Lorette C. Javois

Head activator does not qualitatively alter head morphology in regenerates ofHydra oligactis

SummaryThe characterization of head activator (HA) as a morphogen capable of increasing the number of tentacles regenerated by hydra was re-examined. Gastric tissue was excised from HA-treated whole animals and allowed to regenerate. At the cellular level the differentiation of head-specific ectodermal epithelial cells was monitored by quantifying monoclonal antibody, CP8, labeling. This labeling has been correlated with a rise in head activation potential and the determination of tissue to form head structures (Javois et al. 1986). At the morphological level tentacle number was monitored. HA-treated regenerates began the head patterning processes and evaginated tentacles sooner than controls but did not produce extra tentacles. The kinetics of CP8 labeling did not reveal major differences between treated and control regenerates after the initiation of head-specific epithelial cell differentiation. HA appeared to act more like a growth factor stimulating the differentiation of head-specific cell types rather than a morphogen which altered head morphology. An additional aspect of the study examined axial-specific effects of HA on the initiation and extent of head-specific epithelial cell differentiation. The cellular response of ectodermal epithelial cells to HA was dependent on their original axial location. More CP8+ tissue differentiated in regenerates of apical as opposed to mid-gastric origin.

Simultaneous effects of head activator on the dynamics of apical and basal regeneration in Hydra vulgaris (formerly Hydra attenuata)

Previous studies have demonstrated that head activator (HA), an 11 amino acid peptide, stimulates head-specific differentiation processes in hydra. Additionally, HA enhances the differentiation of interstitial cells into nerve cells. This study investigated the effects of exogenous synthetic HA on the dynamics of both apical and basal regeneration in a piece of tissue excised from the body column of treated animals which comprised one-eighth of the original animal. The dynamics of apical and basal regeneration were monitored using the monoclonal antibody TS19. This antibody binds to apical and basal ectodermal tissue very early in the process of regeneration, before morphological structures are evident. Labeling is ultimately localized to the tentacles of the head and a ring above the basal disc. Thus, TS19 is a useful tool for analyzing the dynamics of both apical and basal patterning processes in the same regenerate simultaneously. Quantification of TS19 positive areas on regenerates over a time course of 72 hr revealed that HA treatment accelerated and amplified the dynamics of both apical and basal TS19 labeling. The specific basal effect was novel and was demonstrated to occur in the absence of a determined head independently of new nerve cell differentiation. It is proposed that the basal effect was the result of growth factor-like activity of HA.

Patterning of the head in hydra as visualized by a monoclonal antibody: II. The initiation and localization of head structures in regenerating pieces of tissue☆

The body column of hydra is polarized such that a new head will regenerate from the apical end when both extremities are removed. This is due to a graded property of the tissue termed the head activation gradient. The aim of the experiments presented here was to determine what events connect a two-dimensional segment of the activation gradient in an isolated piece of tissue with the formation of a head structure at a particular location. To this end, tissue pieces with three different shapes were excised and analyzed during and after regeneration. The most apical tissue of each piece was labeled with the DNA-intercalating dye, DAPI, and the area where developmental changes were occurring was monitored using the monoclonal antibody CP8 (Javois et al., 1986). First, it was shown that polarity of regeneration was maintained regardless of the fraction of body length included in the excised pieces. Second, while head structures usually formed from the original apical tissue, they could be located anywhere in the regenerate. This was an effect of the healing process which shaped the apical edge differently in different pieces. Third, early CP8 binding occurred in similarly shaped areas suggesting that patterning events were initiated in a contiguous manner wherever apical tissue was located. And finally, not all of the CP8-marked tissue successfully formed structures. Apparently some regions were favored to continue the patterning process, and these in turn extinguished the process in neighboring regions.

Interactions between the foot and the head patterning systems in Hydra vulgaris.

The Cnidarian, hydra, is an appealing model system for studying the basic processes underlying pattern formation. Classical studies have elucidated much basic information regarding the role of development gradients, and theoretical models have been quite successful at describing experimental results. However, most experiments and computer simulations have dealt with isolated patterning events such as the dynamics of head regeneration. More global events such as interactions among the head, bud, and foot patterning systems have not been extensively addressed. The characterization of monoclonal antibodies with position-specific labeling patterns and the recent cloning and characterization of genes expressed in position-specific manners now provide the tools for investigating global interactions between patterning systems. In particular, changes in the axial positional value gradient may be monitored in response to experimental perturbation. Rather than studying isolated patterning events, this approach allows us to study patterning over the entire animal. The studies reported here focus on interactions between the foot and the head patterning systems in Hydra vulgaris following induction of a foot in close proximity to a head, axial grafting of a foot closer to the head, or doubling the amount of basal tissue by lateral grafting of an additional peduncle-foot onto host animals. Resulting positional value changes as monitored by antigen (TS19) and gene (ks1 and CnNK-2) expression were assessed in the foot, head, and intervening tissue. The results of the experiments indicate that positional values changed rapidly, in a matter of hours, and that there were reciprocal interactions between the foot and the head patterning systems. Theoretical interpretations of the results in the form of computer simulations based on the reaction-diffusion model are presented and predict many, but not all, of the experimental observations. Since the lateral grafting experiment cannot, at present, be simulated, it is discussed in light of what has been learned from the axial grafting experiments and their simulations.

Fluorescent Labeling of Surface or Intracellular Antigens in Whole-Mounts

During the last century, before the advent of microtomes capable of producing thin, even sections, whole-mounts and thick sections were used to study the peripheral nervous system. Tissues were either viewed directly, if sufficiently thin, or further separated into layers by acid maceration (1). Initially, histochemical staining, particularly methylene blue (2), was applied to wholemounts to characterize neuronal cell types, distributions, and innervation of various organs. In more recent times precise innervations were traced through the use of histochemical techniques specific for neuronal components. Histofluorescence techniques were introduced by Falk (3) for the localization of nonadrenaline-containing nerves, and the discovery of neuropeptides led to the application of immunohistochemical techniques in the late 1970s and early 1980s (4,5). Most recently, the application of monoclonal antibody technology has opened a new era in research and diagnosis such that in situ normal and pathological tissue constituents may be detected in whole-mounts ranging from tissues to entire organisms.