Pegasus, the 'atypical' Ikaros family member, influences left-right asymmetry and regulates pitx2 expression.

Members of the Ikaros family of zinc-finger transcription factors have been shown to be critical for immune and blood cell development. However, the role of the most divergent family member, Pegasus, has remained elusive, although it shows conservation to invertebrate Hunchback proteins that influence embryonic patterning through regulation of homeodomain genes. Zebrafish was employed as a relevant model to investigate the function of Pegasus since it possesses a single pegasus orthologue with high homology to its mammalian counterparts. During zebrafish embryogenesis pegasus transcripts were initially maternally-derived and later replaced by zygotic expression in the diencephalon, tectum, hindbrain, thymus, eye, and ultimately the exocrine pancreas and intestine. Morpholino-mediated knockdown of the zebrafish pegasus gene resulted in disrupted left-right asymmetry of the gut and pancreas. Molecular analysis indicated that zebrafish Pegasus localised to the nucleus in discrete non-nucleolar structures and bound the 'atypical' DNA sequence GN3GN2G, confirming its presumed role as a transcriptional regulator. In vivo transcriptome analysis identified candidate target genes, several of which encoded homeodomain transcription factors. One of these, pitx2, implicated in left-right asymmetry, possessed appropriate 'atypical' Pegasus binding sites in its promoter. Knockdown of Pegasus affected both the level and asymmetry of pitx2 expression, as well as disrupting the asymmetry of the lefty2 and spaw genes, explaining the perturbed left-right patterning in pegasus morphants. Collectively these results provide the first definitive insights into the in vivo role of Pegasus, supporting the notion that it acts as a broader regulator of development, with potential parallels to the related invertebrate Hunchback proteins.

Evolution of the Ikaros gene family: implications for the origins of adaptive immunity.

Members of the Ikaros family of transcription factors are important for immune system development. Analysis of Ikaros-related genes from a range of species suggests the Ikaros family derived from a primordial gene, possibly related to the present-day protostome Hunchback genes. This duplicated before the divergence of urochordates to produce two distinct lineages: one that generated the Ikaros factor-like (IFL) 2 genes of urochordates/lower vertebrates and the Pegasus genes of higher vertebrates, and one that generated the IFL1 genes of urochordates/lower vertebrates, the IKFL1 and IKFL2 genes of agnathans and the remaining four Ikaros members of higher vertebrates. Expansion of the IFL1 lineage most likely occurred via the two intervening rounds of whole genome duplication. A proposed third whole genome duplication in teleost fish produced a further increase in complexity of the gene family with additional Pegasus and Eos members. These findings question the use of IFL sequences as evidence for the existence of adaptive immunity in early chordates and vertebrates. Instead, this study is consistent with a later emergence of adaptive immunity coincident with the appearance of the definitive lymphoid markers Ikaros, Aiolos, and Helios.

Mtx2 directs zebrafish morphogenetic movements during epiboly by regulating microfilament formation.

The homeobox transcription factor Mtx2 is essential for epiboly, the first morphogenetic movement of gastrulation in zebrafish. Morpholino knockdown of Mtx2 results in stalling of epiboly and lysis due to yolk rupture. However, the mechanism of Mtx2 action is unknown. The role of mtx2 is surprising as most mix/bix family genes are thought to have roles in mesendoderm specification. Using a transgenic sox17-promoter driven EGFP line, we show that Mtx2 is not required for endoderm specification but is required for correct morphogenetic movements of endoderm and axial mesoderm. During normal zebrafish development, mtx2 is expressed at both the blastoderm margin and in the zebrafish equivalent of visceral endoderm, the extra-embryonic yolk syncytial layer (YSL). We show that formation of the YSL is not Mtx2 dependent, but that Mtx2 directs spatial arrangement of YSL nuclei. Furthermore, we demonstrate that Mtx2 knockdown results in loss of the YSL F-actin ring, a microfilament structure previously shown to be necessary for epiboly progression. In summary, we propose that Mtx2 acts within the YSL to regulate morphogenetic movements of both embryonic and extra-embryonic tissues, independently of cell fate specification.

Characterization of the zebrafish matrix metalloproteinase 9 gene and its developmental expression pattern.

Members of the matrix metalloproteinase (MMP) family are important for the remodeling of the extracellular matrix in a number of biological processes including a variety of immune responses. Two members of the family, MMP2 and MMP9, are highly expressed in specific myeloid cell populations in which they play a role in the innate immune response. To further expand the repertoire of molecular reagents available to study zebrafish myeloid cell development, the matrix metalloproteinase 9 (mmp9) gene from this organism has been identified and characterized. The encoded protein is 680 amino acids with high homology to known MMP9 proteins, particularly those of other teleost fish. Maternal transcripts of mmp9 are deposited in the oocyte and dispersed throughout the early embryo. These are replaced by specific zygotic transcripts in the notochord from 12h post fertilization (hpf) and also transiently in the anterior mesoderm from 14 to 16h post fertilization. From 24h post fertilization, mmp9 expression was detected in a population of circulating white blood cells that are distinct from macrophages, and which migrate to the site of trauma, and so likely represent zebrafish heterophils. In the adult, mmp9 expression was most prominent in the splenic cords, a site occupied by mature myeloid cells in other species. These results suggest that mmp9 will serve as a useful marker of mature myeloid cells in the zebrafish.

Harnessing zebrafish for the study of white blood cell development and its perturbation.

Considerable progress has been made in understanding the molecular basis of normal white blood cell development and its perturbation in disease through the use of clinical studies and traditional animal and cell line models. Despite this, however, many questions are still being answered and white blood cell disorders, including leukemia and lymphoma, remain a significant health problem. The zebrafish (Danio rerio) has emerged as a powerful alternative vertebrate model for the study of development and disease. We review the recent application of zebrafish to the study of white blood cell development and its disruption, particularly leukemogenesis. Such studies have highlighted the overall conservation of these processes throughout vertebrates, and establish zebrafish as a useful experimental model. This organism is now poised to make an important contribution to our understanding of the underlying genetic control of white blood cell development and its disruption, as well as the identification of new therapeutic agents.