Building on a solid foundation: SAR and QSAR as a fundamental strategy to reduce animal testing.

The development of more efficient, ethical, and effective means of assessing the effects of chemicals on human health and the environment was a lifetime goal of Gilman Veith. His work has provided the foundation for the use of chemical structure for informing toxicological assessment by regulatory agencies the world over. Veith's scientific work influenced the early development of the SAR models in use today at the US Environmental Protection Agency. He was the driving force behind the Organisation for Economic Co-operation and Development QSAR Toolbox. Veith was one of a few early pioneers whose vision led to the linkage of chemical structure and biological activity as a means of predicting adverse apical outcomes (known as a mode of action, or an adverse outcome pathway approach), and he understood at an early stage the power that could be harnessed when combining computational and mechanistic biological approaches as a means of avoiding animal testing. Through the International QSAR Foundation he organized like-minded experts to develop non-animal methods and frameworks for the assessment of chemical hazard and risk for the benefit of public and environmental health. Avoiding animal testing was Gil's passion, and his work helped to initiate the paradigm shift in toxicology that is now rendering this feasible.

Ikaros expression as a marker for lymphoid progenitors during zebrafish development.

The Ikaros gene encodes a transcription factor that, in mice, has been shown to be essential for the correct differentiation of B and T lymphocytes and is expressed in all cells of the lymphoid lineage, including pluripotent hematopoietic stem cells. During development in zebrafish, Ikaros expression begins in lateral mesoderm, and continues in the intermediate cell mass (ICM), which is derived from lateral mesoderm and has been shown to generate primitive hematopoietic precursors. Cells expressing Ikaros were then seen on the ventral side of the dorsal aorta, known to be a location of definitive hematopoietic precursors in birds and mammals. Ikaros-expressing cells were also found in the pharyngeal region, near the forming thymus. Later, such cells were seen in the pronephros, the site of hematopoiesis in adult fish. The timing of appearance of Ikaros-expressing cells suggests that, similar to other vertebrates, lymphocytes in the thymus arise from hematopoietic tissue located near the dorsal aorta or in the ICM.

Early hematopoiesis and developing lymphoid organs in the zebrafish.

In zebrafish, the transparent and rapidly developing embryo and the potential for genetic screening offer a unique opportunity to investigate the early development of the vertebrate immune system. Here we describe the initial appearance of various blood lineages and the nature of accumulating hematopoietic tissue in the thymus and kidney, the main lymphoid organs of adult teleosts. The ultrastructure of the first site of hematopoiesis, the intermediate cell mass (ICM), is described from the 5-somite stage, about 11.5 hours post-fertilization (hpf) until 24 hpf. The ICM gives rise to the primitive erythroid lineage, which accounts for all circulating erythrocytes for the first 4 days pf. From 24 to 72 hpf, a few developing granulocytes are seen close to the yolk sac walls and in the caudal axial vein. The heart, previously proposed as an early blood-forming organ in zebrafish, did not contain hematopoietic cells. The thymic primordium, consisting of two layers of epithelial cells, appears at 60 hpf. By 65 hpf, it is colonized by immature lymphoblasts. The thymus gradually accumulates lymphocytes, and the lymphocytes and epithelial cells progressively differentiate for 3 weeks pf. At 96 hr, the pronephros contains hematopoietic cells, mainly developing erythrocytes and granulocytes. The amount of renal hematopoietic tissue increases rapidly; however, lymphocytes were not detected until 3 weeks pf.

Zebrafish angiogenesis: a new model for drug screening.

Angiogenesis is necessary for tumor growth, making inhibition of vessel formation an excellent target for cancer therapy. Current assays for angiogenesis, however, are too complex to be practical for drug screening. Here, we demonstrate that the zebrafish is a viable whole animal model for screening small molecules that affect blood vessel formation. Blood vessel patterning is highly characteristic in the developing zebrafish embryo and the subintestinal vessels (SIVs) can be stained and visualized microscopically as a primary screen for compounds that affect angiogenesis. Small molecules added directly to the fish culture media diffuse into the embryo and induce observable, dose-dependent effects. To evaluate the zebrafish as a model, we used two angiogenesis inhibitors, SU5416 and TNP470, both of which have been tested in mammalian systems. Both compounds caused a reduction in vessel formation when introduced to zebrafish embryos prior to the onset of angiogenesis. Short duration (1 h) exposure of SU5416 was sufficient to block new angiogenic and vasculogenic vessel formation. In contrast, TNP470 required continuous exposure to block SIV formation and had no apparent effect on vasculogenic vessel formation. To ascertain whether blood vessels in the zebrafish embryo respond to angiogenic compounds, we introduced human VEGF into embryos. Injection of VEGF caused an observable increase in SIV formation.

Expression of zebrafish rag genes during early development identifies the thymus.

Recent experiments have demonstrated that zebrafish is a vertebrate in which it is possible to carry out large-scale mutagenic screens to identify genes involved in specific developmental pathways. To follow development of the immune system in zebrafish, we have analyzed the expression of the recombination activating genes, rag1 and rag2, which we have previously isolated and characterized. These genes catalyze the rearrangement of immunoglobulin genes in immature B lymphocytes and of T cell receptor genes in immature T lymphocytes and are therefore appropriate markers to follow the development of organs containing these cells. By whole-mount in situ hybridization, we detected expression of both rag genes in a paired organ in the head, beginning on the fourth day after fertilization. Histological examination of this organ indicated that it corresponds to the thymus, as described for other fish, an organ that has not previously been identified in zebrafish. By histological analysis, the thymus primordium appears at 54 hr but does not enlarge significantly until 30 hr later. The thymus continues to enlarge and reaches its mature histological organization at 1 month. The pronephros, the major hematopoietic organ in the adult fish, begins to develop hematopoietic tissue about 2 weeks after fertilization. By 1 month, mature lymphocytes are distinguishable in the tissue surrounding renal tubules. Lymphocytes appear in the kidney too late for screening by whole-mount in situ hybridization; however, the pattern of rag1 expression in the thymus forms the basis of an assay for mutations affecting development of the thymus or its constituent lymphocytes.

Characterization and expression of the recombination activating genes (rag1 and rag2) of zebrafish.

The closely linked recombination activating genes, rag1 and rag2, encode components of the recombinase involved in V(D)J recombination of the immunoglobulin and T-cell receptor genes. These genes are expressed together exclusively in immature lymphocytes and are useful markers for following the development of lymphoid tissues. We cloned the rag locus of the zebrafish Danio rerio and sequenced the open reading frames of the rag1 and rag2 genes. Although the gene organization is similar to that in other species, the rag1 gene is unusual in possessing two introns within the coding region. In another teleost, the rainbow trout, the rag1 gene is interrupted by a single intron. Introns are not present in the rag1 gene of any other species examined to date. Expression of both rag1 and rag2 begins late in embryonic development, on day 4, by northern RNA blot analysis. Expression of rag1 was detected in the adult zebrafish thymus, pronephros, mesonephros, and ovary. This pattern of expression is consistent with previous histological studies of adult teleosts, which implicate the kidney as the major site of hematopoiesis and the thymus as the major lymphocyte-containing organ.

Sequence and expression pattern of J chain in the amphibian, Xenopus laevis.

We have determined the cDNA sequence encoding J chain, a polypeptide accessory molecule associated with polymeric Ig, from the anuran amphibian, Xenopus laevis (South African clawed frog). The translated polypeptide consists of 164 amino acid residues, including the signal sequence, and is somewhat longer than the corresponding sequence in mouse and cow, the two mammalian species in which the signal sequence of J chain has been determined. J chain in several mammalian species (human, mouse, cow and rabbit) has eight Cys residues. In the human chain, two of these Cys residues, the second and third in the sequence, have been shown to form disulfide bridges to heavy chains in IgM or IgA; the remaining Cys residues form intrachain disulfide bonds. The Xenopus J chain contains only seven of these Cys residues. Ser is found at the position corresponding to the third Cys in mammalian J chains. Northern blot analysis, performed on RNA isolated from various organs of 3-month old frogs, indicated that the highest level of expression was in the intestine. Transcripts corresponding to J chain were also detected in the spleen and at very low levels in the thymus.

A complex regulatory element from the yeast gene ENO2 modulates GCR1-dependent transcriptional activation.

The GCR1 gene product is required for maximal transcription of many yeast genes including genes encoding glycolytic enzymes. Transcription of the yeast enolase gene ENO2 is reduced 50-fold in strains carrying a gcr1 null mutation. cis-acting sequences that are sufficient for GCR1-dependent regulation of ENO2 expression were identified by using an enhancerless CYC1 promoter which is not normally dependent on GCR1 for expression. A 60-bp ENO2 sequence that was sufficient to provide high-level, GCR1-dependent transcriptional activation of the CYC1 promoter was identified. This 60-bp element could be subdivided into a 30-bp sequence containing a novel RAP1-binding site and a GCR1-binding site which did not activate CYC1 transcription and a 30-bp sequence containing a novel enhancer element that conferred moderate levels of GCR1-independent transcriptional activation. The 60-bp CGCR1-dependent upstream activator sequence is located immediately downstream from previously mapped overlapping binding sites for the regulatory proteins ABFI and RAP1. Evidence is presented that the overlapping ABFI- and RAP1-binding sites function together with sequences that bind GCR1 and RAP1 to stage transcriptional activation of ENO2 expression.

Multiple factors bind the upstream activation sites of the yeast enolase genes ENO1 and ENO2: ABFI protein, like repressor activator protein RAP1, binds cis-acting sequences which modulate repression or activation of transcription.

Binding sites for three distinct proteins were mapped within the upstream activation sites (UAS) of the yeast enolase genes ENO1 and ENO2. Sequences that overlapped the UAS1 elements of both enolase genes bound a protein which was identified as the product of the RAP1 regulatory gene. Sequences within the UAS2 element of the ENO2 gene bound a second protein which corresponded to the ABFI (autonomously replicating sequence-binding factor) protein. A protein designated EBF1 (enolase-binding factor) bound to sequences which overlapped the UAS2 element in ENO1. There was a good correlation among all of the factor-binding sites and the location of sequences required for UAS activity identified by deletion mapping analysis. This observation suggested that the three factors play a role in transcriptional activation of the enolase genes. UAS elements which bound the RAP1 protein or the ABFI protein modulated glucose-dependent induction of ENO1 and ENO2 expression. The ABFI-binding site in ENO2 overlapped sequences required for UAS2 activity in wild-type strains and for repression of ENO2 expression in strains carrying a null mutation in the positive regulatory gene GCR1. These latter results showed that the ABFI protein, like the RAP1 protein, bound sequences required for positive as well as negative regulation of gene expression. These observations strongly suggest that the biological functions of the RAP1 and ABFI proteins are similar.

Isolation and physical characterization of three essential conidiation genes from Aspergillus nidulans.

We cloned and characterized three genes from Aspergillus nidulans, designated brlA, abaA, and wetA, whose activities are required to complete different stages of conidiophore development. Inactivation of these genes causes major abnormalities in conidiophore morphology and prevents expression of many unrelated, developmentally regulated genes, without affecting the expression of nonregulated genes. The three genes code for poly(A)+ RNAs that begin to accumulate at different times during conidiation. The brlA- and abaA-encoded RNAs accumulate specifically in cells of the conidiophore. The wetA-encoded RNA accumulates in mature conidia. Inactivation of the brlA gene prevents expression of the abaA and wetA genes, whereas inactivation of the abaA gene prevents expression of the wetA gene. Our results confirm genetic predictions as to the temporal and spatial patterns of expression of these genes and demonstrate that these patterns are specified at the level of RNA accumulation.