A glial cell arises from an additional division within the mechanosensory lineage during development of the microchaete on the Drosophila notum.

We have used different cell markers to trace the development of the sensory cells of the thoracic microchaete. Our results dictate a revision in the currently accepted model for cell lineage within the mechanosensory bristle. The sensory organ progenitor divides to form two secondary progenitors: PIIa and PIIb. PIIb divides first to give rise to a tertiary progenitor-PIII and a glial cell. This is followed by division of PIIa to form the shaft and socket cells as described before. PIII expresses high levels of Elav and low levels of Prospero and divides to produce neuron and sheath. Its sibling cell expresses low Elav and high Prospero and is recognized by the glial marker, Repo. This cell migrates away from the other cells of the lineage following differentiation. The proposed modification in lineage has important implications for previous studies on sibling cell fate choice and cell fate specification in sensory systems.     

Combinatorial expression of Prospero,Seven-up,and Elav identifies progenitor cell types during sense-organ differentiation in the Drosophila antenna

The Drosophila antenna has a diversity of chemosensory organs within a single epidermal field. We have some idea from recent studies of how the three broad categories of sense-organs are specified at the level of progenitor choice. However, little is known about how cell fates within single sense-organs are specified. Selection of individual primary olfactory progenitors is followed by organization of groups of secondary progenitors, which divide in a specific order to form a differentiated sensillum. The combinatorial expression of Prospero Elav, and Seven-up allows us to distinguish three secondary progenitor fates. The lineages of these cells have been established by clonal analysis and marker distribution following mitosis. High Notch signaling and the exclusion of these markers identifies PIIa; this cell gives rise to the shaft and socket. The sheath/neuron lineage progenitor PIIb, expresses all three markers; upon division, Prospero asymmetrically segregates to the sheath cell. In the coeloconica, PIIb undergoes an additional division to produce glia. PIIc is present in multiinnervated sense-organs and divides to form neurons. An understanding of the lineage and development of olfactory sense-organs provides a handle for the analysis of how olfactory neurons acquire distinct terminal fates.     

RanGAP‐mediated nucleocytoplasmic transport of Prospero regulates neural stem cell lifespan in Drosophila larval central brain

By the end of neurogenesis in Drosophila pupal brain neuroblasts (NBs), nuclear Prospero (Pros) triggers cell cycle exit and terminates NB lifespan. Here, we reveal that in larval brain NBs, an intrinsic mechanism facilitates import and export of Pros across the nuclear envelope via a Ran‐mediated nucleocytoplasmic transport system. In rangap mutants, the export of Pros from the nucleus to cytoplasm is impaired and the nucleocytoplasmic transport of Pros becomes one‐way traffic, causing an early accumulation of Pros in the nuclei of the larval central brain NBs. This nuclear Pros retention initiates NB cell cycle exit and leads to a premature decrease of total NB numbers. Our data indicate that RanGAP plays a crucial role in this intrinsic mechanism that controls NB lifespan during neurogenesis. Our study may provide insights into understanding the lifespan of neural stem cells during neurogenesis in other organisms.     

Isolation, purification, and properties of Penicillium charlesii alkaline protease.

A serine protease with a pH optimum from 7 to 9 and activity over the range of pH 3 to 10 was isolated and purified from culture filtrates of Penicillium charlesii 16 days after inoculation. The enzyme was purified by the following sequence of procedures: (i) gel permeation chromatography through Sephacryl S-200, (ii) DEAE-Sepharose anion-exchange chromatography, and (iii) fast protein liquid chromatography (FPLC) over Superose 12. Anion-exchange chromatography separated the protease activity into a major activity (protease PII, 82%) and two minor activities (proteases PI and PIII, 10 and 8%, respectively, of the total activity). Protease PII has a molecular mass of 44 kilodaltons. Purified preparations of this enzyme are susceptible to autodegradation. FPLC of heat-treated PII gave one major species (PIIa), whereas untreated enzyme resulted in three species (PIIb, PIIc, and PIId). PIIb and PIIc also catalyzed the hydrolysis of protein (hide powder azure). PIIb and PIIc were in the molecular mass range of 10 to 20 kilodaltons. Protease PII is completely inhibited by phenylmethylsulfonyl fluoride (PMSF). The protease has primary substrate specificity for phenylalanyl or arginyl amino acyl residues attached to amines. The enzyme has amidase, but no esterase activity toward similar synthetic substrates such as occurs with trypsinlike microbial serine proteases. The addition of PMSF (final concentration, 10(-4) M) to 1- and 2-day-old cultures of P. charlesii inhibited the production of extracellular peptidophosphogalactomannan (pPGM) by 41 and 34%, respectively, and inhibited the alkaline protease activity by 85%. These results suggest that the production and release of pPGM may be affected by alkaline protease.     

Drosophila homologs of mammalian TNF/TNFR-related molecules regulate segregation of Miranda/Prospero in neuroblasts

During neuroblast (NB) divisions, cell fate determinants Prospero (Pros) and Numb, together with their adaptor proteins Miranda (Mira) and Partner of Numb, localize to the basal cell cortex at metaphase and segregate exclusively to the future ganglion mother cells (GMCs) at telophase. In inscuteable mutant NBs, these basal proteins are mislocalized during metaphase. However, during anaphase/telophase, these mutant NBs can partially correct these earlier localization defects and redistribute cell fate determinants as crescents to the region where the future GMC "buds" off. This compensatory mechanism has been referred to as "telophase rescue". We demonstrate that the Drosophila homolog of the mammalian tumor-necrosis factor (TNF) receptor-associated factor (DTRAF1) and Eiger (Egr), the homolog of the mammalian TNF, are required for telophase rescue of Mira/Pros. DTRAF1 localizes as an apical crescent in metaphase NBs and this apical localization requires Bazooka (Baz) and Egr. The Mira/Pros telophase rescue seen in inscuteable mutant NBs requires DTRAF1. Our data suggest that DTRAF1 binds to Baz and acts downstream of Egr in the Mira/Pros telophase rescue pathway.     

Wasp, the Drosophila Wiskott-Aldrich syndrome gene homologue, is required for cell fate decisions mediated by Notch signaling

Wiskott-Aldrich syndrome proteins, encoded by the Wiskott-Aldrich syndrome gene family, bridge signal transduction pathways and the microfilament-based cytoskeleton. Mutations in the Drosophila homologue, Wasp (Wsp), reveal an essential requirement for this gene in implementation of cell fate decisions during adult and embryonic sensory organ development. Phenotypic analysis of Wsp mutant animals demonstrates a bias towards neuronal differentiation, at the expense of other cell types, resulting from improper execution of the program of asymmetric cell divisions which underlie sensory organ development. Generation of two similar daughter cells after division of the sensory organ precursor cell constitutes a prominent defect in the Wsp sensory organ lineage. The asymmetric segregation of key elements such as Numb is unaffected during this division, despite the misassignment of cell fates. The requirement for Wsp extends to additional cell fate decisions in lineages of the embryonic central nervous system and mesoderm. The nature of the Wsp mutant phenotypes, coupled with genetic interaction studies, identifies an essential role for Wsp in lineage decisions mediated by the Notch signaling pathway.     

In vivo birthdating by BAPTISM reveals that trigeminal sensory neuron diversity depends on early neurogenesis

Among sensory systems, the somatic sense is exceptional in its ability to detect a wide range of chemical, mechanical and thermal stimuli. How this sensory diversity is established during development remains largely elusive. We devised a method (BAPTISM) that uses the photoconvertible fluorescent protein Kaede to simultaneously analyze birthdate and cell fate in live zebrafish embryos. We found that trigeminal sensory ganglia are formed from early-born and late-born neurons. Early-born neurons give rise to multiple classes of sensory neurons that express different ion channels. By contrast, late-born neurons are restricted in their fate and do not form chemosensory neurons expressing the ion channel TrpA1b. Accordingly, larvae lacking early-born neurons do not respond to the TrpA1b agonist allyl isothiocyanate. These results indicate that the multimodal specification and function of trigeminal sensory ganglia depends on the timing of neurogenesis.     

Origins of inner ear sensory organs revealed by fate map and time-lapse analyses

The inner ear develops from a simple ectodermal thickening called the otic placode into a labyrinth of chambers which house sensory organs that sense sound and are used to maintain balance. Although the morphology and function of the sensory organs are well characterized, their origins and lineage relationships are virtually unknown. In this study, we generated a fate map of Xenopus laevis inner ear at otic placode and otocyst stages to determine the developmental origins of the sensory organs. Our lineage analysis shows that all regions of the otic placode and otocyst can give rise to the sensory organs of the inner ear, though there were differences between labeled quadrants in the range of derivatives formed. A given region often gives rise to cells in multiple sensory organs, including cells that apparently dispersed from anterior to posterior poles and vice versa. These results suggest that a single sensory organ arises from cells in different parts of the placode or otocyst and that cell mixing plays a large role in ear development. Time-lapse videomicroscopy provides further evidence that cells from opposite regions of the inner ear mix during the development of the inner ear, and this mixing begins at placode stages. Lastly, bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta (TGF-beta) family, is expressed in all sensory organs of the frog inner ear, as it is in the developing chicken ear. Inner ear fate maps provide a context for interpreting gene expression patterns and embryological manipulations.     

Sensillum development in the absence of cell division: the sensillum phenotype of the Drosophila mutant string

We have investigated sensillum development in Drosophila embryos homozygous for mutations in the locus string (stg). In these embryos, cell division is blocked following blastoderm formation. This permits a study of the differentiative fate of undivided precursor cells, in particular those giving rise to the larval sensory organs (sensilla). Of the different cell fates normally represented in the sensilla (i.e., sensory neuron, thecogen cell, trichogen cell, tormogen cell, glia cell), only the phenotype of sensory neurons is expressed morphologically in stg embryos, suggesting that the neuronal fate predominates over the fates of the nonneuronal accessory cells. Consistent with this finding, the P element-lacZ insertion A1-2nd-29, which is a marker for trichogen and tormogen cells in the wild-type embryo, is not expressed in the body wall of the stg embryo. Some sensillum precursor cells appear to express a mixed fate in stg mutants: They express antigens (recognized by the monoclonal antibodies 22C10 and 21A6) which in the wild-type appear in separate cells (sensory neurons and thecogen cell, respectively). The differentiation of undivided cells in stg embryos is not restricted to the peripheral nervous system; in all types of tissues analyzed in this study (e.g., epidermis, intestine, muscle, CNS), precursor cells express characteristics normally exhibited by their progeny.     

The metalloproteinase inhibitor Reck is essential for zebrafish DRG development

The neural crest is a migratory, multipotent cell lineage that contributes to myriad tissues, including sensory neurons and glia of the dorsal root ganglia (DRG). To identify genes affecting cell fate specification in neural crest, we performed a forward genetic screen for mutations causing DRG deficiencies in zebrafish. This screen yielded a mutant lacking all DRG, which we named sensory deprived (sdp). We identified a total of four alleles of sdp, all of which possess lesions in the gene coding for reversion-inducing cysteine-rich protein containing Kazal motifs (Reck). Reck is an inhibitor of metalloproteinases previously shown to regulate cell motility. We found reck function to be both necessary for DRG formation and sufficient to rescue the sdp phenotype. reck is expressed in neural crest cells and is required in a cell-autonomous fashion for appropriate sensory neuron formation. In the absence of reck function, sensory neuron precursors fail to migrate to the position of the DRG, suggesting that this molecule is crucial for proper migration and differentiation.     

Intrinsic regulation of enteroendocrine fate by Numb

How terminal cell fates are specified in dynamically renewing adult tissues is not well understood. Here we explore terminal cell fate establishment during homeostasis using the enteroendocrine cells (EEs) of the adult Drosophila midgut as a paradigm. Our data argue against the existence of local feedback signals, and we identify Numb as an intrinsic regulator of EE fate. Our data further indicate that Numb, with alpha‐adaptin, acts upstream or in parallel of known regulators of EE fate to limit Notch signaling, thereby facilitating EE fate acquisition. We find that Numb is regulated in part through its asymmetric and symmetric distribution during stem cell divisions; however, its de novo synthesis is also required during the differentiation of the EE cell. Thus, this work identifies Numb as a crucial factor for cell fate choice in the adult Drosophila intestine. Furthermore, our findings demonstrate that cell‐intrinsic control mechanisms of terminal cell fate acquisition can result in a balanced tissue‐wide production of terminally differentiated cell types.     

Neural Crest Cell Fate

The neural crest, the intriguing cell population that gives rise to a panoply of derivatives in the vertebrate embryo, including the mesenchymal structures in the head, melanocytes and most of the peripheral nervous system, still proves to be an important yet enigmatic developmental cell population to study with applications in stem cell biology, cancer biology and clinical medicine. Albeit our knowledge base is rich due to a strong history of experimentation, the fact that we have yet to decipher so many key aspects of neural crest cell (NCC) behavior speaks to the challenging complexity of this transient yet vital cell population. With the advent of new fluorescent tracing techniques, we have reexamined the migratory behaviors and ultimate fate of ventrally migrating avian NCCs within a late wave of emigration and identified a subpopulation of lineally restricted NCCs who migrate to the contralateral dorsal root ganglia (DRG) and therein give rise to mitotically active progenitor cells that ultimately produce the majority of the nociceptive sensory neurons in the DRG. These data provide evidence for the fate prespecification of subsets of NCCs while still resident in the neural tube.     

Sprouty2, a mouse deafness gene, regulates cell fate decisions in the auditory sensory epithelium by antagonizing FGF signaling

The auditory sensory epithelium (organ of Corti), where sound waves are converted to electrical signals, comprises a highly ordered array of sensory receptor (hair) cells and nonsensory supporting cells. Here, we report that Sprouty2, which encodes a negative regulator of signaling via receptor tyrosine kinases, is required for normal hearing in mice, and that lack of SPRY2 results in dramatic perturbations in organ of Corti cytoarchitecture: instead of two pillar cells, there are three, resulting in the formation of an ectopic tunnel of Corti. We demonstrate that these effects are due to a postnatal cell fate transformation of a Deiters' cell into a pillar cell. Both this cell fate change and hearing loss can be partially rescued by reducing Fgf8 gene dosage in Spry2 null mutant mice. Our results provide evidence that antagonism of FGF signaling by SPRY2 is essential for establishing the cytoarchitecture of the organ of Corti and for hearing.     

Canonical Wnt signaling is required for ophthalmic trigeminal placode cell fate determination and maintenance

Cranial placodes are ectodermal regions that contribute extensively to the vertebrate peripheral sensory nervous system. The development of the ophthalmic trigeminal (opV) placode, which gives rise only to sensory neurons of the ophthalmic lobe of the trigeminal ganglion, is a useful model of sensory neuron development. While key differentiation processes have been characterized at the tissue and cellular levels, the signaling pathways governing opV placode development have not. Here we tested in chick whether the canonical Wnt signaling pathway regulates opV placode development. By introducing a Wnt reporter into embryonic chick head ectoderm, we show that the canonical pathway is active in Pax3+ opV placode cells as, or shortly after, they are induced to express Pax3. Blocking the canonical Wnt pathway resulted in the failure of targeted cells to adopt or maintain an opV fate, as assayed by the expression of various markers including Pax3, FGFR4, Eya2, and the neuronal differentiation markers Islet1, neurofilament, and NeuN, although, surprisingly, it led to upregulation of Neurogenin2, both in the opV placode and elsewhere in the ectoderm. Activating the canonical Wnt signaling pathway, however, was not sufficient to induce Pax3, the earliest specific marker of the opV placode. We conclude that canonical Wnt signaling is necessary for normal opV placode development, and propose that other molecular cues are required in addition to Wnt signaling to promote cells toward an opV placode fate.     

Alcohol impairs astrogliogenesis by stem cells in rodent neurospheres

Neurosphere cultures derived from fetal brain regions can proliferate in response to exogenous growth factors such as basic fibroblast growth factor (bFGF) and give rise to undifferentiated precursor cells that form a floating neurosphere. In this study, neurospheres generated from the ganglionic eminence region of embryonic day 15 (E15) rat embryos were treated in the presence or absence of ethanol. We found that such neurospheres respond to environmental toxins such as alcohol and still retain the multi-potential capability of differentiation into neuronal and glial cell types. Ethanol at high concentration (50 mM) affected proliferation, gliogenesis and neurogenesis, although the most profound effect was observed on glial phenotype. Our findings suggest that extrinsic agents, such as alcohol can alter intrinsic cellular mechanisms of stem cell fate choices contributing to altered neurogenesis and gliogenesis during central nervous system (CNS) maturation, which might in part be responsible for defective astroglial and neuronal functions in fetal alcohol syndrome (FAS).