Linking specification to differentiation:From proneural genes to the regulation of ciliogenesis

Much of developmental biology is concerned with the processes by which cells become committed to particular fates in a regulated fashion, whereas cell biology addresses, among other things, the variety of differentiated forms and functions that cells can acquire. One open question is how the regulators of the former process lead to attainment of the latter. “High-level” regulators of cell fate specification include the proneural factors, which drive cells to commit as precursors in the sensory nervous system. Recent research has concentrated on the gene expression events downstream of proneural factor function. Here we summarize this research and describe our own research that has provided clear links between a proneural factor, atonal and the cell biological program of ciliogenesis, which is a central aspect of sensory neuron differentiation

The function and regulation of the bHLH gene, cato, in Drosophila neurogenesis

Abstract Background bHLH transcription factors play many roles in neural development. cousin of atonal (cato) encodes one such factor that is expressed widely in the developing sensory nervous system of Drosophila. However, nothing definitive was known of its function owing to the lack of specific mutations. Results We characterised the expression pattern of cato in detail using newly raised antibodies and GFP reporter gene constructs. Expression is predominantly in sensory lineages that depend on the atonal and amos proneural genes. In lineages that depend on the scute proneural gene, cato is expressed later and seems to be particularly associated with the type II neurons. Consistent with this, we find evidence that cato is a direct target gene of Atonal and Amos, but not of Scute. We generated two specific mutations of cato. Mutant embryos show several defects in chordotonal sensory lineages, most notably the duplication of the sensory neuron, which appears to be caused by an extra cell division. In addition, we show that cato is required to form the single chordotonal organ that persists in atonal mutant embryos. Conclusions We conclude that although widely expressed in the developing PNS, cato is expressed and regulated very differently in different sensory lineages. Mutant phenotypes correlate with cato's major expression in the chordotonal sensory lineage. In these cells, we propose that it plays roles in sense organ precursor maintenance and/or identity, and in controlling the number of cell divisions in the neuronal branch of the lineage arising from these precursors.</p

Structural insights into the activity regulation of full-length non-structural protein 1 from SARS-CoV-2

Non-structural protein 1 (Nsp1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major virulence factor and thus an attractive drug target. The last 33 amino acids of Nsp1 have been shown to bind within the mRNA entry tunnel of the 40S ribosomal subunit, shutting off host gene expression. Here, we report the solution-state structure of full-length Nsp1, which features an α/β fold formed by a six-stranded, capped β-barrel-like globular domain (N-terminal domain [NTD]), flanked by short N-terminal and long C-terminal flexible tails. The NTD has been found to be critical for 40S-mediated viral mRNA recognition and promotion of viral gene expression. We find that in free Nsp1, the NTD mRNA-binding surface is occluded by interactions with the acidic C-terminal tail, suggesting a mechanism of activity regulation based on the interplay between the folded NTD and the disordered C-terminal region. These results are relevant for drug-design efforts targeting Nsp1

Requirement for EGF receptor signalling in neural recruitment during formation of Drosophila chordotonal sense organ clusters

AbstractBackground:Drosophila proneural genes act in the process of selecting neural precursors from undifferentiated ectoderm. The proneural gene atonal is required for the development of precursors of both chordotonal organs (stretch receptors) and photoreceptors. Although these types of sensory element are dissimilar in structure and function, they both occur as organized arrays of neurons. Previous studies have shown that clustering of photoreceptors involves local recruitment, and that signalling by the Drosophila epidermal growth factor receptor (DER) pathway is involved in the recruitment process. We present evidence that a similar mechanism is required for the clustering of embryonic chordotonal organs.Results: We have examined the expression patterns of atonal and genes of the DER pathway in wild-type and mutant backgrounds. Expression of atonal was restricted to a subset of the atonal-requiring chordotonal precursors, which we call founder precursors. The remaining precursors required DER signalling for their selection. Signalling by the founder precursors was initiated by atonal activating, directly or indirectly, rhomboid expression in these cells. Signalling by these founder precursors then provoked a response in the surrounding ectodermal cells, as shown by the activation of expression of the DER target genes pointed and argos. The signal and response then led to recruitment of some of the ectodermal cells to the chordotonal precursor cell fate. DER hyperactivation by misexpression of rhomboid resulted in excessive chordotonal precursor recruitment.Conclusions: Increased numbers of chordotonal precursors are recruited by homeogenetic induction involving signalling via DER from founder precursors to surrounding ectodermal cells. We suggest that the reason chordotonal organs and photoreceptors share a requirement for the proneural gene atonal is that this gene activates a common pathway leading to neural aggregation

Strongly truncated Dnaaf4 plays a conserved role in Drosophila ciliary dynein assembly as part of an R2TP-like co-chaperone complex with Dnaaf6

Axonemal dynein motors are large multi-subunit complexes that drive ciliary movement. Cytoplasmic assembly of these motor complexes involves several co-chaperones, some of which are related to the R2TP co-chaperone complex. Mutations of these genes in humans cause the motile ciliopathy, Primary Ciliary Dyskinesia (PCD), but their different roles are not completely known. Two such dynein (axonemal) assembly factors (DNAAFs) that are thought to function together in an R2TP-like complex are DNAAF4 (DYX1C1) and DNAAF6 (PIH1D3). Here we investigate the Drosophila homologues, CG14921/Dnaaf4 and CG5048/Dnaaf6. Surprisingly, Drosophila Dnaaf4 is truncated such that it completely lacks a TPR domain, which in human DNAAF4 is likely required to recruit HSP90. Despite this, we provide evidence that Drosophila Dnaaf4 and Dnaaf6 proteins can associate in an R2TP-like complex that has a conserved role in dynein assembly. Both are specifically expressed and required during the development of the two Drosophila cell types with motile cilia: mechanosensory chordotonal neurons and sperm. Flies that lack Dnaaf4 or Dnaaf6 genes are viable but with impaired chordotonal neuron function and lack motile sperm. We provide molecular evidence that Dnaaf4 and Dnaaf6 are required for assembly of outer dynein arms (ODAs) and a subset of inner dynein arms (IDAs)

Multiple enhancers contribute to spatial but not temporal complexity in the expression of the proneural gene, amos

BACKGROUND: The regulation of proneural gene expression is an important aspect of neurogenesis. In the study of the Drosophila proneural genes, scute and atonal, several themes have emerged that contribute to our understanding of the mechanism of neurogenesis. First, spatial complexity in proneural expression results from regulation by arrays of enhancer elements. Secondly, regulation of proneural gene expression occurs in distinct temporal phases, which tend to be under the control of separate enhancers. Thirdly, the later phase of proneural expression often relies on positive autoregulation. The control of these phases and the transition between them appear to be central to the mechanism of neurogenesis. We present the first investigation of the regulation of the proneural gene, amos. RESULTS: Amos protein expression has a complex pattern and shows temporally distinct phases, in common with previously characterised proneural genes. GFP reporter gene constructs were used to demonstrate that amos has an array of enhancer elements up- and downstream of the gene, which are required for different locations of amos expression. However, unlike other proneural genes, there is no evidence for separable enhancers for the different temporal phases of amos expression. Using mutant analysis and site-directed mutagenesis of potential Amos binding sites, we find no evidence for positive autoregulation as an important part of amos control during neurogenesis. CONCLUSION: For amos, as for other proneural genes, a complex expression pattern results from the sum of a number of simpler sub-patterns driven by specific enhancers. There is, however, no apparent separation of enhancers for distinct temporal phases of expression, and this correlates with a lack of positive autoregulation. For scute and atonal, both these features are thought to be important in the mechanism of neurogenesis. Despite similarities in function and expression between the Drosophila proneural genes, amos is regulated in a fundamentally different way from scute and atonal

The dynamics of protein localisation to restricted zones within Drosophila mechanosensory cilia

The Drosophila chordotonal neuron cilium is the site of mechanosensory transduction. The cilium has a 9 + 0 axoneme structure and is highly sub-compartmentalised, with proximal and distal zones harbouring different TRP channels and the proximal zone axoneme also being decorated with axonemal dynein motor complexes. The activity of the dynein complexes is essential for mechanotransduction. We investigate the localisation of TRP channels and dynein motor complexes during ciliogenesis. Differences in timing of TRP channel localisation correlate with order of construction of the two ciliary zones. Dynein motor complexes are initially not confined to their target proximal zone, but ectopic complexes beyond the proximal zone are later cleared, perhaps by retrograde transport. Differences in transient distal localisation of outer and inner dynein arm complexes (ODAs and IDAs) are consistent with previous suggestions from unicellular eukaryotes of differences in processivity during intraflagellar transport. Stable localisation depends on the targeting of their docking proteins in the proximal zone. For ODA, we characterise an ODA docking complex (ODA-DC) that is targeted directly to the proximal zone. Interestingly, the subunit composition of the ODA-DC in chordotonal neuron cilia appears to be different from the predicted ODA-DC in Drosophila sperm

Revaluating “Germany’s worst street”. Commercial gentrification on Leipzig’s Eisenbahnstraße?

Leipzig’s Eisenbahnstraße in Germany is currently discussed from different points of view. Ethnicity, crime, but also urban growth and revaluation processes are in the center of the discourse. As one of the city’s high streets, the Eisenbahnstraße and its two surrounding quarters show changes in the commercial structure, which are claimed to be gentrification processes. This paper aims to analyze both the process by using the concept of commercial gentrification and its local perception. This is done by mapping current commercial uses in the retail, service and gastronomy sector, categorizing them, and comparing them to secondary data. Apart from that, local stakeholder’s perspectives are evaluated based on 16 structured interviews conducted with shop owners on the one hand, and a survey among 105 passersby on the street on the other hand. The material indicates that the Eisenbahnstraße is currently in an initial phase of commercial gentrification. This is displayed by diversification of supply and demand structures, represented by the arrival of new potential customers, but also by a diversification of business concepts. Apart from that, commercial activities experience a strong increase in the area and have led to spatial dispersion from the high street to neighboring roads. We argue that Leipzig’s Eisenbahnstraße follows partially the East German gentrification path, as until now no displacement is identified, but the case also stands out due to the important role of ethnicity in commercial structures