Neurogenesis of the peripheral nervous system in Drosophila embryos: DNA replication patterns and cell lineages

Cell lineages that give rise to the PNS were studied using the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) to visualized DNA replication immunocytochemically. The precursors of the PNS in the body segments of Drosophila embryos replicate their DNA in a spatially and temporally stereotyped pattern. The sequence of DNA replication within developing sensory organs suggests particular lineage relationships of the cells that constitute a sensory organ, i.e., neuron and associated support cells. In embryos that are mutant for the achaete-scute complex or daughterless, in which most or all of the PNS is missing, no BrdU-labeled cells were found in the appropriate regions, suggesting that these PNS precursors either do not form or fail to replicate. Thus, the BrdU technique allows determination as to whether a mutation affects the PNS precursors or terminal differentiation.     

Molecular markers for identified neuroblasts and ganglion mother cells in the Drosophila central nervous system.

The first step in generating cellular diversity in the Drosophila central nervous system is the formation of a segmentally reiterated array of neural precursor cells, called neuroblasts. Subsequently, each neuroblast goes through an invariant cell lineage to generate neurons and/or glia. Using molecular lineage markers, I show that (1) each neuroblast forms at a stereotyped time and position; (2) the neuroblast pattern is indistinguishable between thoracic and abdominal segments; (3) the development of individual neuroblasts can be followed throughout early neurogenesis; (4) gene expression in a neuroblast can be reproducibly modulated during its cell lineage; (5) identified ganglion mother cells form at stereotyped times and positions; and (6) the cell lineage of four well-characterized neurons can be traced back to two identified neuroblasts. These results set the stage for investigating neuroblast specification and the mechanisms controlling neuroblast cell lineages.     

Spatial and temporal segregation of auditory and vestibular neurons in the otic placode

The otic placode generates the auditory and vestibular sense organs and their afferent neurons; however, how auditory and vestibular fates are specified is unknown. We have generated a fate map of the otic placode and show that precursors for vestibular and auditory cells are regionally segregated in the otic epithelium. The anterior-lateral portion of the otic placode generates vestibular neurons, whereas the posterior-medial region gives rise to auditory neurons. Precursors for vestibular and auditory sense organs show the same distribution. Thus, different regions of the otic placode correspond to particular sense organs and their innervating neurons. Neurons from contiguous domains rarely intermingle suggesting that the regional organisation of the otic placode dictates positional cues to otic neurons. But, in addition, vestibular and cochlear neurogenesis also follows a stereotyped temporal pattern. Precursors from the anterior-lateral otic placode delaminate earlier than those from its medial-posterior portion. The expression of the proneural genes NeuroM and NeuroD reflects the sequence of neuroblast formation and differentiation. Both genes are transiently expressed in vestibular and then in cochlear neuroblasts, while differentiated neurons express Islet1, Tuj1 and TrkC, but not NeuroM or NeuroD. Together, our results indicate that the position of precursors within the otic placode confers identity to sensory organs and to the corresponding otic neurons. In addition, positional information is integrated with temporal cues that coordinate neurogenesis and sensory differentiation.     

Homeotic genes have specific functional roles in the establishment of the Drosophila embryonic peripheral nervous system.

The Drosophila embryonic peripheral nervous system (PNS) contains segment-specific spatial patterns of sensory organs which derive from the ectoderm. Many studies have established that the homeotic genes of Drosophila control segment specific characteristics of the epidermis, and more recently these genes have also been shown to control gut morphogenesis through their expression in the visceral mesoderm (Tremml, G. and Bienz, M. (1989), EMBO J. 8, 2677-2685). We report here the roles of homeotic genes in establishing the spatial patterns of sensory organs in the embryonic PNS. The PNS was examined in embryos homozygous for mutations in the homeotic genes Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B) with antibodies that label specific subsets of sensory organs. Our results suggest that the homeotic genes have specific roles in establishing the correct spatial patterns of sensory organs in their normal domains of expression. In addition, we also report the effects of ectopic expression of the homeotic genes labial (lab), Deformed (Dfd), Scr, Antp or Ubx on the normal development of sensory organs in the embryonic PNS. Interestingly, while previous studies have concluded that ectopic expression of the homeotic genes Dfd, Scr and Antp has no effect on the segmental identity of the abdominal segments, our results demonstrate that this is not true. We show that ectopic expression of these genes does result in the disruption of the developing PNS in the abdomen. Our results are suggestive of a role for the homeotic gene products in regulating genes which are necessary for generating sensory progenitor cells in the developing PNS.     

A hidden program in Drosophila peripheral neurogenesis revealed: fundamental principles underlying sensory organ diversity

How is cell fate diversity reliably achieved during development? Insect sensory organs have been a favorable model system for investigating this question for over 100 years. They are constructed using defined cell lineages that generate a maximum of cell diversity with a minimum number of cell divisions, and display tremendous variety in their morphologies, constituent cell types, and functions. An unexpected realization of the past 5 years is that very diverse sensory organs in Drosophila are produced by astonishingly similar cell lineages, and that their diversity can be largely attributed to only a small repertoire of developmental processes. These include changes in terminal cell differentiation, cell death, cell proliferation, cell recruitment, cell-cell interactions, and asymmetric segregation of cell fate determinants during mitosis. We propose that most Drosophila sensory organs are built from an archetypal lineage, and we speculate about how this stereotyped pattern of cell divisions may have been built during evolution.     

BMP and Delta/Notch signaling control the development of amphioxus epidermal sensory neurons: insights into the evolution of the peripheral sensory system

The evolution of the nervous system has been a topic of great interest. To gain more insight into the evolution of the peripheral sensory system, we used the cephalochordate amphioxus. Amphioxus is a basal chordate that has a dorsal central nervous system (CNS) and a peripheral nervous system (PNS) comprising several types of epidermal sensory neurons (ESNs). Here, we show that a proneural basic helix-loop-helix gene (Ash) is co-expressed with the Delta ligand in ESN progenitor cells. Using pharmacological treatments, we demonstrate that Delta/Notch signaling is likely to be involved in the specification of amphioxus ESNs from their neighboring epidermal cells. We also show that BMP signaling functions upstream of Delta/Notch signaling to induce a ventral neurogenic domain. This patterning mechanism is highly similar to that of the peripheral sensory neurons in the protostome and vertebrate model animals, suggesting that they might share the same ancestry. Interestingly, when BMP signaling is globally elevated in amphioxus embryos, the distribution of ESNs expands to the entire epidermal ectoderm. These results suggest that by manipulating BMP signaling levels, a conserved neurogenesis circuit can be initiated at various locations in the epidermal ectoderm to generate peripheral sensory neurons in amphioxus embryos. We hypothesize that during chordate evolution, PNS progenitors might have been polarized to different positions in various chordate lineages owing to differential regulation of BMP signaling in the ectoderm.     

Early events in the development of Drosophila peripheral nervous system

1. We have analysed the development of the larval PNS of Drosophila, with the aim of understanding the genetic programme that underlies this development. 2. The achaete-scute gene complex (AS-C), which is required for the development of the adult PNS, is also necessary for the larval PNS. The analysis of different AS-C lesions shows that the larval PNS results from the superimposition of two independent subpatterns, each of which depends on one AS-C gene. 3. The analysis of the two subpatterns reveals hidden homologies between the very different arrangements of sense organs observed on different segments, suggesting that the initial pattern is the same in all segments and is later modified in the different segments. 4. The early arrangement of sensory mother cells can be visualised in a special transgenic line, A37. In this line the initial repetitive pattern inferred above can be directly observed. Furthermore this line makes it possible to decide whether a given mutation acts on the very early steps of the PNS development (determination) or at later stages (differentiation). 5. The line A37 has been used to show that mutations that reduce the PNS such as AS-C- or da- alter the very first steps of the process, while mutations which result in a hypertrophied PNS such as N seem to alter a subsequent step. We end up with an overview of the genetic operations that generate the arrangement of sense organs and sensory neurons.     

Morphological differentiation of the embryonic peripheral neurons in Drosophila

Summary The stereotyped segmental and dorso-ventral organization of the peripheral nervous system (PNS) of Drosophila embryos allows the identification of all the neurons in the body wall. Distinct classes of neurons are distinguishable according to their location, the targets they innervate, the particular shape of their dendrites and their cell size. Those neurons innervating external sensory structures (es) and chordotonal organs (ch) have single dendrites and have been previously described (Ghysen et al. 1986; Dambly-Chaudiere and Ghysen 1986; Campos-Ortega and Hartenstein 1985). We describe here the identity and morphological features of three other classes of neurons in the body segments which have multiple dendrites (md neurons): 1) neurons that give rise to elaborate dendritic arborisations (da neurons); 2) neurons that have bipolar dendrites (bd neurons); 3) neurons that arborize around particular tracheal branches (td neurons). The thoracic hemisegment (T2 and T3) contains 13 da, one bd, one td, 21 es and four ch neurons; the abdominal hemisegment (A1 to A7) contains 14 da, three bd, three td, 15 es and eight ch neurons. The arrangement of the segmented peripheral neurons is highly invariant and provides a favorable assay system for the genetic analysis of neurodevelopment.     

A genetic screen for elements of the network that regulates neurogenesis in Drosophila

The development of external sensory organs on the notum of Drosophila is promoted by the proneural genes achaete and scute. Their activity defines proneural cell clusters in the wing imaginal disc. Ectopic expression, under control of the GAL4 system, of the proneural gene lethal of scute (l'sc) causes the development of ectopic bristles. Persistent ectopic expression of l'sc is not sufficient to impose a neural fate on any given cell. This implies that mutual inhibition, mediated by the Notch signaling pathway, occurs among the cells of the ectopic proneural cluster. Consequently, the dominant, quantifiable phenotype associated with ectopic expression of l'sc is modified by mutations in genes known to be involved in neurogenesis. This phenotype has been utilized to screen for dominant enhancers and suppressors that modify the number of ectopic bristles. In this way, about 100 000 progeny of EMS or X-ray-treated flies have been analyzed to identify autosomal genes involved in regulation of the neural fate. In addition 1200 chromosomes carrying lethal P-element insertions were screened for modifiers. Besides mutations in genes expected to modify the phenotype, we have isolated mutations in six genes not known so far to be involved in neurogenesis. Received: 20 September 1997 / Accepted: 8 October 1997     

A genetic screen for elements of the network that regulates neurogenesis in Drosophila

The development of external sensory organs on the notum of Drosophila is promoted by the proneural genes achaete and scute. Their activity defines proneural cell clusters in the wing imaginal disc. Ectopic expression, under control of the GAL4 system, of the proneural gene lethal of scute (l'sc) causes the development of ectopic bristles. Persistent ectopic expression of l'sc is not sufficient to impose a neural fate on any given cell. This implies that mutual inhibition, mediated by the Notch signaling pathway, occurs among the cells of the ectopic proneural cluster. Consequently, the dominant, quantifiable phenotype associated with ectopic expression of l'sc is modified by mutations in genes known to be involved in neurogenesis. This phenotype has been utilized to screen for dominant enhancers and suppressors that modify the number of ectopic bristles. In this way, about 100 000 progeny of EMS or X-ray-treated flies have been analyzed to identify autosomal genes involved in regulation of the neural fate. In addition 1200 chromosomes carrying lethal P-element insertions were screened for modifiers. Besides mutations in genes expected to modify the phenotype, we have isolated mutations in six genes not known so far to be involved in neurogenesis.     

P-Element Mutations Affecting Embryonic Peripheral Nervous System Development in Drosophila Melanogaster

The Drosophila embryonic peripheral nervous system (PNS) is an excellent model system to study the molecular mechanisms governing neural development. To identify genes controlling PNS development, we screened 2000 lethal P-element insertion strains. The PNS of mutant embryos was examined using the neural specific marker MAb 22C10, and 92 mutant strains were retained for further analysis. Genetic and cytological analysis of these strains shows that 42 mutations affect previously isolated genes that are known to be required for PNS development: longitudinals lacking (19), mastermind (15), numb (4), big brain (2), and spitz (2). The remaining 50 mutations were classified into 29 complementation groups and the P-element insertions were cytologically mapped. The mutants were classified in five major classes on the basis of their phenotype: gain of neurons, loss of neurons, organizational defects, pathfinding defects and morphological defects. Herein we report the preliminary phenotypic characterization of each of these complementation groups as well as the embryonic lacZ expression pattern of each P-element strain. Our analysis indicates that in most of the P-element insertion strains, the lacZ reporter gene is not expressed in the developing PNS.     

Context-dependent utilization of Notch activity in Drosophila glial determination

During Drosophila neurogenesis, glial differentiation depends on the expression of glial cells missing (gcm). Understanding how glial fate is achieved thus requires knowledge of the temporal and spatial control mechanisms directing gcm expression. A recent report showed that in the adult bristle lineage, gcm expression is negatively regulated by Notch signaling ( Van De Bor, V. and Giangrande, A. (2001). Development 128, 1381-1390). Here we show that the effect of Notch activation on gliogenesis is context-dependent. In the dorsal bipolar dendritic (dbd) sensory lineage in the embryonic peripheral nervous system (PNS), asymmetric cell division of the dbd precursor produces a neuron and a glial cell, where gcm expression is activated in the glial daughter. Within the dbd lineage, Notch is specifically activated in one of the daughter cells and is required for gcm expression and a glial fate. Thus Notch activity has opposite consequences on gcm expression in two PNS lineages. Ectopic Notch activation can direct gliogenesis in a subset of embryonic PNS lineages, suggesting that Notch-dependent gliogenesis is supported in certain developmental contexts. We present evidence that POU-domain protein Nubbin/PDM-1 is one of the factors that provide such context.     

Comparison of the generic neuronal differentiation and neuron subtype specification functions of mammalian achaete-scute and atonal homologs in cultured neural progenitor cells

In the vertebrate peripheral nervous system, the proneural genes neurogenin 1 and neurogenin 2 (Ngn1 and Ngn2), and Mash1 are required for sensory and autonomic neurogenesis, respectively. In cultures of neural tube-derived, primitive PNS progenitors NGNs promote expression of sensory markers and MASH1 that of autonomic markers. These effects do not simply reflect enhanced neuronal differentiation, suggesting that both bHLH factors also specify neuronal identity like their Drosophila counterparts. At high concentrations of BMP2 or in neural crest stem cells (NCSCs), however, NGNs like MASH1 promote only autonomic marker expression. These data suggest that that the identity specification function of NGNs is more sensitive to context than is that of MASH1. In NCSCs, MASH1 is more sensitive to Notch-mediated inhibition of neurogenesis and cell cycle arrest, than are the NGNs. Thus, the two proneural genes differ in other functional properties besides the neuron subtype identities they can promote. These properties may explain cellular differences between MASH1- and NGN-dependent lineages in the timing of neuronal differentiation and cell cycle exit.     

Genes involved in the development of the peripheral nervous system of Drosophila

The development of the peripheral nervous system (PNS) requires the activity of a number of genes. The neurogenic and the proneural genes are necessary in the earliest phase; their mutations lead to hyperplasia and partial or total elimination of the PNS respectively. Some of these mutations also affect other developmental processes. Other mutations affect later events: cut transforms one type of sensory organ into another; numb alters the fate of the components of a single sensory organ. We will describe the effects of the best studied mutations on PNS development and discuss the possible role of the wild type genes.     

Transgenic mice expressing the <Emphasis Type="Italic">Peripherin-EGFP</Emphasis> genomic reporter display intrinsic peripheral nervous system fluorescence

The development of homologous recombination methods for the precise modification of bacterial artificial chromosomes has allowed the introduction of disease causing mutations or fluorescent reporter genes into human loci for functional studies. We have introduced the EGFP gene into the human PRPH-1 locus to create the Peripherin-EGFP (hPRPH1-G) genomic reporter construct. The hPRPH1-G reporter was used to create transgenic mice with an intrinsically fluorescent peripheral nervous system (PNS). During development, hPRPH1-G expression was concomitant with the acquisition of neuronal cell fate and growing axons could be observed in whole embryo mounts. In the adult, sensory neurons were labeled in both the PNS and central nervous system, while motor neurons in the spinal cord had more limited expression. The fusion protein labeled long neuronal processes, highlighting the peripheral circuitry of hPRPH1-G transgenic mice to provide a useful resource for a range of neurobiological applications.