Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells.

The homeobox gene tinman and the nuclear receptor gene seven-up are expressed in mutually exclusive dorsal vessel cells in Drosophila, however, the physiological reason for this distinction is not known. We demonstrate that tin and svp-lacZ expression persists through the larval stage to the adult stage in the same pattern of cells expressing these genes in the embryo. In the larva, six pairs of Svp-expressing cells form muscular ostia, which permit hemolymph to enter the heart for circulation, however, more anterior Svp-expressing cells form the wall of the dorsal vessel. During pupation, the adult heart forms from a chimera of larval and imaginal muscle fibers. The portion of the dorsal vessel containing the larval ostia is histolyzed and the anterior Svp-expressing cells metamorphose into imaginal ostia. This is the first demonstration that the significant molecular diversity of cardial cells identified in the embryonic heart correlates with the formation of physiologically and functionally distinct muscle cells in the animal. Furthermore, our experiments define the cellular changes that occur as the larval heart is remodeled into an imaginal structure in an important model organism.     

Slit coordinates cardiac morphogenesis in Drosophila

Slit is a secreted guidance cue that conveys repellent or attractive signals from target and guidepost cells. In Drosophila, responsive cells express one or more of three Robo receptors. The cardial cells of the developing heart express both Slit and Robo2. This is the first report of coincident expression of a Robo and its ligand. In slit mutants, cardial cell alignment, polarization and uniform migration are disrupted. The heart phenotype of robo2 mutants is similar, with fewer migration defects. In the guidance of neuronal growth cones in Drosophila, there is a phenotypic interaction between slit and robo heterozygotes, and also with genes required for Robo signaling. In contrast, in the heart, slit has little or no phenotypic interaction with Robo-related genes, including Robo2, Nck2, and Disabled. However, there is a strong phenotypic interaction with Integrin genes and their ligands, including Laminin and Collagen, and intracellular messengers, including Talin and ILK. This indicates that Slit participates in adhesion or adhesion signaling during heart development.     

Taste perception and coding in Drosophila

BACKGROUND: Discrimination between edible and contaminated foods is crucial for the survival of animals. In Drosophila, a family of gustatory receptors (GRs) expressed in taste neurons is thought to mediate the recognition of sugars and bitter compounds, thereby controlling feeding behavior. RESULTS: We have characterized in detail the expression of eight Gr genes in the labial palps, the fly's main taste organ. These genes fall into two distinct groups: seven of them, including Gr66a, are expressed in 22 or fewer taste neurons in each labial palp. Additional experiments show that many of these genes are coexpressed in partially overlapping sets of neurons. In contrast, Gr5a, which encodes a receptor for trehalose, is expressed in a distinct and larger set of taste neurons associated with most chemosensory sensilla, including taste pegs. Mapping the axonal targets of cells expressing Gr66a and Gr5a reveals distinct projection patterns for these two groups of neurons in the brain. Moreover, tetanus toxin-mediated inactivation of Gr66a- or Gr5a-expressing cells shows that these two sets of neurons mediate distinct taste modalities-the perception of bitter (caffeine) and sweet (trehalose) taste, respectively. CONCLUSION: Discrimination between two taste modalities-sweet and bitter-requires specific sets of gustatory receptor neurons that express different Gr genes. Unlike the Drosophila olfactory system, where each neuron expresses a single olfactory receptor gene, taste neurons can express multiple receptors and do so in a complex Gr gene code that is unique for small sets of neurons.     

The Hox gene abdominal-A specifies heart cell fate in the Drosophila dorsal vessel

The Drosophila melanogaster dorsal vessel is a linear organ that pumps blood through the body. Blood enters the dorsal vessel in a posterior chamber termed the heart, and is pumped in an anterior direction through a region of the dorsal vessel termed the aorta. Although the genes that specify dorsal vessel cell fate are well understood, there is still much to be learned concerning how cell fate in this linear tube is determined in an anteroposterior manner, either in Drosophila or in any other animal. We demonstrate that the formation of a morphologically and molecularly distinct heart depends crucially upon the homeotic segmentation gene abdominal-A (abd-A). abd-A expression in the dorsal vessel was detected only in the heart, and overexpression of abd-A induced heart fate in the aorta in a cell-autonomous manner. Mutation of abd-A resulted in a loss of heart-specific markers. We also demonstrate that abd-A and sevenup co-expression in cardial cells defined the location of ostia, or inflow tracts. Other genes of the Bithorax Complex do not appear to participate in heart specification, although high level expression of Ultrabithorax is capable of inducing a partial heart fate in the aorta. These findings for the first time demonstrate a specific involvement for Hox genes in patterning the muscular circulatory system, and suggest a mechanism of broad relevance for animal heart patterning.     

Spire, an actin nucleation factor, regulates cell division during Drosophila heart development

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, we demonstrate that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, we determines that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. We conclude that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis.     

Applications of mRNA injections for analyzing cell lineage and asymmetric cell divisions during segmentation in the leech Helobdella robusta

Synthetic mRNAs can be injected to achieve transient gene expression even for 'non-model' organisms in which genetic approaches are not feasible. Here, we have used this technique to express proteins that can serve as lineage tracers or reporters of cellular events in embryos of the glossiphoniid leech Helobdella robusta (phylum Annelida). As representatives of the proposed super-phylum Lophotrochozoa, glossiphoniid leeches are of interest for developmental and evolutionary comparisons. Their embryos are suitable for microinjection, but no genetic approaches are currently available. We have injected segmentation stem cells (teloblasts) with mRNAs encoding nuclear localized green fluorescent protein (nGFP) and its spectral variants, and have used tandem injections of nGFP mRNA followed by antisense morpholino oligomer (AS MO), to label single blast cell clones. These techniques permit high resolution cell lineage tracing in living embryos. We have applied them to the primary neurogenic (N) lineage, in which alternate segmental founder cells (nf and ns blast cells) contribute distinct sets of progeny to the segmental ganglia. The nf and ns blast cell clones exhibit strikingly different cell division patterns: the increase in cell number within the nf clone is roughly linear, while that in the ns clone is almost exponential. To analyze spindle dynamics in the asymmetric divisions of individual blast cells, we have injected teloblasts with mRNA encoding a tau::GFP fusion protein. Our results show that the asymmetric divisions of n blast cells result from a posterior shift of both the spindle within the cell and the midbody within the mitotic spindle, with differential regulation of these processes between nf and ns.     

Seven up acts as a temporal factor during two different stages of neuroblast 5-6 development

Drosophila embryonic neuroblasts generate different cell types at different time points. This is controlled by a temporal cascade of Hb→Kr→Pdm→Cas→Grh, which acts to dictate distinct competence windows sequentially. In addition, Seven up (Svp), a member of the nuclear hormone receptor family, acts early in the temporal cascade, to ensure the transition from Hb to Kr, and has been referred to as a 'switching factor'. However, Svp is also expressed in a second wave within the developing CNS, but here, the possible role of Svp has not been previously addressed. In a genetic screen for mutants affecting the last-born cell in the embryonic NB5-6T lineage, the Ap4/FMRFamide neuron, we have isolated a novel allele of svp. Expression analysis shows that Svp is expressed in two distinct pulses in NB5-6T, and mutant analysis reveals that svp plays two distinct roles. In the first pulse, svp acts to ensure proper downregulation of Hb. In the second pulse, which occurs in a Cas/Grh double-positive window, svp acts to ensure proper sub-division of this window. These studies show that a temporal factor may play dual roles, acting at two different stages during the development of one neural lineage.     

The heterotrimeric protein Go is required for the formation of heart epithelium in Drosophila.

The gene encoding the alpha subunit of the Drosophila Go protein is expressed early in embryogenesis in the precursor cells of the heart tube, of the visceral muscles, and of the nervous system. This early expression coincides with the onset of the mesenchymal-epithelial transition to which are subjected the cardial cells and the precursor cells of the visceral musculature. This gene constitutes an appropriate marker to follow this transition. In addition, a detailed analysis of its expression suggests that the cardioblasts originate from two subpopulations of cells in each parasegment of the dorsal mesoderm that might depend on the wingless and hedgehog signaling pathways for both their determination and specification. In the nervous system, the expression of Goalpha shortly precedes the beginning of axonogenesis. Mutants produced in the Goalpha gene harbor abnormalities in the three tissues in which the gene is expressed. In particular, the heart does not form properly and interruptions in the heart epithelium are repeatedly observed, henceforth the brokenheart (bkh) name. Furthermore, in the bkh mutant embryos, the epithelial polarity of cardial cells was not acquired (or maintained) in various places of the cardiac tube. We predict that bkh might be involved in vesicular traffic of membrane proteins that is responsible for the acquisition of polarity.     

Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output

Adult stem cells modulate their output by varying between symmetric and asymmetric divisions, but have rarely been observed in living intact tissues. Germline stem cells (GSCs) in the Drosophila testis are anchored to somatic hub cells and were thought to exclusively undergo oriented asymmetric divisions, producing one stem cell that remains hub-anchored and one daughter cell displaced out of the stem cell-maintaining micro-environment (niche). We developed extended live imaging of the Drosophila testis niche, allowing us to track individual germline cells. Surprisingly, new wild-type GSCs are generated in the niche during steady-state tissue maintenance by a previously undetected event we term 'symmetric renewal', where interconnected GSC-daughter cell pairs swivel such that both cells contact the hub. We also captured GSCs undergoing direct differentiation by detaching from the hub. Following starvation-induced GSC loss, GSC numbers are restored by symmetric renewals. Furthermore, upon more severe (genetically induced) GSC loss, both symmetric renewal and de-differentiation (where interconnected spermatogonia fragment into pairs while moving towards then establishing contact with the hub) occur simultaneously to replenish the GSC pool. Thus, stereotypically oriented stem cell divisions are not always correlated with an asymmetric outcome in cell fate, and changes in stem cell output are governed by altered signals in response to tissue requirements.     

Generation and early differentiation of glial cells in the first optic ganglion of Drosophila melanogaster.

We have examined the generation and development of glial cells in the first optic ganglion, the lamina, of Drosophila melanogaster. Previous work has shown that the growth of retinal axons into the developing optic lobes induces the terminal cell divisions that generate the lamina monopolar neurons. We investigated whether photoreceptor ingrowth also influences the development of lamina glial cells, using P element enhancer trap lines, genetic mosaics and birthdating analysis. Enhancer trap lines that mark the differentiating lamina glial cells were found to require retinal innervation for expression. In mutants with only a few photoreceptors, only the few glial cells near ingrowing axons expressed the marker. Genetic mosaic analysis indicates that the lamina neurons and glial cells are readily separable, suggesting that these are derived from distinct lineages. Additionally, BrdU pulse-chase experiments showed that the cell divisions that produce lamina glia, unlike those producing lamina neurons, are not spatially or temporally correlated with the retinal axon ingrowth. Finally, in mutants lacking photoreceptors, cell divisions in the glial lineage appeared normal. We conclude that the lamina glial cells derive from a lineage that is distinct from that of the L-neurons, that glia are generated independently of photoreceptor input, and that completion of the terminal glial differentiation program depends, directly or indirectly, on an inductive signal from photoreceptor axons.     

Diverged and conserved aspects of heart formation in a spider

Heart development exhibits some striking similarities between vertebrates and arthropods, for example in both cases the heart develops as a linear tube from mesodermal cells. Furthermore, the underlying molecular pathways exhibit a significant number of similarities between vertebrates and the fruit fly Drosophila, suggesting a common origin of heart development in the last common ancestor of flies and vertebrates. However, there is hardly any molecular data from other animals. Here we show that many of the key genes are also active in heart development in the spider Cupiennius salei. Spiders belong to the chelicerates and are distantly related to insects with respect to the other arthropods. The tinman/Nkx2.5 ortholog is the first gene to be specifically expressed in the presumptive spider heart, like in flies and vertebrates. We also show that tinman is expressed in a similar way in the beetle Tribolium castaneum. Taken together this demonstrates that tinman has a conserved role in the specification of the arthropod heart. In addition, we analyzed the expression of other heart genes (decapentaplegic, Wnt5, H15, even-skipped, and Mef2 ) in Cupiennius. The expression of these genes suggests that the genetic pathway of heart development may be largely conserved among arthropods. However, a major difference is seen in the earlier expression of the even-skipped gene in the developing spider heart compared with Drosophila, implying that the role of even-skipped in heart formation might have changed during arthropod evolution. The most striking finding, however, is that in addition to the dorsal tissue of the fourth walking leg segment and the opisthosomal segments, we discovered tinman-expressing cells that arise from a position dorsal to the cephalic lobe and that contribute to the anterior dorsal vessel. In contrast to the posterior heart tissue, these cells do not express the other heart genes. The spider heart thus is composed of two distinct populations of cells.     

Asymmetric Numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts

Stem cells and neuroblasts derived from mouse embryos undergo repeated asymmetric cell divisions, generating neural lineage trees similar to those of invertebrates. In Drosophila, unequal distribution of Numb protein during mitosis produces asymmetric cell divisions and consequently diverse neural cell fates. We investigated whether a mouse homologue m-numb had a similar role during mouse cortical development. Progenitor cells isolated from the embryonic mouse cortex were followed as they underwent their next cell division in vitro. Numb distribution was predominantly asymmetric during asymmetric cell divisions yielding a beta-tubulin III(-) progenitor and a beta-tubulin III(+) neuronal cell (P/N divisions) and predominantly symmetric during divisions producing two neurons (N/N divisions). Cells from the numb knockout mouse underwent significantly fewer asymmetric P/N divisions compared to wild type, indicating a causal role for Numb. When progenitor cells derived from early (E10) cortex undergo P/N divisions, both daughters express the progenitor marker Nestin, indicating their immature state, and Numb segregates into the P or N daughter with similar frequency. In contrast, when progenitor cells derived from later E13 cortex (during active neurogenesis in vivo) undergo P/N divisions they produce a Nestin(+) progenitor and a Nestin(-) neuronal daughter, and Numb segregates preferentially into the neuronal daughter. Thus during mouse cortical neurogenesis, as in Drosophila neurogenesis, asymmetric segregation of Numb could inhibit Notch activity in one daughter to induce neuronal differentiation. At terminal divisions generating two neurons, Numb was symmetrically distributed in approximately 80% of pairs and asymmetrically in 20%. We found a significant association between Numb distribution and morphology: most sisters of neuron pairs with symmetric Numb were similar and most with asymmetric Numb were different. Developing cortical neurons with Numb had longer processes than those without. Numb is expressed by neuroblasts and stem cells and can be asymmetrically segregated by both. These data indicate Numb has an important role in generating asymmetric cell divisions and diverse cell fates during mouse cortical development.