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LCIA (low CO2-inducible protein A) is a chloroplast envelope protein associated with the CO2-concentrating mechanism of the green alga Chlamydomonas reinhardtii. LCIA is postulated to be a HCO3– channel, but previous studies were unable to show that LCIA was actively transporting bicarbonate in planta. Therefore, LCIA activity was investigated more directly in two heterologous systems: an Escherichia coli mutant (DCAKO) lacking both native carbonic anhydrases and an Arabidopsis mutant (βca5) missing the plastid carbonic anhydrase βCA5. Neither DCAKO nor βca5 can grow in ambient CO2 conditions, as they lack carbonic anhydrase-catalyzed production of the necessary HCO3– concentration for lipid and nucleic acid biosynthesis. Expression of LCIA restored growth in both systems in ambient CO2 conditions, which strongly suggests that LCIA is facilitating HCO3– uptake in each system. To our knowledge, this is the first direct evidence that LCIA moves HCO3– across membranes in bacteria and plants. Furthermore, the βca5 plant bioassay used in this study is the first system for testing HCO3– transport activity in planta, an experimental breakthrough that will be valuable for future studies aimed at improving the photosynthetic efficiency of crop plants using components from algal CO2-concentrating mechanisms.

A long-held goal of synthetic biology has been the transfer of a bacterial nitrogen-fixation pathway into plants to reduce the use of chemical fertiliser on crops such as rice, wheat and maize. There are three classes of bacterial nitrogenases, named after their unique metalloclusters containing either Mo-, V- or Fe, that convert N2 gas to ammonia. Relative to the Mo-nitrogenase the Fe-nitrogenase is not as efficient for catalysis but has less complex genetic and metallocluster requirements, features that may be preferable for engineering into crops. Here we report the successful targeting of bacterial Fe-nitrogenase proteins, AnfD, AnfK, AnfG and AnfH, to plant mitochondria. When expressed as a single protein AnfD was mostly insoluble in plant mitochondria, but coexpression of AnfD with AnfK greatly improved its solubility. Using affinity-based purification of mitochondrially expressed AnfK or AnfG we were able to demonstrate a strong interaction of AnfD with AnfK and a weaker interaction of AnfG with AnfDK. This work establishes that the structural components of the Fe-nitrogenase can be engineered into plant mitochondria and form a complex, which will be a requirement for function. This report outlines the first use of Fe-nitrogenase genes within a plant as a preliminary step towards engineering an alternative nitrogenase into crops.

New classes of astronomical objects are often discovered serendipitously. The enormous data volumes produced by recent high-time resolution, radio-telescope surveys imply that efficient algorithms are required for a discovery. Such algorithms are usually tuned to detect specific, known sources. Existing data sets therefore likely contain unknown astronomical sources, which will remain undetected unless algorithms are developed that can detect a more diverse range of signals. We present the Single-dish PARKES data challenge for finding the uneXpected (SPARKESX), a compilation of real and simulated high-time resolution observations. SPARKESX comprises three mock surveys from the Parkes "Murriyang" radio telescope. A broad selection of simulated and injected expected signals (such as pulsars, fast radio bursts), poorly characterised signals (plausible flare star signatures) and unknown unknowns are generated for each survey. The goal of this challenge is to aid in the development of new algorithms that can detect a wide-range of source types. We show how successful a typical pipeline based on the standard pulsar search software, PRESTO, is at finding the injected signals. The dataset is publicly available at https://doi.org/10.25919/fd4f-0g20., Accepted for publication in MNRAS. 18 pages, 8 figures, 4 table

Six cycles of recurrent selection for early shoot vigour in wheat resulted in significant increases in leaf width and shoot biomass. Here, in replicated controlled-environment studies, the effect of early shoot vigour on root biomass, rhizosheath size, root hair length, and cell size in the roots and leaves was examined across different cycles of selection. Increased shoot vigour was associated with greater root biomass, larger rhizosheath size, and longer root hairs. Our findings demonstrate that rhizosheath size was a reliable surrogate for root hair length in this germplasm. Examination of the root epidermis revealed that the ‘cell body’ of the trichoblasts (hair-forming cells) and the atrichoblasts (non-hair-forming cells) decreased in size as shoot vigour increased. Therefore, in higher vigour germplasm, longer root hairs emerged from smaller trichoblasts so that total trichoblast volume (root hair plus cell body) was generally similar regardless of shoot vigour. Similarly, the sizes of the four main cell types on the leaf epidermis became progressively smaller as shoot vigour increased, which also increased stomatal density. The relationship between shoot vigour and root traits is considered, and the potential contribution of below-ground root traits to performance and competitiveness of high vigour germplasm is discussed.

Plant oil production has been increasing continuously in the past decade. There has been significant investment in the production of high biomass plants with elevated oil content. We recently showed that the expression of Arabidopsis thaliana WRI1 and DGAT1 genes increase oil content by up to 15% in leaf dry weight tissue. However, triacylglycerols in leaf tissue are subject to degradation during senescence. In order to better package the oil, we expressed a series of lipid droplet proteins isolated from bacterial and plant sources in Nicotiana benthamiana leaf tissue. We observed further increases in leaf oil content of up to 2.3‐fold when we co‐expressed Sesamum indicum Oleosin L with AtWRI1 and AtDGAT1. Biochemical assays and lipid droplet visualization with confocal microscopy confirmed the increase in oil content and revealed a significant change in the size and abundance of lipid droplets.

Cocos nucifera (coconut), a member of the Arecaceae family, is an economically important woody palm that is widely grown in tropical and subtropical regions. The coconut palm is well known for its ability to accumulate large amounts of oil, approximately 63% of the seed weight. Coconut oil varies significantly from other vegetable oils as it contains a high proportion of medium-chain fatty acids (MCFA; 85%). The unique composition of coconut oil raises interest in understanding how the coconut palm produces oil of a high saturated MCFA content, and if such an oil profile could be replicated via biotechnology interventions. Although some gene discovery work has been performed there is still a significant gap in the knowledge associated with coconut’s oil production pathways. In this study, a de novo transcriptome was assembled for developing coconut endosperm to identify genes involved in the synthesis of lipids, particularly triacylglycerol. Of particular interest were thioesterases, acyltransferases and oleosins because of their involvement in the processes of releasing fatty acids for assembly, esterification of fatty acids into glycerolipids and protecting oils from degradation, respectively. It is hypothesized that some of these genes may exhibit a strong substrate preference for MCFA and hence may assist the future development of vegetable oils with an enriched MCFA composition. In this study, we identified and confirmed functionality of five candidate genes from the gene families of interest. This study will benefit future work in areas of increasing vegetable oil production and the tailoring of oil fatty acid compositions.

Summary Synthesis and accumulation of the storage lipid triacylglycerol in vegetative plant tissues has emerged as a promising strategy to meet the world's future need for vegetable oil. Sorghum (Sorghum bicolor) is a particularly attractive target crop given its high biomass, drought resistance and C4 photosynthesis. While oilseed‐like triacylglycerol levels have been engineered in the C3 model plant tobacco, progress in C4 monocot crops has been lagging behind. In this study, we report the accumulation of triacylglycerol in sorghum leaf tissues to levels between 3 and 8.4% on a dry weight basis depending on leaf and plant developmental stage. This was achieved by the combined overexpression of genes encoding the Zea mays WRI1 transcription factor, Umbelopsis ramanniana UrDGAT2a acyltransferase and Sesamum indicum Oleosin‐L oil body protein. Increased oil content was visible as lipid droplets, primarily in the leaf mesophyll cells. A comparison between a constitutive and mesophyll‐specific promoter driving WRI1 expression revealed distinct changes in the overall leaf lipidome as well as transitory starch and soluble sugar levels. Metabolome profiling uncovered changes in the abundance of various amino acids and dicarboxylic acids. The results presented here are a first step forward towards the development of sorghum as a dedicated biomass oil crop and provide a basis for further combinatorial metabolic engineering.

Membrane proteins that are imported into chloroplasts must be accurately routed in order to establish and maintain the highly differentiated membranes characteristic of these organelles. Little is known about the targeting information or pathways involved, especially in the case of proteins with multiple transmembrane domains. We have studied targeting of the SCY components of the two SEC translocases in chloroplasts. SCY1 and SCY2 share a similar, highly conserved structure with 10 transmembrane domains, but are targeted to different membranes: the thylakoids and inner envelope, respectively. We used protoplast transfections and a confocal microscopy imaging assay in combination with a domain-swapping approach to investigate sorting pathways and identify important targeting elements in these proteins. We show that the N-terminal region of SCY1 contains targeting determinants that allow SCY1 to be recruited to the signal-recognition particle pathway. In addition, substituting the N-terminal region of SCY1 for the N-terminal region of SCY2 causes SCY2 to be displaced out of the inner envelope. The region of SCY2 that contains transmembrane domains 3 and 4 is necessary for localization to the inner envelope and may serve as a membrane anchor, enhancing the integration of other transmembrane domains via either stop-transfer or post-import mechanisms.

Reef-building corals form symbiotic relationships with dinoflagellates of the genus Symbiodinium. Symbiodinium are genetically and physiologically diverse, and corals may be able to adapt to different environments by altering their dominant Symbiodinium phylotype. Notably, each coral species associates only with specific Symbiodinium phylotypes, and consequently the diversity of symbionts available to the host is limited by the species specificity. Currently, it is widely presumed that species specificity is determined by the combination of cell-surface molecules on the host and symbiont. Here we show experimental evidence supporting a new model to explain at least part of the specificity in coral–Symbiodinium symbiosis. Using the laboratory model Aiptasia–Symbiodinium system, we found that symbiont infectivity is related to cell size; larger Symbiodinium phylotypes are less likely to establish a symbiotic relationship with the host Aiptasia. This size dependency is further supported by experiments where symbionts were replaced by artificial fluorescent microspheres. Finally, experiments using two different coral species demonstrate that our size-dependent-infection model can be expanded to coral–Symbiodinium symbiosis, with the acceptability of large-sized Symbiodinium phylotypes differing between two coral species. Thus the selectivity of the host for symbiont cell size can affect the diversity of symbionts in corals.

Neurachne is the only known grass lineage containing closely related C3, C3-C4 intermediate, and C4 species, making it an ideal taxon with which to study the evolution of C4 photosynthesis in the grasses. To begin dissecting the molecular changes that led to the evolution of C4 photosynthesis in this group, the complementary DNAs encoding four distinct β-carbonic anhydrase (CA) isoforms were characterized from leaf tissue of Neurachne munroi (C4), Neurachne minor (C3-C4), and Neurachne alopecuroidea (C3). Two genes (CA1 and CA2) each encode two different isoforms: CA1a/CA1b and CA2a/CA2b. Transcript analyses found that CA1 messenger RNAs were significantly more abundant than transcripts from the CA2 gene in the leaves of each species examined, constituting ∼99% of all β-CA transcripts measured. Localization experiments using green fluorescent protein fusion constructs showed that, while CA1b is a cytosolic CA in all three species, the CA1a proteins are differentially localized. The N. alopecuroidea and N. minor CA1a isoforms were imported into chloroplasts of Nicotiana benthamiana leaf cells, whereas N. munroi CA1a localized to the cytosol. Sequence analysis indicated an 11-amino acid deletion in the amino terminus of N. munroi CA1a relative to the C3 and C3-C4 proteins, suggesting that chloroplast targeting of CA1a is the ancestral state and that loss of a functional chloroplast transit peptide in N. munroi CA1a is associated with the evolution of C4 photosynthesis in Neurachne spp. Remarkably, this mechanism is homoplastic with the evolution of the C4-associated CA in the dicotyledonous genus Flaveria, although the actual mutations in the two lineages differ.

Flaveria bidentis carbonic anhydrase 3 catalyses the first step in C4 photosynthesis, with its cognate gene containing an element that shares homology and function with the C4Flaveria MEM1 motif., The first two reactions of C4 photosynthesis are catalysed by carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPC) in the leaf mesophyll (M) cell cytosol. Translatome experiments using a tagged ribosomal protein expressed under the control of M and bundle-sheath (BS) cell-specific promoters showed transcripts encoding CA3 from the C4 species Flaveria bidentis were highly enriched in polysomes from M cells relative to those of the BS. Localisation experiments employing a CA3-green fluorescent protein fusion protein showed F. bidentis CA3 is a cytosolic enzyme. A motif showing high sequence homology to that of the Flaveria M expression module 1 (MEM1) element was identified approximately 2 kb upstream of the F. bidentis and F. trinervia ca3 translation start sites. MEM1 is located in the promoter of C4Flaveria ppcA genes, which encode the C4-associated PEPC, and is necessary for M-specific expression. No MEM1-like sequence was found in the 4 kb upstream of the C3 species F. pringlei ca3 translation start site. Promoter–reporter fusion experiments demonstrated the region containing the ca3 MEM1-like element also directs M-specific expression. These results support the idea that a common regulatory switch drives the expression of the C4Flaveria ca3 and ppcA1 genes specifically in M cells.

Global population growth is projected to outpace plant-breeding improvements in major crop yields within decades. To ensure future food security, multiple creative efforts seek to overcome limitations to crop yield. Perhaps the greatest limitation to increased crop yield is photosynthetic inefficiency, particularly in C3 crop plants. Recently, great strides have been made toward crop improvement by researchers seeking to introduce the cyanobacterial CO2-concentrating mechanism (CCM) into plant chloroplasts. This strategy recognises the C3 chloroplast as lacking a CCM, and being a primordial cyanobacterium at its essence. Hence the collection of solute transporters, enzymes, and physical structures that make cyanobacterial CO2-fixation so efficient are viewed as a natural source of genetic material for C3 chloroplast improvement. Also we highlight recent outstanding research aimed toward the goal of introducing a cyanobacterial CCM into C3 chloroplasts and consider future research directions.

In plants, stable expression is arguably the method of choice to test transgene function but it is a slow and labor-intensive process. This bottleneck generally limits the number of transgenes that can be tested, and as such hinders construct optimization. In the face of this challenge, transient expression in Nicotiana benthamiana leaves has emerged as a powerful screening platform to test gene expression, as well as subcellular distribution and function of many proteins within a week. This system relies on the infiltration of Agrobacterium tumefaciens (Agrobacterium) carrying DNA of interest into the leaf air spaces of N. benthamiana plants. Agrobacterium rapidly transforms the plant cells and the leaves can be analyzed within a few days. Investigating the subcellular localization of a protein of interest often relies on its fusion to a fluorescent tag. While the amount of accumulation of such fusion proteins can often be gauged by observing the fluorescence of the tag at the whole-leaf level, subcellular protein distribution is best determined in protoplasts extracted from transformed leaves. Here I present a simple and effective method to transform N. benthamiana leaves with Agrobacterium and to prepare protoplasts from these leaves to characterize the subcellular localization of proteins of interest.

In plants, stable expression is arguably the method of choice to test transgene function but it is a slow and labor-intensive process. This bottleneck generally limits the number of transgenes that can be tested, and as such hinders construct optimization. In the face of this challenge, transient expression in Nicotiana benthamiana leaves has emerged as a powerful screening platform to test gene expression, as well as subcellular distribution and function of many proteins within a week. This system relies on the infiltration of Agrobacterium tumefaciens (Agrobacterium) carrying DNA of interest into the leaf air spaces of N. benthamiana plants. Agrobacterium rapidly transforms the plant cells and the leaves can be analyzed within a few days. Investigating the subcellular localization of a protein of interest often relies on its fusion to a fluorescent tag. While the amount of accumulation of such fusion proteins can often be gauged by observing the fluorescence of the tag at the whole-leaf level, subcellular protein distribution is best determined in protoplasts extracted from transformed leaves. Here I present a simple and effective method to transform N. benthamiana leaves with Agrobacterium and to prepare protoplasts from these leaves to characterize the subcellular localization of proteins of interest.

Lipid droplets (LDs) are composed of a monolayer of phospholipids (PLs), surrounding a core of non-polar lipids that consist mostly of triacylglycerols (TAGs) and to a lesser extent diacylglycerols. In this study, lipidome analysis illustrated striking differences in non-polar lipids and PL species between LDs derived from Triadica sebifera seed kernels and mesocarp. In mesocarp LDs, the most abundant species of TAG contained one C18:1 and two C16:0 and fatty acids, while TAGs containing three C18 fatty acids with higher level of unsaturation were dominant in the seed kernel LDs. This reflects the distinct differences in fatty acid composition of mesocarp (palmitate-rich) and seed-derived oil (α-linoleneate-rich) in T. sebifera. Major PLs in seed LDs were found to be rich in polyunsaturated fatty acids, in contrast to those with relatively shorter carbon chain and lower level of unsaturation in mesocarp LDs. The LD proteome analysis in T. sebifera identified 207 proteins from mesocarp, and 54 proteins from seed kernel, which belong to various functional classes including lipid metabolism, transcription and translation, trafficking and transport, cytoskeleton, chaperones, and signal transduction. Oleosin and lipid droplets associated proteins (LDAP) were found to be the predominant proteins associated with LDs in seed and mesocarp tissues, respectively. We also show that LDs appear to be in close proximity to, a number of organelles including the endoplasmic reticulum, mitochondria, peroxisomes and Golgi apparatus. This comparative study between seed and mesocarp LDs may bridge some of the fundamental gaps in knowledge andshed some light on develop an integrated picturethe structure of plant LDs by significantly addressing the limited and improve our understanding of their structure, their functionality and cellular metabolic networks in oleaginous plant tissues.

Photorespiration is an energy-intensive process that recycles 2-phosphoglycolate, a toxic product of the Rubisco oxygenation reaction. The photorespiratory pathway is highly compartmentalized, involving the chloroplast, peroxisome, cytosol, and mitochondria. Though the soluble enzymes involved in photorespiration are well characterized, very few membrane transporters involved in photorespiration have been identified to date. In this work, Arabidopsis thaliana plants containing a T-DNA disruption of the bile acid sodium symporter BASS6 show decreased photosynthesis and slower growth under ambient, but not elevated CO2. Exogenous expression of BASS6 complemented this photorespiration mutant phenotype. In addition, metabolite analysis and genetic complementation of glycolate transport in yeast showed that BASS6 was capable of glycolate transport. This is consistent with its involvement in the photorespiratory export of glycolate from Arabidopsis chloroplasts. An Arabidopsis double knockout line of both BASS6 and the glycolate/glycerate transporter PLGG1 (bass6, plgg1) showed an additive growth defect, an increase in glycolate accumulation, and reductions in photosynthetic rates compared with either single mutant. Our data indicate that BASS6 and PLGG1 partner in glycolate export from the chloroplast, whereas PLGG1 alone accounts for the import of glycerate. BASS6 and PLGG1 therefore balance the export of two glycolate molecules with the import of one glycerate molecule during photorespiration.

Stem cells need to balance self-renewal and differentiation for correct tissue development and homeostasis. Defects in this balance can lead to developmental defects or tumor formation. In recent years, mRNA splicing has emerged as an important mechanism regulating cell fate decisions. Here we address the role of the evolutionarily conserved splicing co-factor Barricade (Barc)/Tat-SF1/CUS2 in

The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.

Stem cells need to balance self-renewal and differentiation for correct tissue development and homeostasis. Defects in this balance can lead to developmental defects or tumor formation. In recent years, mRNA splicing has emerged as an important mechanism regulating cell fate decisions. Here we address the role of the evolutionarily conserved splicing co-factor Barricade (Barc)/Tat-SF1/CUS2 in Drosophila neural stem cell (neuroblast) lineage formation. We show that Barc is required for the generation of neurons during Drosophila brain development by ensuring correct neural progenitor proliferation and differentiation. Barc associates with components of the U2 small nuclear ribonucleoprotein (snRNP) complex, and its depletion causes alternative splicing in the form of intron retention in a subset of genes. Using bioinformatics analysis and a cell culture-based splicing assay, we found that Barc-dependent introns share three major traits: they are short, GC rich and have weak 3′ splice sites. Our results show that Barc, together with the U2 snRNP complex, plays an important role in regulating neural stem cell lineage progression during brain development and facilitates correct splicing of a subset of introns.

In Neurachne, the loss of the sequence encoding a functional chloroplast transit peptide from an ancestral C3 carbonic anhydrase is associated with the evolution of C4 photosynthesis.

Correct subcellular targeting is crucial for protein function. Protein location can be visualized in vivo by fusion to a fluorescent protein (FP). Nevertheless, despite intense engineering efforts, most FPs are dim or completely quenched at low pH (, Short Abstract In vivo comparison of fluorescent proteins with various degrees of in vitro sensitivity to low pH identifies tags suitable for tracking proteins in cell walls under dynamic conditions.

Protoxylem plays an important role in the hydraulic function of vascular systems of both herbaceous and woody plants, but relatively little is known about the processes underlying the maintenance of protoxylem function in long-lived tissues. In this study, embolism repair was investigated in relation to xylem structure in two cushion plant species, Azorella macquariensis and Colobanthus muscoides, in which vascular water transport depends on protoxylem. Their protoxylem vessels consisted of a primary wall with helical thickenings that effectively formed a pit channel, with the primary wall being the pit channel membrane. Stem protoxylem was organized such that the pit channel membranes connected vessels with paratracheal parenchyma or other protoxylem vessels and were not exposed directly to air spaces. Embolism was experimentally induced in excised vascular tissue and detached shoots by exposing them briefly to air. When water was resupplied, embolized vessels refilled within tens of seconds (excised tissue) to a few minutes (detached shoots) with water sourced from either adjacent parenchyma or water-filled vessels. Refilling occurred in two phases: (1) water refilled xylem pit channels, simplifying bubble shape to a rod with two menisci; and (2) the bubble contracted as the resorption front advanced, dissolving air along the way. Physical properties of the protoxylem vessels (namely pit channel membrane porosity, hydrophilic walls, vessel dimensions, and helical thickenings) promoted rapid refilling of embolized conduits independent of root pressure. These results have implications for the maintenance of vascular function in both herbaceous and woody species, because protoxylem plays a major role in the hydraulic systems of leaves, elongating stems, and roots.

An RFX-binding site is shown to be conserved in the promoters of a subset of ciliary genes and a subsequent screen for this site in two Drosophila species identified novel RFX target genes that are involved in sensory ciliogenesis., Background Regulatory factor X (RFX) transcription factors play a key role in ciliary assembly in nematode, Drosophila and mouse. Using the tremendous advantages of comparative genomics in closely related species, we identified novel genes regulated by dRFX in Drosophila. Results We first demonstrate that a subset of known ciliary genes in Caenorhabditis elegans and Drosophila are regulated by dRFX and have a conserved RFX binding site (X-box) in their promoters in two highly divergent Drosophila species. We then designed an X-box consensus sequence and carried out a genome wide computer screen to identify novel genes under RFX control. We found 412 genes that share a conserved X-box upstream of the ATG in both species, with 83 genes presenting a more restricted consensus. We analyzed 25 of these 83 genes, 16 of which are indeed RFX target genes. Two of them have never been described as involved in ciliogenesis. In addition, reporter construct expression analysis revealed that three of the identified genes encode proteins specifically localized in ciliated endings of Drosophila sensory neurons. Conclusion Our X-box search strategy led to the identification of novel RFX target genes in Drosophila that are involved in sensory ciliogenesis. We also established a highly valuable Drosophila cilia and basal body dataset. These results demonstrate the accuracy of the X-box screen and will be useful for the identification of candidate genes for human ciliopathies, as several human homologs of RFX target genes are known to be involved in diseases, such as Bardet-Biedl syndrome.

SummaryIn both vertebrates and insects, neurons typically arise from neural stem cells or terminally dividing intermediate progenitors. Here, we describe another mode of neurogenesis where neural stem cells generate secondary precursors that undergo multiple rounds of self-renewing transit-amplifying divisions. We identify the Posterior Asense-Negative (PAN) neuroblasts, which do not express the transcription factors Asense or Prospero. PAN neuroblasts rely on the segregating determinants Numb and Brat to generate smaller, secondary neuroblasts that in turn give rise to ganglion mother cells (GMCs) and neurons throughout larval development. In brat or numb mutants, misspecified secondary neuroblasts are unable to produce differentiated progeny and initiate tumor-like overgrowth. In prospero mutants, however, tumors arise from GMCs while secondary neuroblasts are correctly specified. Our data describe a transit-amplifying lineage in the Drosophila nervous system and suggest that different vulnerabilities in intermediate cell types can affect the outcome of tumor suppressor loss in stem cell lineages.