7-[(7S)-7-Aza-niumyl-5-aza-spiro-[2.4]hept-5-yl]-8-chloro-6-fluoro-1-[(1S,2R)-2-fluoro-cyclo-prop-yl]-4-oxo-1,4-dihydroquinoline-3-carboxyl-ate methanol monosolvate.

Sitafloxacin is a newly developed fluoro-quinolone anti-bacterial drug. The crystal studied, C(19)H(18)ClF(2)N(3)O(3)·CH(3)OH, consists of one mol-ecule of sitafloxacin and one methanol solvent mol-ecule. The mol-ecule of sitafloxacin is a zwitterion with a protonated primary amine group and a deprotonated carboxylate group. The cyclopropane ring and the CO(2) group make dihedral angles of 79.5 (3) and 35.4 (4)°, respectively, with the fused ring system. The supra-molecular structure is defined by N-H⋯O and O-H⋯O hydrogen bonds.

Cyclic Nrarp mRNA expression is regulated by the somitic oscillator but Nrarp protein levels do not oscillate.

Somites are formed progressively from the presomitic mesoderm (PSM) in a highly regulated process according to a strict periodicity driven by an oscillatory mechanism. The Notch and Wnt pathways are key components in the regulation of this somitic oscillator and data from Xenopus and zebrafish embryos indicate that the Notch-downstream target Nrarp participates in the regulation of both activities. We have analyzed Nrarp/nrarp-a expression in the PSM of chick, mouse and zebrafish embryos, and we show that it cycles in synchrony with other Notch regulated cyclic genes. In the mouse its transcription is both Wnt- and Notch-dependent, whereas in the chick and fish embryo it is simply Notch-dependent. Despite oscillating mRNA levels, Nrarp protein does not oscillate in the PSM. Finally, neither gain nor loss of Nrarp function interferes with the normal expression of Notch-related cyclic genes.

Temporal Notch activation through Notch1a and Notch3 is required for maintaining zebrafish rhombomere boundaries.

In vertebrates, hindbrain is subdivided into seven segments termed rhombomeres and the interface between each rhombomere forms the boundary. Similar to the D/V boundary formation in Drosophila, Notch activation has been shown to regulate the segregation of rhombomere boundary cells. Here we further explored the function of Notch signaling in the formation of rhombomere boundaries. By using bodipy ceramide cell-labeling technique, we found that the hindbrain boundary is formed initially in mib mutants but lost after 24 hours post-fertilization (hpf). This phenotype was more severe in mib(ta52b) allele than in mib(tfi91) allele. Similarly, injection of su(h)-MO led to boundary defects in a dosage-dependent manner. Boundary cells were recovered in mib(ta52b) mutants in the hdac1-deficient background, where neurogenesis is inhibited. Furthermore, boundary cells lost sensitivity to reduced Notch activation from 15 somite stage onwards. We also showed that knockdown of notch3 function in notch1a mutants leads to the loss of rhombomere boundary cells and causes neuronal hyperplasia, indicating that Notch1a and Notch3 play a redundant role in the maintenance of rhombomere boundary.

Genomewide expression analysis in zebrafish mind bomb alleles with pancreas defects of different severity identifies putative Notch responsive genes.

BACKGROUND: Notch signaling is an evolutionarily conserved developmental pathway. Zebrafish mind bomb (mib) mutants carry mutations on mib gene, which encodes a RING E3 ligase required for Notch activation via Delta/Jagged ubiquitylation and internalization. METHODOLOGY/PRINCIPAL FINDINGS: We examined the mib mutants for defects in pancreas development using in situ hybridization and GFP expression analysis of pancreas-specific GFP lines, carried out the global gene expression profile analysis of three different mib mutant alleles and validated the microarray data using real-time PCR and fluorescent double in situ hybridization. Our study showed that the mib mutants have diminished exocrine pancreas and this defect was most severe in mib(ta52b) followed by mib(m132) and then mib(tfi91), which is consistent with the compromised Notch activity found in corresponding mib mutant alleles. Global expression profile analysis of mib mutants showed that there is a significant difference in gene expression profile of wt and three mib mutant alleles. There are 91 differentially expressed genes that are common to all three mib alleles. Through detailed analysis of microarray data, we have identified several previously characterized genes and some putative Notch-responsive genes involved in pancreas development. Moreover, results from real-time PCR and fluorescent double in situ hybridization were largely consistent with microarray data. CONCLUSIONS/SIGNIFICANCE: This study provides, for the first time, a global gene expression profile in mib mutants generating useful genomic resources and providing an opportunity to identify the function of novel genes involved in Notch signaling and Notch-regulated developmental processes.

The characterization of zebrafish antimorphic mib alleles reveals that Mib and Mind bomb-2 (Mib2) function redundantly.

Both mind bomb (mib) and mind bomb-2 (mib2) encode RING E3 ubiquitin ligases that promote Delta ubiquitylation and endocytosis in Notch activation. Detailed morphological and molecular examinations revealed that zebrafish mib(ta52b) (missense mutation in the C-terminal RING Finger (RF), M1013R) and mib(m132) (nonsense mutation resulting in a truncated protein that loses all three RFs, C785stop) are strong and weak antimorphic alleles, respectively, compared to the null allele, mib(tfi91) (nonsense mutation resulting in a truncated protein of only 60 amino acids, Y60stop). Zebrafish mib2 ortholog was identified in this study. Zebrafish Mib and Mib2 are colocalized in transfected cells and function redundantly in regulating Notch signaling in embryos. Mib(ta52b) and Mib(m132) have a dosage-dependent dominant-negative effect, at least, on Mib2, which is a molecular basis for the antimorphic phenotypes. It was also shown that Notch signaling negatively regulates mib expression in a Su(H)-dependent manner, forming a negative feedback loop in modulating Notch activation.

Sequence and embryonic expression of three zebrafish fringe genes: lunatic fringe, radical fringe, and manic fringe.

Drosophila fringe and its homologues in vertebrates code for glycosyltransferases that modify Notch, altering the sensitivity of this receptor protein to its ligands Delta and Serrate and, thereby, playing an essential part in the demarcation of tissue boundaries. We describe the isolation and characterization of three zebrafish (Danio rerio) fringe homologues: lunatic fringe (lfng), radical fringe (rfng), and manic fringe (mfng). In addition to the sites previously described (Prince et al. [2001] Mech. Dev. 105:175-180; Leve et al. [ 2001] Dev. Genes Evol. 211:493-500), lfng is also expressed in the sensory patches of the inner ear. The newly described rfng is expressed in adaxial cells, tectum, rhombomere boundaries, and formed somites, but the expression of mfng is only detectable by reverse transcription-polymerase chain reaction and not by whole-mount in situ hybridization (WISH) during early embryonic development; later, it is expressed in the sensory patches of the ear. In mib mutants, where Notch signaling is defective and rhombomere boundaries fail to form, the rfng expression in hindbrain is almost completely lost. None of the three zebrafish fringe genes is detectably expressed in the posterior presomitic mesoderm, suggesting that, in contrast with chick and mouse, the somitogenesis oscillator in this tissue in the zebrafish does not depend on Fringe activity.

Notch activation regulates the segregation and differentiation of rhombomere boundary cells in the zebrafish hindbrain.

During segmentation of the vertebrate hindbrain, a distinct population of boundary cells forms at the interface between each segment. Little is known regarding mechanisms that regulate the formation or functions of these cells. We have investigated a potential role of Notch signaling and find that in the zebrafish hindbrain, radical fringe is expressed in boundary cells and delta genes are expressed adjacent to boundaries, consistent with a sustained activation of Notch in boundary cells. Mosaic expression experiments reveal that activation of the Notch/Su(H) pathway regulates cell affinity properties that segregate cells to boundaries. In addition, Notch signaling correlates with a delayed neurogenesis at hindbrain boundaries and is required to inhibit premature neuronal differentiation of boundary cells. These findings reveal that Notch activation couples the regulation of location and differentiation in hindbrain boundary cells. Such coupling may be important for these cells to act as a stable signaling center.