Overview
The long-term interest of our lab is the evolution and development of the immune system. Our current goal is to understand the early development of cells in the lymphocytic lineage and of the organs in which these cells differentiate. Despite a wealth of information about later stages, little is known about early steps in the differentiation of B and T lymphocytes, including commitment to the lymphocytic lineages and homing of lymphocytic progenitors to the thymus. We are also interested in utilizing the emerging information about the zebrafish genome to describe the genetic loci encoding antigen-specific receptors and to analyze the expression and function of the genes within these loci.

Research Summary
The zebrafish, Danio rerio, offers unique opportunities for investigating early events in vertebrate development. Most organs are formed by five days and, since the fish remain transparent for about the first two weeks, the development of organ systems can be followed visually. The transparency of the developing fish also facilitates the examination of gene expression in intact fish by whole-mount in situ hybridization, as well as the identification of mutants.

We are analyzing the development of lymphoid cells and organs in the zebrafish. Our initial strategy was to clone genes whose expression is needed for B and T cell differentiation such as the highly conserved rag genes, which are required for V(D)J recombination, Igμ, which encodes the antigen-specific receptor on B cells; and TCRα , which encodes one chain of the receptor on a major subset of T lymphocytes, the TCRαβ cells. We follow gene expression by in situ hybridization, either by the whole-mount technique or on sections.

Rag1 and TCRα were found to be expressed in the thymus at about four days, signaling the onset of T cell differentiation. No other site for rag1 expression was noted, nor were lymphoid cells detected in the pronephros, the presumed site for B cell differentiation in teleosts, until three weeks. These findings suggested that B lymphopoiesis may be delayed in zebrafish, relative to T lymphopoiesis. However, VDJ rearrangements, which occur as B lymphocytes differentiate, were detected in genomic DNA extracted from whole zebrafish by day 4. Igm transcripts were detected in RNA derived from whole fish by day 7. These findings indicated that cells of the B lineage are present well before any lymphocytes can be detected in the pronephros and posed the challenge of identifying sites where B cells are localized in the period between four days and three weeks.

Reexamination of fish for early sites of rag1 expression revealed, in addition to the thymus, a small stained spot in the right dorsal region of the abdomen, consistent in location with the pancreas. The identity of the stained organ as the pancreas was supported by staining with an insulin probe. In situ hybridization on sections of 10-day-old fish revealed Igμ, as well as rag1, staining in a region surrounding an islet of Langerhans. Both genes were expressed in the pronephros beginning at 19 days, in agreement with the appearance of lymphoid cells in this organ at about this time. There was no evidence for expression in the liver, the site for B cell development in fetal mouse and human.

In adult zebrafish, Igμ and rag1 expression are prominent in the pronephros and mesonephros, consistent with previous observations of the role of the teleost kidney in B cell development and in antibody production. Igμ-expressing cells were also found in the intestine as well as in the mesentery along the intestine, near vessels, apparently commingled with pancreatic tissue. The zebrafish spleen contains mostly erythrocytes, but Igμ expression is also seen, especially in older fish.

In collaboration with the laboratory of Shuo Lin, we prepared a construct in which the gene encoding green fluorescent protein is driven by the rag1 promoter. The thymus is fluorescent in the living transgenic fish. The availability of these fish will facilitate studies of early thymic development as well as the identification of mutants lacking the thymus.

We have explored the expression of another gene, Ikaros, that is needed for the development B and T lymphocytes. In mice, this gene is expressed in all cells of the lymphoid lineage, including mature B and T cells, as well as in hematopoietic stem cells. We determined sites of Ikaros expression in zebrafish beginning on the first day. Expression of the Ikaros gene may be a guide for identifying progenitor cells that differentiate into lymphocytes. Zebrafish embryos are ideal for lineage-tracing as their transparency facilitates labeling cells at different locations and following their migration.

The zebrafish genome sequencing project is at an advanced stage, with completion expected in 2005. We have identified the Ig heavy chain (IgH) locus (175 kb) and its constituent V, D, J and C genes. Although only two heavy chain isotypes have so far been described in teleosts, Igμ and Igδ, the zebrafish locus was found to contain an additional isotype, which we have designated Igζ (ζ, for first identified in zebrafish), which is not closely related to any known Ig. As in Igμ, 4 exons encode the secreted form of Igζ, but the membrane form is generated by splicing to a cryptic site in exon 4, rather than to the end of exon 3, as in the membrane form of zebrafish and other teleost Igμ. During development, Igζ is more highly expressed than is Igμ. In adults, Igζ is expressed in the kidney and thymus, the primary lymphoid organs in teleosts, whereas Igμ is also expressed at peripheral sites, especially in the spleen.

In contrast to the translocon configuration of the IgH locus, teloest IgL genes are organized in a clustered arrangement, with groups of closely linked V, J and C gene segments. However, few details about gene arrangement are available. We are also utilizing the sequence information from the zebrafish genome to identify all light chain gene segments and establish their organization. We anticipate that the information we obtain will be applicable to light chain loci in other teleosts. In addition to VJC clusters, there are groups of 3 or 4 V segments, an isolated V segment and mixtures of segments, seemingly in no regular pattern. The transcriptional orientation of many of the V segments is opposite to that of the J and C segments, implying that VJ rearrangement is by inversion. We are examining rearrangement of light chain gene segments by examining their expression as cDNA. We have found that a V segment can rearrange to a distant, as well as to an adjacent, J-C gene. That rearrangement is not confined to nearby gene segments, together with the occurrence of inversional rearrangement in either direction, demonstrates the potential for extensive V-J combinatorial diversity of the zebrafish light chain genes.

In ongoing experiments we are identifying other genes that have important roles in the immune system, e.g., genes encoding T cell receptor γ (TCRγ) and terminal deoxynucleotidyl transferase (TdT). TCRγ is a constituent of the antigen-specific receptor expressed by the TCRγδ population of T cells. The function of TCRγδ cells in humans and mice is not well understood and the advantages of zebrafish in analyzing gene expression may be helpful in elucidating the role of these cells. TdT catalyzes the addition of non-templated or “N” nucleotides at VDJ junctions, thereby enhancing sequence diversity of V genes. The expression of this gene, as well as sequence diversification, apparently increase as mice mature, but evidence to date suggests that this may not be the case for all species. Following TdT expression during the lifetime of the zebrafish may clarify this matter.

Selected Publications
Danilova, N., Bussmann, J., Jekosch, K. and Steiner, L.A. The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z. Nat. Immunol.6:295-302 (2005).

Danilova , N., Visel, A., Willett, C.E., and Steiner. L.A. Expression of the winged helix/forkhead gene, foxn4, during zebrafish development. Brain Res Dev Brain Res. 153: 115-119 (2004).

Steiner, L. A., Danilova, N. and Willett, C.E. . The immune system in zebrafish, in Molecular Biology of B Cells, 3rd edition, F. W. Alt, T.Honjo, M.S. Neuberger, ed. Elsevier Academic Press, London (2004).

Danilova, N., Hohman, V.S., Sacher, F., Ota, T., Willett, C.E., and Steiner, L.A. T cells and the thymus in developing zebrafish. Dev. Comp. Immunol. 28:757-769 (2004).

Danilova, N. and Steiner, L.A. B cells develop in the zebrafish pancreas. Proc. Natl. Acad. Sci. USA 99:13711-6 (2002).

Willett, C.E., Kawasaki, H., Amemiya, C.T., Lin, S., and Steiner, L.A. Ikaros expression as a marker for lymphoid progenitors during zebrafish development. Dev. Dyn. 222: 694-698 (2001).

Willett, C.E., Zapata, A., Hopkins, N., and Steiner, L.A. Expression of zebrafish rag genes during early development identifies the thymus. Dev. Biol. 182: 331-341 (1997).

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