Hans R. Bode - US grants

How a multipotent cell becomes committed to a particular differentiation pathway is not known. The long term goal of this research is to understand the controls governing the proliferation and differentiation behavior of a multipotent stem cell, the interstitial cell of hydra. At this stage, the approach is to elucidate the cell-cell interactions regulating each of the several differentiations the interstitial cell can undergo. It is known that the interstitial cell population contains multipotent stem cells and cells committed to a particular differentiation. There is increasing evidence that for this system, and every other system with a multipotent cell, there are cells with intermediate differentiation capacities. Thus, any cell-cell interaction, or cell-molecule interaction thought to affect the multipotent cell, may yield ambiguous results as it may affect a derivative cell instead. Therefore to accurately work out the controls effecting the multipotent cell, a precise knowledge of the complexity of the population in terms of number of cell types with different differentiation capabilities and of the cell lineage relationships among them is necessary. The goals of this proposal are to do that for the interstitial cell system of hydra with the following approach: (1) Monoclonal antibodies that bind specifically to I-cells, to subsets of I-cells, to subsets of I-cells and differentiation intermediates and/or products will be sought. (2) Subsets of I-cells, each defined by a monoclonal antibody, will be examined in pairs to determine their relatedness. This will lead to a map of nearest neighbors which will tentatively be a map of the cell lineage relationships. (3) Using the defining antibody, cells of each subset will be isolated and their differentiation and proliferation capacities examined. This information will be used to validate the relationships described in the map. An understanding of the cellular interactions controlling the differentiation of multipotent cells in basic to an understanding of some classes of defects that occur during human development.

Hydra attenuata, a freshwater coelenterate, has the ability to regenerate its entire form from a fragment of body tissue, without the necessity of cell division or DNA synthesis. The cells change their fates and take part in the formation of a new pattern, which is regulated to fit into the reduced tissue. There are seveal aspects of the pattern formation process which will be examined using grafting, transplantation, and regeneration experiments. The roles of inhibition as the "inhibitor" of an autocatalytic patterning reaction, and the size sensor which limits the reaction so as to maintain proportions will be reevaluated. The specific source of inhibition in the head, and the mechanism of transmission will be examined. The kinetics of proprotioning in the head structures and in the bsal disc will be followed during regeneration to determine if they are consistent with the kinetics of inhibition return. The ability of inhibition to modulate proportions when at a high level will also be determined. The interactions between the head and foot during simultaneous regeneration will be examined to determine if there is a single patterning system of both or two separate systems which cross-react. Finally, the possibility of an adhesivity gradient in the body column, which is a force in its shapin, will be considered.

How a pluripotent cell becomes committed to a specific differentiation pathway is a central question in developmental biology. The overall objective is to understand such commitment decisions by studying the interstitial lineages of hydra. The multipotent stem cell of this system gives rise to four classes of differentiation products: neurons, nematocytes, secretory cells and gametes. In previous segments of this work an extensive body of information has been gained about the stem cells and the four classes of products at the cellular level. In this proposed segment, the focus will be on the commitment decisions that occur during neuron differentiation. Two are known. One is the restriction of the multipotent stem cell to neuron differentiation, and the other is a decision late in the pathway as to neuron phenotype. The aims here are three-fold. [1] To complete an understanding of the neuron lineage in order to determine if there are any more stages where a decision as to cell type is made. This will be done by marking individual interstitial cells and determining the differentiated cells they produce. [2] Neuron phenotype determination is position-dependent in hydra. For one subset of neurons this effect is mediated by the phosphatidylinositol pathway. The aim is to identify the signal that is responsible for the position-dependent effect, and determine if it is acting directly or indirectly on the neurons. Grafting experiments involving tissue with a variety of cell compositions coupled with diacylglycerol treatments will determine if the effect is direct or indirect. Examination of a number of peptides and proteins known to affect neuron behavior or be associated with neurons in hydra will determine if any is the signal. [3] To identify genes involved in the commitment decisions. The approach is to determine if specific genes involved in neuron differentiation decisions in both vertebrates and arthropods are present in hydra. These will be identified using PCR, and the isolation and sequencing of corresponding cDNAs. Expression patterns of the gene at the mRNA and protein level will determine if they affect the neuron lineage. The initial candidates will be members of the achaete-scute family of genes.

cell differentiation; hematopoietic stem cells; Cnidaria;

This proposal is a request for funds to defray travel costs for a number of investigators (faculty, post-doctoral fellows and graduate students) to attend the 3rd International Workshop on Hydra and Hydractinia Development. The workshop will be held July 5-9, 1989 in Gunzburg, West Germany. %%% This meeting has proved a valuable venue for workers in this rather small field to exchange ideas, results and newly developed methods. Several collaborations have arisen as a result of previous meetings.

Bode 9723660 During the last 10-15 years a great deal has been learned about the genes and processes that regulate the development of an embryo into a functioning animal. One of the most interesting aspects of the accumulating knowledge is that a common set of regulatory genes involved in a common, or related, set of mechanisms underlie many of the developmental processes of the metazoans studied so far. These common features raise interesting and important questions as to how early during evolution did these genes arise? Were they already present in the Ur-metazoan? What developmental mechanisms and developmental processes did they participate in? And, to what extent were the mechanisms and processes invented early and conserved through animal evolution? One approach to these issues is to study these genes and their functions in organisms that belong to groups of animals that arose very early during metazoan evolution, such as the cnidaria, whose members include jellyfish, sea anemones, and the freshwater hydra. Of these, hydra, is particularly useful since the developmental processes (pattern formation, control of the proliferation and differentiation of the several cell types) are well-understood. Further, a number of genes known to regulate developmental events in many vertebrates and invertebrates have also been isolated, and in part, characterized in hydra. The overall aim of the proposed work is to determine if the genes and mechanisms underlying the development of the head of hydra are similar to those found in the development of the heads of vertebrates and insects. One such gene has already been found. Cnotx a hydra homologue, of the members of the Otx family has been characterized and shown to be involved in the development of the tentacles, a particular structure of the hydra head. There is also evidence that homologues of the Dlx and Emx families exist in hydra, or a close marine relative. Genes of these families play important roles in the patterning of the head in vertebrates and arthropods. The detailed aims are two. One is to examine in the role of the Coots gene in patterning of the hydra head in detail as well as to investigate its role in cell cycle traverse, a role that preliminary evidence suggests it has. These efforts will be pursued with a variety of approaches including examining alterations of expression patterns during tissue manipulations and functional studies. The other is to isolate and characterize the role of other genes involved in head development in hydra. The focus will be on genes of the families mentioned above.

Accumulating evidence indicates is that a common set of regulatory genes involved in a common, or related, set of mechanisms underlie many of the developmental processes of the metazoans studied so far. How early did these genes arise during metazoan evolution and to what extent has their function been conserved? A useful approach to these questions is to study such genes in a cnidarian, the first group of animals to have a defined body plan. Among the cnidaria, hydra is particularly useful as the developmental processes are extensively understood. The overall aim of the proposed work is to determine if the genes and mechanisms underlying axis formation and axial patterning in hydra are similar to those found vertebrates and arthropods. The four detailed aims are the following.

In hydra, axis formation and axial patterning can be studied during the process of bud formation, the animal's asexual form of reproduction. Two of the aims focus on clarifying aspects of bud formation. [1] Of the three cell lineages in hydra, the two epithelial lineages [ectoderm and endoderm] play an obvious role. A possible role of the interstitial cell lineage will be investigated by manipulations that alter or eliminate this lineage. [2] A number of homologues of axial patterning genes found in vertebrates and Drosphila have been isolated from hydra and show to be expressed during bud formation. More direct evidence of their particular roles will be investigated by introducing anti-sense oligonucleotides against such genes into budding tissue.

The other two aims focus on the isolation of new genes involved in axial patterning in hydra. One approach will be to look for homologues of genes involved in dorsal-ventral patterning, which is in part, highly conserved among vertebrates and Drosophila. The other involves using the induction of a 2nd axis in frog embryos to identify hydra genes involved in axial patterning. Any genes isolated with either procedure will be examined for their role in axial patterning in hydra by examining their expression patterns in a variety of developmental situations as well as using the anti-sense approach to gain more direct information.

The results should extend the understanding of the evolution of axis formation as well as how animals with a single axis evolved into animals with more than one axis.

DESCRIPTION (adapted from investigator's abstract): To gain an understanding of the function of specific genes, simple model systems often have the advantage that the gene has fewer functions and the gene family of which it is a member has fewer members, thereby minimizing redundancy. One approach is to use a system in which the developing tissues and regions can be manipulated in a variety of ways resulting in alterations of the expression and activity of an introduced transgene leading to a clearer and/or more detailed understanding of the role of the gene. Hydra, a cnidarian, is particularly useful for this approach to studying gene function. The animal has a simple body plan with few cell types. The processes governing pattern formation, cell fate decisions, and tissue growth are well understood at a tissue and cell level, These processes are constantly active in the adult hydra due to the tissue dynamics of the adult. The adult animal is amenable to a variety of manipulations which are useful for exploring the role of a particular gene. Many genes which regulate these developmental processes in more complex organisms have been isolated from hydra. For most of these genes the expression patterns are simple. For a number of these genes, their normal patterns coupled with alterations in expression due to tissue manipulations indicate they have functions analogous to their homologues in vertebrates. The focus of this proposal will be the generation of transgenic hydra. Two approaches will be tried towards stably introducing transgenes into the hydra genome. One makes use of two different retroviruses, each with a vsv-g coat protein that permits infection of a wide variety of organisms from amoeba to mammals. Initial experiments indicate that when hydra embryos at the blastula stage are injected, a small fraction of the resulting hatchlings contain the viral genome suggesting stable integration. The second approach will involve use of a transposable elements with a broad host range specificity [amoeba to insects]. Should either or both of these approaches work, efforts will be focused on optimizing the procedure for introducing the transgenes.

0120591
Bode

It is becoming increasingly clear that understanding how an organism interacts with its environment will require a complete accounting of the genes which make up the organism and the expression patterns of the genes under various conditions. Members of the phylum Cnidaria (jellyfish, corals, sea anemones, and hydras) are key members of their environments, yet very little is known about their genetic makeup. To address this problem, a collection of cloned DNAs representing the messenger RNA populations (cDNAs) of two well-studied cnidarians will be generated. The two organisms that will be used are the freshwater cnidarian Hydra and the colonial marine cnidarian Podocoryne. Bacteria containing the cloned cDNAs will be robotically arrayed to generate archives of the clones. Sequence data will be obtained from approximately 50,000 of the cDNA clones from each organism. The resulting sequence data will be analyzed in various ways using bioinformatic computing tools. The analyses will provide information on what genes are present in cnidarians and how those genes are evolutionarily related to those in animals which diverged more recently than cnidarians, such as vertebrates and insects. Such information will be very valuable for defining the processes by which multicellular animals have evolved. The availability of cloned cDNA sets from two model cnidarians will also make it possible to examine the expression of large numbers of genes in these organisms using the technique of DNA array analysis. In particular it will be possible to identify genes whose expression changes when the organisms are placed under conditions which reflect those present during periods of environmental stress (e.g. elevated temperature). Genes whose expression levels change under stress conditions in the laboratory may be useful tools for monitoring the health of cnidarians (e.g. corals) in their natural setting.

0130375
Hans Bode

During the last 20 years a great deal has been learned about the genes and the molecular mechanisms that regulate the processes underlying the development of an embryo into a functioning animal. One of the most interesting aspects of the accumulating knowledge is that similar sets of genes and mechanisms underlie similar developmental processes in many of the diverse animals studied so far. These common features raise a number of important questions. When during animal evolution did these genes arise? Were they already present in the earliest metazoan? To what extent has the function of a particular gene, or pathway of genes been conserved throughout metazoan evolution?
One approach to these issues is to study these genes and their functions in organisms that belong to groups of animals that arose very early during metazoan evolution. One of the earliest groups is the Cnidaria, whose members include jellyfish, sea anemones, and the freshwater hydra. Of these, hydra, is particularly useful since the developmental processes are well-understood.
The overall aim of the proposed work is to determine the extent that the genes and mechanisms underlying axis formation in hydra are similar to those found vertebrates and arthropods. Though hydra, and all cnidarians, have a single axis compared to the two axes [anterior-posterior and dorsal-ventral] in bilaterians, a number of genes known to be involved in axis formation in vertebrates and arthropods play similar roles in hydra.
A specific problem that needs to be addressed concerns the genes and mechanisms involved in the initiation of axis formation. In vertebrates, a region termed the organizer plays a central role. Recently, a region of the hydra head, the hypostome, has been shown to have clear similarities with this vertebrate structure. Thus, the focus of this proposal is to isolate genes from the hydra organizer, determine their roles, and determine to what extent the same or similar genes are involved in hydra and vertebrate organizers as well as arthropod organizing regions.
The common approach is to look for homologues of genes that function in the organizer activity of a vertebtate or the fruit fly, Drosophila. This approach has been very successful but is slow and tedious. DNA array analysis provides a means for identifying a subset of genes among several thousand that are candidates for roles in organizer activity in hydra. Sequence analysis will reveal which are homologues of known genes, which belong to a specific class of gene and which are novel genes. Thereafter, the role of each of these genes will be analyzed by examining their expression patterns during the development and functioning of the organizer region as well as with the use of a functional assay.
The information gained will provide (a) a more detailed understanding of the organizer in hydra, (b) a more detailed understanding of the molecular evolution of this structure, (c) and possibly the identification of novel genes that may generally be involved in organizer development and activity.