Search

The anatomy and developmental dynamics of the nerve ring in the hypostome of Hydra oligactis were examined immunocytochemically with an antiserum against a neuropeptide and with neuron-specific monoclonal antibodies. The nerve ring is unique in the mesh-like nerve net of hydra. It is a distinct neuronal complex consisting of a thick nerve bundle running circumferentially at the border between the hypostome and tentacle zone. Immunostaining showed that the nerve ring was heterogeneous and contained at least four different subsets of neurons. During head regeneration and budding, the nerve ring appeared only after the nerve net of ganglion and sensory cells had formed. Every epithelial cell is continuously displaced with neurons toward either head or foot in an adult hydra. However, the ectoderm in the immediate vicinity of, and including, the nerve ring constitutes a stationary zone that is not displaced. Tissue immediately above this zone is displaced toward the tip of the hypostome, while tissue below is displaced along the tentacles. Correspondingly, the production of new neurons in the ring as measured by their differentiation kinetics is much slower than in surrounding areas. Thus, the nerve ring is static and stable in contrast to the dynamic features of the nerve net of hydra.

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.

Part I. Antibody Preparation. Overview of Antibody Use in Immunocytochemistry, Su-Yau Mao, Lorette C. Javois, and Ute M. Kent. Purification of Antibodies Using Ammonium Sulfate Fractionation or Gel Filtration, Ute M. Kent. Purification of Antibodies Using Ion-Exchange Chromatography, Ute M. Kent. Purification of Antibodies Using Affinity Chromatography, Ute M. Kent. Purification of Antibodies Using Protein A-Sepharose and FPLC, Ute M. Kent. Conjugation of Fluorochromes to Antibodies, Su-Yau Mao.Biotinylation of Antibodies, Su-Yau Mao. Part II. Tissue Preparation for light Microscopic Analyses. Overview of Cell Fixatives and Cell Membrane Permeants, Melissa A. Melan. Preparation of Frozen Sections for Analysis, Gary L. Bratthauer. Processing of Cytological Specimens, Gary L. Bratthauer. Processing of Tissue Culture Cells, Gary L. Bratthauer. Processing of Tissue Specimens, Gary L. Bratthauer. Antigen Retrieval for Immunohistochemical Reactions in Routinely Processed Paraffin Sections, Laszlo Krenacs, Tibor Krenacs, and Mark Raffeld. Part III. Light Microscopic Detection Systems. Overview of Fluorochromes, J. Michael Mullins. Direct Immunofluorescent Labeling of Cells, Lorette C. Javois. Fluorescence Labeling of Surface Antigens on Attached or Suspended Tissue-Culture Cells, Mark C. Willingham. Fluorescence Labeling of Intracellular Antigens in Attached or Suspended Tissue-Culture Cells, Mark C. Willingham. Fluorescent Labeling of Surface or Intracellular Antigens in Whole-Mounts, Lorette C. Javois. TUNEL Assay for Apoptotic Cells, Virginia M. Heatwole. Overview of Fluorescence Analysis with the Confocal Microscope, Liana Harvath. Overview of Laser Microbeam Applications as Related to Antibody Targeting, P. Scott Pine. Overview of Fluorescence Photomicography, Lorette C. Javois and J. Michael Mullins. Overview of Antigen Detection Through Enzymatic Activity, Gary L. Bratthauer. The Peroxidase-Antiperoxidase (PAP) Method and Other All-Immunologic Detection Methods, Gary L. Bratthauer. The Avidin Biotin Complex (ABC) Method and Other Avidin Biotin Binding Methods,Gary L. Bratthauer. Avidin Biotin Labeling of Cellular Antigens in Cryostat Sectioned Tissue, Mark Raffeld and Elaine S. Jaffe. Multiple Antigen Immunostaining Procedures, Laszlo Krenacs, Tibor Krenacs, and Mark Raffeld. ELISA on Attachment Dependent or Suspension Grown Cells, Julie Ann Brent. Use of Immunogold with Silver Enhancement ,Constance Oliver. Part IV. Fluorescence Activated Cell Sorter (FACS) Analyses. Overview of Flow Cytometry and Fluorescent Probes for Cytometry, Robert E. Cunningham. Tissue Disaggregation, Robert E. Cunningham. Indirect Immunofluorescent Labeling of Viable Cells, Robert E. Cunningham. Indirect Immunofluorescent Labeling of Fixed Cells, Robert E. Cunningham. Fluorescent Labeling of DNA, Robert E. Cunningham. Deparaffinization and Processing of Pathologic Material, Robert E. Cunningham. Assay for Phagocytosis, Liana Harvath and Douglas A. Terle. Assay for Filamentous Actin, Liana Harvath. Assay for Chemoattractant Binding, Liana Harvath, Robert R. Aksamit, and Robert E. Cunningham. Assay for Oxidative Metabolism, Liana Harvath and Douglas A. Terle.Part V. Colloidal Gold Detection Systems For Electron Microscopic Analyses. Fixation and Embedding, Constance Oliver and Maria Celia Jamur. Preparation of Colloidal Gold, Constance Oliver. Conjugation of Colloidal Gold to Proteins, Constance Oliver. Colloidal Gold/Streptavidin Methods, Constance Oliver. Pre-Embedding Labeling Methods, Constance Oliver. Postembedding Labeling Methods, Constance Oliver. Part VI. Applications For Nucleic Acid Localization By In Situ Hybridization. Overview of In Situ Hybridization, Roland M. Nardone. In Situ Hybridization to Human Chromosomes of an Alkaline Phosphatase-Labeled Centromeric Probe, Roland M. Nardone. Fluorescence In Situ Hybridization Using Whole Chromosome Library Probes, Roland M. Nardone. In Situ RT-PCR and Hybridization Techniques, Vishakha Thaker. Part VII. Special Applications For the Clinical Laboratory. Overview of the Clinical Immunohistochemistry Laboratory: Regulations and Troubleshooting Guidelines, Patricia A. Fetsch and Andrea Abati. Overview of Immunohistochemical Approaches to the Differential Diagnosis of Tumors,Dennis M. Frisman. Overview of Automated Immunostainers, Gilbert E. Herman, Edna A. Elfont, and Alton D. Floyd.

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.

The 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.

The dynamics of the early patterning processes leading to the regeneration of a head in tissue excised from the body column of Hydra oligactis were examined by using a monoclonal antibody, CP8. This antibody displays position-specific binding, labeling the head ectodermal epithelial cells. During regeneration of a head, antibody labeling is present well before morphological signs of the head, at a time correlated with the determination of the tissue (Javois et al., Dev. Biol., 117:607-618, '86). By quantifying antibody labeling during regeneration of three different pieces of tissue excised from the body column, it was found that the dynamics of the early patterning processes as visualized by CP8 labeling varied. The pattern of labeling observed as well as the spread of labeled tissue suggested that the amount and geometry of apical tissue in the regenerate played a critical role in the patterning processes. Contrary to the labeling pattern observed in heads which formed during bud development or which regenerated following decapitation (Javois et al., '86), not all the CP8+ tissue was confined to the head structures in these regenerates. Several alternative explanations for this surprising result are presented. The usefulness of these data in refining pattern formation models by more explicitly constraining their parameters is discussed.

The formation of supernumerary limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. Different “wedges” (ectodern and mesoderm) of posterior donor right wing bud (stage 21) were transplanted to a slit made in stage 20–23 host right wing buds. Donor posterior tissue was transplanted to an anterior position in a host wing bud or, as a control, to the same position as its position of origin. Transplanting different wedges of posterior tissue to the same anterior host position results in wings with supernumerary structures, and different extra structures form depending on the position of origin of the donor tissue. The identification of extra limb structures formed was based on the skeletal and integumentary patterns of resulting wings and the pattern of muscles as seen in serial sections of resulting limbs. The results of experiments presented here are considered in light of current models that have been used to describe the formation of supernumerary limb structures by the embryonic chick limb bud.

The formation of duplicated wing skeletal elements and/or extra wing muscles was studied by juxtaposing normally nonadjacent embryonic chick wing bud cells. A wedge of right or left stage 21 wing bud ectoderm and mesoderm was inserted in a slit made in a host stage 20 to 22 right wing bud at the same anteroposterior position as its position of origin. The distal edge of the donor wedge and host wing bud were aligned with each other. Donor tissue was grafted into a host wing bud in one of the following four axial relationships: both the anteroposterior and dorsoventral axes corresponded with each other ( aadd ); only the anteroposterior axes were opposed ( apdd ); only the dorsoventral axes were opposed ( aadv ); both the anteroposterior and dorsoventral axes were opposed ( apdv ). Of the 63 wings resulting from the control aadd operation and the 45 wings from the apdd operation, only 12 wings had a duplicated skeletal element; of the 69 wings sectioned from these two groups of operations, only one had an extra muscle. However, of the wings resulting from the aadv and apdv operations (48 and 52 cases, respectively), 23 had a duplicated skeletal element; of the 54 wings sectioned from these operations, 43 wings had one to four extra muscles. Furthermore, when the aadv operation was performed with a wedge of donor quail wing bud ectoderm and mesoderm or mesoderm alone, supernumerary muscles formed in these chimeric wings and they were made up of donor quail and host chick cells or only donor quail cells.

The relationship between populations of nerve cells defined by two monoclonal antibodies was investigated in Hydra oligactis. A population of sensory nerve cells localized in the head (hypostome and tentacles) is identified by the binding of antibody JD1. A second antibody, RC9, binds ganglion cells throughout the animal. When the nerve cell precursors, the interstitial cells, are depleted by treatment with hydroxyurea or nitrogen mustard, the JD1+ nerve cells are lost as epithelial tissue is sloughed at the extremities. In contrast, RC9+ nerve cells remain present in all regions of the animal following treatment with either drug. When such hydra are decapitated to initiate head regeneration, the new head tissue formed is again free of JD1+ sensory cells but does contain RC9+ ganglion cells. Our studies indicate that (1) nerve cells are passively displaced with the epithelial tissue in hydra, (2) JD1+ sensory cells do not arise by the conversion of body column nerve cells that are displaced into the head, whereas RC9+ head nerve cells can originate in the body column, (3) formation of new JD1+ sensory cells requires interstitial cell differentiation. We conclude from these results that the two populations defined by these antibodies are incorporated into the h ad via different developmental pathways and, therefore, constitute distinct nerve cell lineages.

A monoclonal antibody, termed JD1, was generated that bound to a subset of the nerve cells in the hypostome and tentacles of Hydra oligactis. Using a whole-mount technique the spatial pattern of the subset of nerve cells and their processes could be clearly visualized using indirect immunofluorescence. The subset largely corresponds to the epidermal sensory cells. Using the same technique the development of the pattern during head regeneration and budding was examined. The appearance of the nerve cells coincides with the formation of both the tentacles and hypostome. When head regeneration does not occur, JD1+ cells do not appear suggesting the differentiation of JD1+ cells is an integral event in head formation dependent on antecedent patterning processes.

The relationship between the position transplanted in a host limb bud, the orientation of a graft in a host limb bud, and the extra limb structures formed was studied by juxtaposing normally nonadjacent embryonic chick wing bud tissue. In one series of transplantation operations, two different wedges (ectoderm and mesoderm) of stage 21 right donor posterior wing bud tissue were transplanted to the middle of a host stage 20 to 22 right wing bud such that the dorsal-ventral polarity of the graft and host were the same or reversed. The results of these transplantation operations show that the formation of supernumerary limb structures depends on the position of origin of the donor tissue, the anterior-posterior position transplanted in a host limb bud, and the orientation of the graft in the host limb bud. In a second series of transplantation operations, the relationship between the proximodistal position where posterior donor tissue is transplanted in an anterior host site and the extra structures formed was studied. A wedge of posterior stage 21 right wing bud tissue was transplanted to an anterior proximal or anterior distal site of a stage 22 to 24 host right wing bud. The results of these transplantation operations show that when the donor tissue is transplanted to an anterior proximal position in a host wing bud, then limbs with only a duplicated humerus result, whereas, when transplanted to an anterior distal position, then limbs with a duplicated forearm element and extra digits result.

Publisher Summary In a mature animal or plant, the differentiated state of a cell is generally quite stable. The cell has a distinct morphology—a characteristic set of macromolecules that are involved in a particular physiological function—and usually does not undergo cell division. This state persists throughout the lifetime of the cell, which can coincide with the life-span of the organism as with a muscle cell, or be much shorter, as in the red blood cell. This pattern suggests that a cell once having traversed a differentiation pathway and having reached the end state is now fixed in its structure and function. However, ablation experiments have revealed that this is not always the case. For a number of cell types the stability of the differentiated state is not fixed, but is only metastable. In some tissues, such as the liver, partial ablation results in the resumption of cell division of the remaining cells. Here, quiescent cells do not alter their cell type, but undergo a behavior not usually associated with the final differentiated state. The existence of cell type conversion, or transdifferentiation, in which a cell undergoes a change in its physiological function with accompanying structural changes clearly demonstrates that the differentiated state is not always immutable.