Fibronectin regulates epithelial organization during myocardial migration in zebrafish.

Several genes have been implicated in heart tube formation, yet we know little about underlying cellular mechanisms. We analyzed the cellular architecture of the migrating myocardial precursors, and find that they form coherent epithelia that mature as they move medially. Mutant analyses indicate that the cardia bifida locus natter (nat) is required for the integrity of the myocardial epithelia. We positionally cloned nat and show that it encodes Fibronectin. During myocardial migration, Fibronectin is deposited at the midline between the endoderm and endocardial precursors, and laterally around the myocardial precursors. Further analyses show that Fibronectin deposition at the midline is required for the timely migration of myocardial precursors, but dispensable for the migration process itself. In the complete absence of Fibronectin, adherens junctions between myocardial precursors do not form properly, suggesting that cell-substratum interactions are required for epithelial organization. These data suggest that myocardial migration is dependent on epithelial integrity.

The Mix family homeodomain gene bonnie and clyde functions with other components of the Nodal signaling pathway to regulate neural patterning in zebrafish.

Mix family homeodomain proteins, such as Xenopus Mixer and zebrafish Bonnie and clyde (Bon), have been shown to regulate the formation of the endoderm and are likely to be transcriptional mediators of Nodal signaling. Here, we show that, in addition to its previously described role in endoderm formation, Bon also regulates the anteroposterior patterning of the neuroectoderm. bon-mutant embryos exhibit an anterior reduction of the neural plate. By using targeted injection of antisense morpholino oligonucleotides, we demonstrate that Bon is required in the axial mesoderm for anterior neural development. Consistent with these results, bon-mutant embryos show defects in axial mesoderm gene expression starting at mid-gastrulation stages. In addition, genetic analyses demonstrate a functional interaction during neural patterning between bon and two components of the Nodal signaling pathway, the nodal-related gene squint (sqt) and forkhead box H1 [foxh1; mutant locus schmalspur (sur)]. bon-/-;sqt-/- and bon-/-;sur-/- embryos exhibit neural patterning defects that are much more severe than those seen in the single mutants, suggesting that these genes function in parallel in this process. We also show that the severity of the neural patterning defects in the single- and double-mutant embryos correlates with the degree of reduction in expression of the Wnt antagonist gene dickkopf 1. Furthermore, bon-/-;sqt-/- and bon-/-;sur-/- embryos exhibit identical morphological and gene expression defects, suggesting, in part, that bon, sqt and sur (foxh1) play overlapping roles in neural patterning. Taken together, these results provide evidence for a complex genetic network in which bon functions both downstream of, and possibly in parallel to, Nodal signaling to regulate neural patterning via the modulation of mesendodermal gene expression.

Down-regulation of TDT transcription in CD4(+)CD8(+) thymocytes by Ikaros proteins in direct competition with an Ets activator.

Ikaros is a unique regulator of lymphopoiesis that associates with pericentromeric heterochromatin and has been implicated in heritable gene inactivation. Binding and competition experiments demonstrate that Ikaros dimers compete with an Ets activator for occupancy of the lymphocyte-specific TdT promoter. Mutations that selectively disrupt Ikaros binding to an integrated TdT promoter had no effect on promoter function in a CD4(+)CD8(+) thymocyte line. However, these mutations abolished down-regulation on differentiation, providing evidence that Ikaros plays a direct role in repression. Reduced access to restriction enzyme cleavage suggested that chromatin alterations accompany down-regulation. The Ikaros-dependent down-regulation event and the observed chromatin alterations appear to precede pericentromeric repositioning. Current models propose that the functions of Ikaros should be disrupted by a small isoform that retains the dimerization domain and lacks the DNA-binding domain. Surprisingly, in the CD4(+)CD8(+) thymocyte line, overexpression of a small Ikaros isoform had no effect on differentiation or on the pericentromeric targeting and DNA-binding properties of Ikaros. Rather, the small isoform assembled into multimeric complexes with DNA-bound Ikaros at the pericentromeric foci. The capacity for in vivo multimer formation suggests that interactions between Ikaros dimers bound to the TdT promoter and those bound to pericentromeric repeat sequences may contribute to the pericentromeric repositioning of the inactive gene.

The zebrafish bonnie and clyde gene encodes a Mix family homeodomain protein that regulates the generation of endodermal precursors.

Vertebrate endoderm development has recently become the focus of intense investigation. In this report, we first show that the zebrafish bonnie and clyde (bon) gene plays a critical early role in endoderm formation. bon mutants exhibit a profound reduction in the number of sox17-expressing endodermal precursors formed during gastrulation, and, consequently, a profound reduction in gut tissue at later stages. The endodermal precursors that do form in bon mutants, however, appear to differentiate normally indicating that bon is not required at later steps of endoderm development. We further demonstrate that bon encodes a paired-class homeodomain protein of the Mix family that is expressed transiently before and during early gastrulation in both mesodermal and endodermal progenitors. Overexpression of bon can rescue endodermal gene expression and the formation of a gut tube in bon mutants. Analysis of a newly identified mutant allele reveals that a single amino acid substitution in the DNA recognition helix of the homeodomain creates a dominant interfering form of Bon when overexpressed. We also show through loss- and gain-of-function analyses that Bon functions exclusively downstream of cyclops and squint signaling. Together, our data demonstrate that Bon is a critical transcriptional regulator of early endoderm formation.