical to the gradients in their complete counterparts. The changes of the two potentials occurring after head removal in the epithelial 105 animals were also similar to those in the complete 105 animals. However, the postdecapitation changes in the epithelial reg-16 polyps were different from those in complete reg-16 polyps. The changes in the epithelial reg-16 animals were similar to those observed in wild-type hydra while those of complete reg-16 polyps were highly abnormal. These observations suggest that the phenotypic expression of the genetic defect present in the reg-16 mutant strain is attenuated when the interstitial cell lineage is eliminated from its tissue. The role of the interstitial cell lineage in head regeneration and the nature of the defect present in the reg-16 strain are discussed based on the observations made in this and previous related studies.

Temporal pattern of posterior positional identity in mouse limb buds.

This study describes the temporal pattern of posterior positional identity in mouse limb bud cells. To do this wedges of tissue from the posterior edge of mouse limb buds at various stages (limb stages: Wanek et al., 1989b. J. Exp. Zool. 249, 41-49) were grafted to the anterior edge of a host chick embryo wing bud. Grafts of mouse posterior cells are able to induce the formation of supernumerary digits every time when they are taken from buds from stage 3 through stage 6. At stage 7, the frequency declines and by stage 8 the chick cells no longer respond. The results indicate a change in tissue properties at stage 7, which progresses by stage 8 to the point at which posterior positional identity is no longer detectable by this assay. These temporal changes in this aspect of limb pattern formation can be used as an additional criterion to guide the identification of genes involved in the specification of posterior positional identity.

Effects of localized application of transforming growth factor beta 1 on developing chick limbs.

The effects of exogenous transforming growth factor beta (TGF-beta) on chick limb development in vivo were studied by implanting carriers of TGF-beta 1 into developing wing buds. Agarose beads were soaked in solutions containing TGF-beta 1 and implanted into wing buds at stages 18 to 27. Localized application of TGF-beta 1 to distal regions of the wing bud caused specific skeletal elements in the limb to be reduced or absent. The particular proximal-distal limb element affected depended on the stage at which the bead was implanted. Position of the bead in the anterior-posterior axis also influenced the pattern of affected structures. Experiments in which TGF-beta 1 beads were implanted and then removed at 24- and 48-hr intervals indicate that there are specific periods during which a skeletal element appears to be sensitive to the effects of exogenous TGF-beta 1. In a few cases, beads placed in proximal positions in later staged limbs resulted in formation of ectopic cartilage near the bead. These results suggest that exposure to exogenous TGF-beta 1 in vivo influences the development of skeletal elements in the chick limb in a stage- and position-dependent manner.

Conversion by retinoic acid of anterior cells into ZPA cells in the chick wing bud.

In recent years there has been considerable interest in the role of retinoic acid (RA) in vertebrate-limb pattern formation. When RA is applied to the anterior of the chick wing bud, a mirror-image duplication of the limb pattern develops that is identical to the pattern resulting from grafts of posterior tissue (zone of polarizing activity, or ZPA). It has been proposed that position along the anterior-posterior axis in the chick limb is specified by a gradient of a diffusible factor produced by the ZPA. The ZPA-mimicking action of RA has led to the hypothesis that exogenously applied RA acts by providing graded spatial information across the anterior-posterior limb axis. An alternative interpretation is that RA changes anterior cells into ZPA cells, which in turn provide the actual pattern-duplicating stimulus; there is already some preliminary evidence that this occurs. A hybrid interpretation has also been suggested whereby ZPA cells are formed in response to RA exposure and then begin to release retinoids that act as graded spatial cues. We have used a functional assay to test anterior chick wing-bud cells for ZPA activity after exposure to RA. The results of our studies indicate that the action of RA is to change anterior cells into ZPA cells. Further, our results indicate that it is unlikely that RA-treated anterior cells then begin producing RA in such a way as to provide a graded positional signal.

A staging system for mouse limb development.

A series of 15 stages of development for the mouse limb bud have been defined, spanning the time from the first appearance of the limb bud to the completion of limb outgrowth. The stages are based on changes in the morphology of the limb in living preparations. The development and regression of the apical ectodermal ridge (AER) as well as the development of the skeletal structures are also described. This staging system has been developed in response to the need to standardize in situ experimental analyses of the mouse limb bud. Comparable stages of the commonly used chick wing and mouse whole embryo systems are presented.

Mammalian limb bud development: in situ fate maps of early hindlimb buds.

Fate maps of the developing mouse hindlimb bud have been constructed for the first time using exo utero surgical techniques and carbon particle injections. Such fate maps demonstrate that the limb develops in a proximal to distal manner as a result of distal expansion. The anterior-posterior extent of the limb bud develops asymmetrically with the posterior half giving rise to slightly more of the digit pattern (digits 3-5) than the anterior half (digits 1 and 2). We found no evidence for the occurrence of extensive cellular rearrangements during limb development, and the free limb bud appears to give rise to only zeugo- and autopodial elements with the stylopod arising in the body wall proximal to the bud. These results are consistent with our current understanding of limb development in lower vertebrates and also provide detailed information that will be useful for future limb studies in mammals.

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.

Mouse embryos develop normally exo utero.

Surgical procedures are described which permit direct experimental access to postimplantation mouse embryos. These procedures arose from our finding that development proceeds normally to term after embryos have been released from the confines of the uterus, while remaining attached to it via the placenta. Embryos continue to develop exo utero, within the abdominal cavity of the mother, and are capable of surviving a variety of different surgical manipulations. This finding opens the way for experimental analyses of mouse development in vivo.

Duodenal immunoglobulin deficiency in graft versus host disease (GVHD) mice.

The small intestine is a well documented target organ in mouse and human GVHD, and diarrhea is a prominent part of the clinical GVHD syndrome. Although a plethora of systemic immune deficits has been documented in GVHD, the integrity of the small intestinal immune system has not been investigated. A correlation has not been demonstrated between systemic immune dysfuction and the incidence of lymphomas in mouse GVHD survivors. If gastrointestinal immune deficiency exists in mouse GVHD, its possible relationship to GVHD lymphomas, frequently abdominal. should be investigated. GVHD was produced in newborn BLA (C57 BL/Ka females x BALB-C males) mice house in a specific pathogen-free environment by the i.p. inoculation of 10(7) male BALB-C spleen cells. Control mice received syngeneic spleen cells. Twenty GVHD and 16 control mice were sacrificed at 3 weeks and specimens of duodenum were removed for routine histologic and immunofluorescent examination. All but one GVHD mouse (95%) had virtually absent duodenal IgA and IgM. Duodenal cellular fluorescence was demonstrated in all controls. A significant duodenal immunoglobulin deficit has been demonstrated in 3-week-old GVHD mice. The relationship of this finding to GVHD diarrhea, wasting, and neoplasia remains to be determined.