The ectodermal control of mesodermal patterns of differentiation in the developing chick wing

The influence of limb ectoderm on the dorso-ventral muscle and skeletal patterns in the chick wing was studied by recombining stage 14-21 limb mesoderm with the same stage ectoderm in dorso-ventrally reversed orientation. Recombinants grafted to the flank of host embryos were allowed to develop for 10 days. Fully developed wings obtained from stage 15-21 donor embryos have at their distal half d-v polarity conforming to the reversed ectoderm and proximally polarity conforming with the mesoderm. The ectodermal effect is generally observed as a bidorsal feather pattern at the autopod and an almost complete d-v reversal of muscle and skeletal patterns. In experimental wings from donor embryos younger than stage 15, the dorso-ventral pattern conforms with the polarity of the limb mesoderm. The results suggest that control of dorso-ventral polarity resides in the mesoderm until the onset of limb development at stage 15. At this stage, the ectoderm acquires dorso-ventral information which it can impose on the mesoderm.     

Ectodermal influence on physiological cell death in the posterior necrotic zone of the chick wing bud

The phenomenon of "programmed cell death" in the posterior necrotic zone (PNZ) of the chick wing bud was reexamined. Prospective PNZs (pPNZs) were excised from stage 18-21 donor wings and observed for signs of necrosis in vitro. Cell death was quantified by a chromium-51 release assay. Prospective PNZs from the youngest donors (stage 18) showed no signs of death above control levels, while necrosis increased in vitro with increasing donor age. Cell death in the PNZ at stage 24 could be inhibited by removing the overlying ridge at stage 20 or 21. These results suggest that cell death in the PNZ is not rigidly determined early in development as previous studies suggest, but remains responsive to the cellular environment until shortly before the cells die.     

The wide-domain carbon catabolite repressor CreA indirectly controls expression of the Aspergillus nidulans xlnB gene, encoding the acidic endo-beta-(1,4)-xylanase X(24)

The Aspergillus nidulans xlnB gene, which encodes the acidic endo-beta-(1,4)-xylanase X(24), is expressed when xylose is present as the sole carbon source and repressed in the presence of glucose. That the mutation creA(d)30 results in considerably elevated levels of xlnB mRNA indicates a role for the wide-domain repressor CreA in the repression of xlnB promoter (xlnBp) activity. Functional analyses of xlnBp::goxC reporter constructs show that none of the four CreA consensus target sites identified in xlnBp are functional in vivo. The CreA repressor is thus likely to exert carbon catabolite repression via an indirect mechanism rather than to influence xlnB expression by acting directly on xlnB.     

The control of the anteroposterior and dorsoventral axes in embryonic chick limbs constructed of dissociated and reaggregated limb-bud mesoderm

In the 3- to 4-day embryonic avian limb bud, a unique zone of mesodermal tissue is located posteriorly at the junction of bud and body wall. Appropriately grafted to a host limb bud, it induces the formation of a supernumerary limb outgrowth from preaxial tissue and determines that its posterior side will face the graft. It is called the zone of polarizing activity (ZPA).When limb-bud mesoderm is isolated, dissociated, reaggregated centrifugally, jacketed in the mesoderm-free hull of another limb bud, and grown as a graft on a host embryo, the recombinant frequently forms a limb-like structure terminating in digits that fail to show differentiation with respect to the anteroposterior axis. When, however, a bit of ZPA tissue is implanted in the recombinant subjacent to the anterior or posterior margin of the ectoderm, the resulting outgrowth shows a characteristic anteroposterior order of digits that corresponds to the placement of the implant, regardless of its relationship with the anteroposterior axis of the ectoderm or of the host embryo.Dorsoventral differentials have been recognized only in limbs formed from reaggregated leg-bud mesoderm. The direction of the dorsoventral axis always corresponds to the original axis of the ectodermal jacket regardless of the orientation of the recombinant on the host.     

Identification,cloning and analysis of theAspergillus niger genepacC,a wide domain regulatory gene responsive to ambient pH

A wide domain regulatory gene implicated in modulating gene expression in response to ambient pH has been cloned and sequenced from the industrially useful filamentous fungusAspergillus niger. This gene,pacC, is able to restore apacC ⁺ phenotype toA. nidulans pacC ^c 11 andpacC ^c 14 mutants with respect to extent of conidiation, conidial pigment intensity and acid phosphatase regulation. ThepacC gene ofA. niger comprises three exons, encodes a three-zinc-finger protein of 677 amino acids, and shows pH-dependent regulation of expression: mRNA levels are elevated under alkaline conditions and considerably reduced under acidic conditions. The occurrence of PacC consensus binding targets within the sequences upstream ofpacC may indicate autoregulation.     

The in vitro maintenance of the apical ectodermal ridge of the chick embryo wing bud: an assay for polarizing activity.

We have devised an in vitro bioassay for limb bud polarizing activity in the chick embryo. This assay has proven to be a relatively quick and effective test for a morphogenetic factor asymmetrically distributed in the limb bud which is capable of maintaining or thickening the apical ectodermal ridge.A small section of the preaxial border of the chick embryo wing bud was cultured alone, with tissue from the posterior border, mid-dorsal or anterior corner of a second donor wing, or from the flank. The tissue from the preaxial border (responding tissue) consisted of mesoderm with overlying ectoderm and apical ectodermal ridge. When the responding tissue was cultured alone, with flank, or with anterior corner limb tissue, the apical ectodermal ridge flattened in 24–36 hr and many macrophages appeared in the underlying mesoderm. When cultured with posterior border limb tissue however, the apical ridge of the responding tissue remained thickened for up to 48 hr., and no macrophages appear in the underlying mesoderm. The behavior of responding tissue was intermediate between these two extremes when cultured with mid-dorsal limb tissue. The morphogenetic activity assayed by this procedure thus seems to be present as a gradient in the wing bud, with activity decreasing from posterior to anterior. Contact with the responding tissue is not required to enable posterior border tissue to elicit ridge thickening and inhibit the cell death.     

Fibroblast growth factor and culture in monolayer rescue mesoderm cells destined to die in the developing avian wing

In an effort to elucidate control mechanisms for developmentally programmed cell death, conditions were sought that rescue the cells destined to die. Three areas of mesodermal cell death in the chick wing were examined: the posterior necrotic zone (PNZ), the opaque patch (OP), and apical mesoderm. The PNZ and OP are areas of normally programmed cell death, whereas the apical mesoderm undergoes cell death only after the overlying apical ectodermal ridge is excised. Cell death in vitro was quantitated using the chromium-release assay. While these tissues undergo apparently normal cell death in organ culture, in monolayer culture almost all are rescued. In addition, the cells are rescued by the addition of fibroblast growth factor to organ cultures. Since fibroblast growth factor is present in decreasing amounts in the limb at this stage of development, normal cell death may occur upon withdrawal of growth factor.