Tbx20-related genes, mid and H15, are required for tinman expression, proper patterning, and normal differentiation of cardioblasts in Drosophila

Tbx20-related T-box genes have been implicated in the regulation of heart development in several vertebrate species. In the present report, we demonstrate that a pair of genes representing Drosophila orthologs of Tbx20, midline (mid) and H15, have important functions during the development of the Drosophila equivalent of the heart, i.e. the dorsal vessel. We show that mid is among the earliest known genes that are specifically expressed in all cardioblasts during early embryogenesis, and H15 expression is subsequently activated in the same cells. Mutant embryos lacking the activity of mid, or both mid and H15, are able to form dorsal vessels with largely normal numbers of cardioblasts and pericardial cells. Furthermore, the mutant cardioblasts express several general cardioblast markers such as Mef2 and Toll at normal levels. However, the expression of tinman (tin), which normally occurs in four out of six cardioblasts in each hemisegment of the dorsal vessel, is almost abolished. Conversely, the expression of the Dorsocross (Doc) T-box genes, which is normally restricted to the two Tin-negative cardioblasts in each hemisegment, is strongly expanded into the majority of cardioblasts in mid mutant and mid+H15-deficient embryos. Altogether, the data from the loss-of-function phenotypes demonstrate that mid, and to a lesser degree H15, have important roles in establishing the metameric patterning of cardioblast identities, but not in specifying cardioblasts as such. Ectopic expression of mid causes ectopic tin expression and, less efficiently, produces extra cardioblasts. We propose that one of the major functions of mid and H15 during cardioblast development is the re-activation of tin expression at a stage when the induction of tin by Dpp in the dorsal mesoderm has ceased. Through this activity, mid and H15 are required for the normal functional diversification of cardioblasts and the expression of tin-dependent terminal differentiation genes within the dorsal vessel.     

A rat homeobox gene, rNKx-2.5, is a homologue of the tinman gene in Drosophila and is mainly expressed during heart development

 We report the cloning of a rat homeobox-containing gene, rNkx-2.5, and investigation of its expression in adult tissues and during embryonic development. The rNkx-2.5 gene is a homologue of the tinman gene in Drosophila. Both genes share an identical TN domain (tinman-like amino-terminal decapeptide) and about 66.7% sequence identity within their homeodomain sequences. In vertebrates, the rNKx-2.5 gene is most closely related to the mouse NKx-2.5 (mNKx-2.5) gene. Coding sequences for both NKx-2.5 genes have 94.1% sequence identity, including three identical conserved domains, the TN, homeo and NK-2 domains (NK-2 specific domain, carboxy-terminal to the homeodomain in vertebrate tinman homologues). The rat NKx-2.5 gene is encoded by a 1.4-kb mRNA and in adult tissues is mainly expressed in heart, with weaker expression in testis, spleen and lung. During embryonic development, expression of rNKx-2.5 is strongly observed in developing heart, specifically in the myocardium of both atrial and ventricular chambers. In addition, rNKx-2.5 expression marks other developing tissues, including tongue, thyroid, stomach, spleen and pulmonary veins. These data show that rNKx-2.5 is expressed in a pattern similar but not identical to that previously observed for mNKx-2.5. Received: 24 February 1997 / Accepted: 23 June 1997     

Drosophila matrix metalloproteinases are required for tissue remodeling,but not embryonic development

The matrix metalloproteinase (MMP) family is heavily implicated in many diseases, including cancer. The developmental functions of these genes are not clear, however, because the >20 mammalian MMPs can be functionally redundant. Drosophila melanogaster has only two MMPs, which are expressed in embryos in distinct patterns. We created mutations in both genes: Mmp1 mutants have defects in larval tracheal growth and pupal head eversion, and Mmp2 mutants have defects in larval tissue histolysis and epithelial fusion during metamorphosis; neither is required for embryonic development. Double mutants also complete embryogenesis, and these represent the first time, to our knowledge, that all MMPs have been disrupted in any organism. Thus, MMPs are not required for Drosophila embryonic development, but, rather, for tissue remodeling.     

Two fission yeast homologs of Drosophila Mei-S332 are required for chromosome segregation during meiosis I and II

BACKGROUND: Meiosis produces haploid gametes from diploid progenitor cells. This reduction is achieved by two successive nuclear divisions after one round of DNA replication. Correct chromosome segregation during the first division depends on sister kinetochores being oriented toward the same spindle pole while homologous kinetochores must face opposite poles. Segregation during the second division depends on retention of sister chromatid cohesion between centromeres until the onset of anaphase II, which in Drosophila melanogaster depends on a protein called Mei-S332 that binds to centromeres. RESULTS: We report the identification of two homologs of Mei-S332 in fission yeast using a knockout screen. Together with their fly ortholog they define a protein family conserved from fungi to mammals. The two identified genes, sgo1 and sgo2, are required for retention of sister centromere cohesion between meiotic divisions and kinetochore orientation during meiosis I, respectively. The amount of meiotic cohesin's Rec8 subunit retained at centromeres after meiosis I is reduced in Deltasgo1, but not in Deltasgo2, cells, and Sgo1 appears to regulate cleavage of Rec8 by separase. Both Sgo1 and Sgo2 proteins localize to centromere regions. The abundance of Sgo1 protein normally declines after the first meiotic division, but extending its expression by altering its 3'UTR sequences does not greatly affect meiosis II. Its mere presence within the cell might therefore be insufficient to protect centromeric cohesion. CONCLUSIONS: A conserved protein family based on Mei-S332 has been identified. The two fission yeast homologs are implicated in meiosis I kinetochore orientation and retention of centromeric sister chromatid cohesion until meiosis II.     

The patterns of wingless,decapentaplegic, and tinman position the Drosophila heart

Two secreted signaling molecules, wingless (wg) and decapentaplegic (dpp), are required to specify the heart in Drosophila. wg and dpp are also required to specify other cell types within the mesoderm and in many other regions of the embryo. Because the spatial patterns of wg and dpp are dynamic, different populations of mesodermal cells are exposed to different combinations of wg and/or dpp at different times. To determine whether the patterns of wg and dpp expression provide unique positional information for the specification of heart precursors, we altered these patterns. Our data suggest that wg and dpp contribute progressively to the elaboration of the expression pattern of the mesoderm-specific homeobox-containing gene tinman (tin), and that the overlap of wg and dpp at an early stage (9) as well as at a later stage (11) in the presence of tin-expressing cells directs cardiac-specific differentiation. Furthermore, ectopic tin expression in the ectoderm at wg/dpp intersects (the primordia of the thoracic imaginal disks) also leads to cardiac-specific differentiation, suggesting that tin confers mesoderm-specificity to the wg/dpp response. We conclude that ectopic heart can be generated by altering the patterns of wg and dpp within the tin-expressing mesoderm, or by ectopic induction of tin within the wg- and dpp-expressing ectoderm.     

Fission yeast Rnf4 homologs are required for DNA repair

We describe two RING finger proteins in the fission yeast Schizosaccharomyces pombe, Rfp1 and Rfp2. We show that these proteins function redundantly in DNA repair. Rfp1 was isolated as a Chk1-interacting protein in a two-hybrid screen and has high amino acid sequence similarity to Rfp2. Deletion of either gene does not cause a phenotype, but a double deletion (rfp1Deltarfp2Delta) showed poor viability and defects in cell cycle progression. These cells are also sensitive to DNA-damaging agents, although they maintained normal checkpoint signaling to Chk1. Rfp1 and Rfp2 are most closely related to human Rnf4, and we showed that Rnf4 can substitute functionally for Rfp1 and/or Rfp2. The double mutants also showed significantly increased levels of protein SUMOylation, and we identified an S. pombe Ulp2/Smt4 homolog that, when overexpressed, reduced SUMO levels and suppressed the DNA damage sensitivity of rfp1Delta rfp2Delta cells.     

Lobe and Serrate are required for cell survival during early eye development in Drosophila

Organogenesis involves an initial surge of cell proliferation, leading to differentiation. This is followed by cell death in order to remove extra cells. During early development, there is little or no cell death. However, there is a lack of information concerning the genes required for survival during the early cell-proliferation phase. Here, we show that Lobe (L) and the Notch (N) ligand Serrate (Ser), which are both involved in ventral eye growth, are required for cell survival in the early eye disc. We observed that the loss-of-ventral-eye phenotype in L or Ser mutants is due to the induction of cell death and the upregulation of secreted Wingless (Wg). This loss-of-ventral-eye phenotype can be rescued by (i) increasing the levels of cell death inhibitors, (ii) reducing the levels of Hid-Reaper-Grim complex, or (iii) reducing canonical Wg signaling components. Blocking Jun-N-terminal kinase (JNK) signaling, which can induce caspase-independent cell death, significantly rescued ventral eye loss in L or Ser mutants. However, blocking both caspase-dependent cell death and JNK signaling together showed stronger rescues of the L- or Ser-mutant eye at a 1.5-fold higher frequency. This suggests that L or Ser loss-of-function triggers both caspase-dependent and -independent cell death. Our studies thus identify a mechanism responsible for cell survival in the early eye.     

Drosophila Bcl-2 proteins participate in stress-induced apoptosis, but are not required for normal development

Many developing tissues require programmed cell death (PCD) for proper formation. In mice and C. elegans, developmental PCD is regulated by the Bcl-2 family of proteins. Two bcl-2 genes are encoded in the Drosophila genome (debcl/dBorg1/Drob-1/dBok and buffy/dBorg2) and previous RNAi-based studies suggested a requirement for these in embryonic development. However, we report here that, despite the fact that many tissues in fruit flies are shaped by PCD, deletion of the bcl-2 genes does not perturb normal development. We investigated whether the fly bcl-2 genes regulate non-apoptotic processes that require caspases, but found these to be bcl-2 gene-independent. However, irradiation of the mutants demonstrates that DNA damage-induced apoptosis, mediated by Reaper, is blocked by buffy and that debcl is required to inhibit buffy. Our results demonstrate that developmental PCD regulation in the fly does not rely upon the Bcl-2 proteins, but that they provide an added layer of protection in the apoptotic response to stress.     

Nkx2.7 and Nkx2.5 Function Redundantly and Are Required for Cardiac Morphogenesis of Zebrafish Embryos

Background

Nkx2.7 is the tinman-related gene, as well as orthologs of Nkx2.5 and Nkx-2.3. Nkx2.7 and Nkx2.5 express in zebrafish heart fields of lateral plate mesoderm. The temporal and spatial expression patterns of Nkx2.7 are similar to those of Nkx2.5, but their functions during cardiogenesis remain unclear.

Methodology/Principal Findings

Here, Nkx2.7 is demonstrated to compensate for Nkx2.5 loss of function and play a predominant role in the lateral development of the heart, including normal cardiac looping and chamber formation. Knocking down Nkx2.5 showed that heart development was normal from 24 to 72 hpf. However, when knocking down either Nkx2.7 or Nkx2.5 together with Nkx2.7, it appeared that the heart failed to undergo looping and showed defective chambers, although embryos developed normally before the early heart tube stage. Decreased ventricular myocardium proliferation and defective myocardial differentiation appeared to result from late-stage up-regulation of bmp4, versican, tbx5 and tbx20, which were all expressed normally in hearts at an early stage. We also found that tbx5 and tbx20 were modulated by Nkx2.7 through the heart maturation stage because an inducible overexpression of Nkx2.7 in the heart caused down-regulation of tbx5 and tbx20. Although heart defects were induced by overexpression of an injection of 150-pg Nkx2.5 or 5-pg Nkx2.7 mRNA, either Nkx2.5 or Nkx2.7 mRNA rescued the defects induced by Nkx2.7-morpholino(MO) and Nkx2.5-MO with Nkx2.7-MO.

Conclusions and Significance

Therefore, we conclude that redundant activities of Nkx2.5 and Nkx2.7 are required for cardiac morphogenesis, but that Nkx2.7 plays a more critical function, specifically indicated by the gain-of-function and loss-of- function experiments where Nkx2.7 is observed to regulate the expressions of tbx5 and tbx20 through the maturation stage.     

eat-2 and eat-18 are required for nicotinic neurotransmission in the Caenorhabditis elegans pharynx

Mutations in eat-2 and eat-18 cause the same defect in C. elegans feeding behavior: the pharynx is unable to pump rapidly in the presence of food. EAT-2 is a nicotinic acetylcholine receptor subunit that functions in the pharyngeal muscle. It is localized to the synapse between pharyngeal muscle and the main pharyngeal excitatory motor neuron MC, and it is required for MC stimulation of pharyngeal muscle. eat-18 encodes a small protein that has no homology to previously characterized proteins. It has a single transmembrane domain and a short extracellular region. Allele-specific genetic interactions between eat-2 and eat-18 suggest that EAT-18 interacts physically with the EAT-2 receptor. While eat-2 appears to be required specifically for MC neurotransmission, eat-18 also appears to be required for the function of other nicotinic receptors in the pharynx. In eat-18 mutants, the gross localization of EAT-2 at the MC synapse is normal, suggesting that it is not required for trafficking. These data indicate that eat-18 could be a novel component of the pharyngeal nicotinic receptor.     

NADPH oxidase homologs are required for normal cell differentiation and morphogenesis in Dictyostelium discoideum

Membrane-associated NADPH oxidase complexes catalyse the production of the superoxide anion radical from oxygen and NADPH. In mammalian systems, NADPH oxidases form a family of at least seven isoforms that participate in host defence and signalling pathways. We report here the cloning and the characterisation of slime mould Dictyostelium discoideum homologs of the mammalian heme-containing subunit of flavocytochrome b (gp91(phox)) (NoxA, NoxB and NoxC), of the small subunit of flavocytochrome b (p22(phox)) and of the cytosolic factor p67(phox). Null-mutants of either noxA, noxB, noxC or p22(phox) show aberrant starvation-induced development and are unable to produce spores. The overexpression of NoxA(myc2) in noxA null strain restores spore formation. Remarkably, the gene alg-2B, coding for one of the two penta EF-hand proteins in Dictyostelium, acts as a suppressor in noxA, noxB, and p22(phox) null-mutant strains. Knockout of alg-2B allows noxA, noxB or p22(phox) null-mutants to return to normal development. However, the knockout of gene encoding NoxC, which contains two penta EF-hands, is not rescued by the invalidation of alg-2B. These data are consistent with a hypothesis connecting superoxide and calcium signalling during Dictyostelium development.