Projection pattern of sensory neurons in the central nervous system of a homeotic mutation of the moth Manduca sexta

Octopod (Octo) is a mutation of the moth Manduca sexta, which transforms the first abdominal segment (A1) in the anterior direction. Mutant animals are characterized by the appearance of homeotic thoracic-like legs on A1. We exploited this mutation to determine what rules might be used in specifying the fates of sensory neurons located on the body surface of larval Manduca. Mechanical stimulation of homeotic leg sensilla did not cause reflexive movements of the homeotic legs, but elicited responses similar to those observed following stimulation of ventral A1 body wall hairs. Intracellular recordings demonstrated that several of the motoneurons in the A1 ganglion received inputs from the homeotic sensory hairs. The responses of these motoneurons to stimulation of homeotic sensilla resembled their responses to stimulation of ventral body wall sensilla. Cobalt fills revealed that the mutation transformed the segmental projection pattern of only the sensory neurons located on the ventral surface of A1, resulting in a greater number with intersegmental projection patterns typical of sensory neurons found on the thoracic body wall. Many of the sensory neurons on the homeotic legs had intersegmental projection patterns typical of abdominal sensory neurons: an anteriorly directed projection terminating in the third thoracic ganglion (T3). Once this projection reached T3, however, it mimicked the projections of the thoracic leg sensory neurons. These results demonstrate that the same rules are not used in the establishment of the intersegmental and leg-specific projection patterns. Segmental identity influences the intersegmental projection pattern of the sensory neurons of Manduca, whereas the leg-specific projections are consistent with a role for positional information in determining their pattern. © 1995 John Wiley & Sons, Inc.     

Distribution of the Sex combs reduced Gene Products in Drosophila melanogaster

The spatial and temporal distribution of RNA and protein encoded by the homeotic Sex combs reduced (Scr) gene were examined during Drosophila development. The gene products are present in the epidermis of both the labial and first thoracic segments as would be predicted from prior genetic studies. However, the pattern in the central nervous system (CNS) and mesoderm is further restricted; the major expression located in the labial neuromere of the CNS and the mesoderm of the first thoracic segment. The spatial restriction within the CNS is correlated with and may be due to a differential timing of expression in the labial and first thoracic ectoderm. The labial ectoderm accumulates the Scr RNA prior to segregation of the neuroblasts while expression in the first thoracic ectoderm occurs after neuroblast segregation. The protein is also observed in the subesophageal ganglia of both larvae and adults, as well as in the labial and first thoracic imaginal discs. Surprisingly, the protein is also present to a lesser extent in second and third thoracic leg discs.     

Neurogenesis in larval stages of the spider crab Hyas araneus (Decapoda,Brachyura): proliferation of neuroblasts in the ventral nerve cord

Larvae of the spider crab Hyas araneus were reared in the laboratory from hatching through to metamorphosis. Neurogenesis was recorded in the ventral nerve cord during development of successive larval stages, zoea 1, zoea 2, megalopa and crab 1. Proliferating cells were detected immunocytochemically after in vivo labelling with 5-bromo-2-deoxyuridine (BrdU) which, as a thymidine analogue, is incorporated into the DNA of dividing cells. Segmental sets of mitotically highly active neuroblasts were found in newly hatched larvae. A dorsal neuroblast, a ventral-median neuroblast, 3–6 anterior-ventral neuroblasts and 1–3 lateral neuroblasts could be distinguished in each thoracic ganglion. Significantly fewer neuroblasts were labelled in the suboesophageal ganglion as compared to the thoracic ganglia. The number of active labelled neuroblasts was high throughout zoea 1 and about 30% of zoea 2 development and then dramatically decreased towards premetamorphosis. In the newly moulted megalopa, only a reduced set of neuroblasts was labelled which ceased dividing within the first few days of megalopa development. There is an indication that, although most ganglion mother cells born by unequal division of neuroblasts may go through their final division at an early stage, certain clusters of ganglion mother cells obviously delay their final mitosis. These results are discussed with regard to neuronal integration which necessarily changes during the course of metamorphosis in relation to the altered behavioural repertoire of the larvae.     

Embryology of the thoracic and abdominal ganglia of the large milkweed bug, Oncopeltus fasciatus (Dallas), (Hemiptera, Lygaeidae)

In this study, the condensation of the three thoracic and 11 abdominal segmental ganglia to form a prothoracic and central nerve mass during embryogenesis is described. During katatrepsis, many changes occur in the organization of these ganglia; this study suggests that some of these changes are caused by mechanical forces acting on the ventral nerve cord at this time. The ventral nerve cord begins its anterior migration and coalescence ten hours after katatrepsis and is completed 63 hours later. The central ganglion is made up of the meso- and metathoracic ganglia and seven abdominal ganglia. Intrasegmental median cord nuclei are shown to form glial elements in the median sagittal plane of the neuropile and in the longitudinal connectives. Intersegmental median cord neuroblasts migrate into the posterior gangliomeres but, apparently, degenerate soon after katatrepsis. Lateral cord cells bordering on the neuropile form a glial investment that surrounds this fiber tract region. Peripheral lateral cord cells are shown to form the cells of the outer ganglionic sheath, the perineurium.     

Ectopic neurons and the organization of insect sensory systems

Summary The transplantation of appendages from one place to another on the body of crickets (Acheta domesticus) has been used to study the similarities and differences between the sensory systems of various ganglia. Mesothoracic legs have been transplanted to the abdomen in place of a cercus and cerci have been transplanted to thoracic leg stumps. After the ectopic sensory neurons had time to regenerate into the CNS, they were stained and their axonal arborizations examined. The results, which were concerned primarily with bristle receptors, revealed that bristle afferents on ectopic cerci arborized in ventral neuropil (the ventralmost association center) and leg afferents arborized in a ventral anterior region of the terminal abdominal ganglion. The results support the idea that each ganglion contains only a few distinct regions of neuropil (probably three), each receiving separate subsets of the afferent projection.The ectopic cerci were also shown to excite interneurons in the thoracic ganglia whose dendrites were located in the most ventral neuropil. These neurons normally respond to thoracic bristle afferents. Thus, the segregation of afferent axons has a correlate in the interneurons they excite.     

Lineage, migration, and morphogenesis of longitudinal glia in the Drosophila CNS as revealed by a molecular lineage marker

Previous studies described three different classes of glial cells in the developing CNS of the early Drosophila embryo that prefigure and ensheath the major CNS axon tracts. Among these are 6 longitudinal glial cells on each side of each segment that overlie the longitudinal axon tracts. Here we use transformant lines carrying a P element containing a 130 bp sequence from the fushi tarazu gene in front of the lacZ reporter gene to direct beta-galactosidase expression in the longitudinal glia. Using this molecular lineage marker, we show that 1 of the "neuroblasts" in each hemisegment is actually a glioblast, which divides once symmetrically, in contrast to the typical asymmetric neuroblast divisions, producing 2 glial cells, which migrate medially and divide to generate the 6 longitudinal glial cells. As with neuroblasts, mutations in Notch and other neurogenic genes lead to supernumerary glioblasts. The results indicate that the glioblast is similar to other neuroblasts; however, the positionally specified fate of this blast cell is to generate a specific lineage of glia rather than a specific family of neurons.     

Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster

Neurogenesis in the ventral CNS of Drosophila was studied using staining with toluidine blue and birth dating of cells monitored by incorporation of bromodeoxyuridine into DNA. The ventral CNS of the larva contains sets of neuronal stem cells (neuroblasts) which are thought to be persistent embryonic neuroblasts. Each thoracic neuromere has at least 47 of these stem cells whereas most abdominal neuromeres possess only 6. They occur in stereotyped locations so that the same neuroblast can be followed from animal to animal. The thoracic neuroblasts begin enlarging at 18-26 hr of larval life, DNA synthesis commences by 31-36 hr, and the first mitoses occur shortly thereafter. Mitotic activity continues through the remainder of larval life with the neuroblasts showing a minimum cell cycle time of less than 55 min during the late third larval instar. By 12 hr after pupariation each neuroblast has produced approximately 100 progeny which are collected with it into a discrete packet. The progeny accumulate in an immature, arrested state and only finish their differentiation into mature neurons with the onset of metamorphosis. Most of the abdominal neuroblasts differ from their thoracic counterparts in their minimum cell cycle time (less than 2 hr) and the duration of proliferation (from about 50 to 90 hr of larval life). Neurons produced during the larval stage account for more than 90% of the cells found in the ventral CNS of the adult.     

Surgical generation of supernumerary appendages for studying neuronal specificity in Drosophila melanogaster

The ability of sensory neurons to establish specific synaptic contacts in the central nervous system (CNS) can be studied by changing the spatial relationship between the periphery and the CNS. In contrast to the genetic displacement of appendages by homoeotic mutations, the surgical approach used in this study allows one to place homologous as well as heterologous appendages to the same site on the body surface. Using an improved technique of “surface transplantation,” we generated supernumerary appendages of any desired type in a particular abdominal position. The sensory axons originating from these grafts enter the CNS through the main abdominal nerve and arborize in the fused abdominal ganglia; many fibers extend also into thoracic centers. In the abdominal ganglia, terminals from dorsal transplants (wings and halteres) stay on the ipsilateral side, whereas terminals from ventral transplants (legs and antennae) distribute ipsi- and contralaterally. The same preference holds true for dorsal and ventral abdominal bristles, respectively, whose projection patterns served as a reference. In thoracic ganglia, axons from dorsal and ventral grafts yield completely different terminal patterns. Dorsal grafts project into the ipsilateral wing center, even in the mutant wingless, in which normal wing afferents are suppressed. In contrast, fibers from ventral grafts often extend along the thoracic midline. These data indicate that sensory axons of homologous appendages on the one hand, and their central targets on the other, share serially repeated surface markers. This may enable sensory fibers to recognize centers of homologous appendages.     

Fate map of the eye-antennal imaginal disc of the tumorous-head mutant of Drosophila melanogaster

In specific genetic backgrounds, a mutation in the tuh-3 gene results in the homeotic transformation of head structures to either leg disc derivatives or structures normally found in the extreme posterior end of wild-type animals. The origins of the homeotic structures were mapped to defined positions in the eye-antennal imaginal disc by transplanting abnormal regions of discs isolated from tuh-3 mutants into host mwh;e4 larvae. These metamorphosed implants were removed and differentiated structures were identified. Of 211 successfully recovered implants, 157 gave rise to homeotic tissue: abdominal tergite, male or female external genitalia and/or leg tissue. Transformations to abdominal tergite occurred primarily in cells taken from the eye region of the compound disc. Male and female genitalia arose most often in implants taken from the antennal portion of the disc, although some tissue taken from the lateral region of the eye disc also gave rise to external genitalia. Leg structures came exclusively from implants from the antennal region of the imaginal disc. These results suggest that cells from within specific regions of the eye-antennal compound disc are constrained in their developmental potential. An obvious constraint observed with this mutation is a dorsal/ventral one: Cells from the eye disc, a dorsal structure, primarily gave rise to other dorsal structures, abdominal tergite tissue. Cells from the antennal disc, a ventrally derived structure, primarily gave rise to other ventral structures including genital tissue and distal leg.     

Genetic dissection of a complex neurological mutant, polyhomeotic, in Drosophila

Null mutations at the polyhomeotic locus of Drosophila produce a complex phenotype during embryogenesis, which includes death of the ventral epidermis, misregulation of homeotic and segmentation gene expression, and global misrouting of CNS axons. It is shown here, through the use of mosaic analyses, double mutant combinations, and in vitro culture experiments, that all aspects of the phenotype with the exception of the axonal phenotype are cell autonomous. The changes in homeotic and segmentation gene expression in the CNS are not caused by death of the ventral epidermis, but are cell autonomous effects which most likely cause changes in neuronal cell identity. The axonal phenotype associated with ph mutations is also independent of epidermal cell death, but may be due to the nonautonomous effects of altered neuronal identities or to death or transformation of some as yet unidentified cell type. Despite the apparent autonomy of the ph mutation, mutant neurons can influence the development of adjacent wild-type neurons, presumably by depriving them of their normal fasciculation partners.     

Homeotic gene expression in the wild-type and a homeotic mutant of the moth Manduca sexta

Antibodies were used to examine the expression patterns of Antennapedia (Antp), Ultrabithorax (Ubx), Ubx and abdominal-A combined(Ubx/abd-A),and Distalless (Dll) in the embryos of the moth Manduca sexta. We found that the spatial and temporal pattern of Antp expression in Manduca was correlated with the anterior migration of two patches of epithelium that include the anterior-most tracheal pits, and with the development of functional spiracles. Ubx expression showed an intricate pattern which suggests complex regulation during development. Throughout Manduca embryogenesis the expression of Ubx/Abd-A and Dll was similar to that reported for other insects. However, there was no apparent reduction in Ubx/Abd-A expression in the Manduca abdominal proleg primordia that expressed Dll. The expression of these four proteins was also examined in embryosof the Manduca homozygous homeotic mutant Octopod (Octo). The Octo mutation results in the transformation of A1 and A2 in the anterior direction, with homeotic legs appearing on A1 and occasionally A2. Our results suggest that in Octo animals there is a reduction in the level of Ubx protein expression throughout its domain. Based on homeotic gene expression in wild-type and mutant Manduca and in other insects, we discuss potential roles of homeotic genes in insect morphological evolution. Received: 21 September 1998 / Accepted: 5 March 1999     

Programmed cell death in the embryonic central nervous system of Drosophila melanogaster

Although programmed cell death (PCD) plays a crucial role throughout Drosophila CNS development, its pattern and incidence remain largely uninvestigated. We provide here a detailed analysis of the occurrence of PCD in the embryonic ventral nerve cord (VNC). We traced the spatio-temporal pattern of PCD and compared the appearance of, and total cell numbers in, thoracic and abdominal neuromeres of wild-type and PCD-deficient H99 mutant embryos. Furthermore, we have examined the clonal origin and fate of superfluous cells in H99 mutants by DiI labeling almost all neuroblasts, with special attention to segment-specific differences within the individually identified neuroblast lineages. Our data reveal that although PCD-deficient mutants appear morphologically well-structured, there is significant hyperplasia in the VNC. The majority of neuroblast lineages comprise superfluous cells, and a specific set of these lineages shows segment-specific characteristics. The superfluous cells can be specified as neurons with extended wild-type-like or abnormal axonal projections, but not as glia. The lineage data also provide indications towards the identities of neuroblasts that normally die in the late embryo and of those that become postembryonic and resume proliferation in the larva. Using cell-specific markers we were able to precisely identify some of the progeny cells, including the GW neuron, the U motoneurons and one of the RP motoneurons, all of which undergo segment-specific cell death. The data obtained in this analysis form the basis for further investigations into the mechanisms involved in the regulation of PCD and its role in segmental patterning in the embryonic CNS.     

Cortical aPKC kinase activity distinguishes neural stem cells from progenitor cells by ensuring asymmetric segregation of Numb

During asymmetric stem cell division, polarization of the cell cortex targets fate determinants unequally into the sibling daughters, leading to regeneration of a stem cell and production of a progenitor cell with restricted developmental potential. In mitotic neural stem cells (neuroblasts) in fly larval brains, the antagonistic interaction between the polarity proteins Lethal (2) giant larvae (Lgl) and atypical Protein Kinase C (aPKC) ensures self-renewal of a daughter neuroblast and generation of a progenitor cell by regulating asymmetric segregation of fate determinants. In the absence of lgl function, elevated cortical aPKC kinase activity perturbs unequal partitioning of the fate determinants including Numb and induces supernumerary neuroblasts in larval brains. However, whether increased aPKC function triggers formation of excess neuroblasts by inactivating Numb remains controversial. To investigate how increased cortical aPKC function induces formation of excess neuroblasts, we analyzed the fate of cells in neuroblast lineage clones in lgl mutant brains. Surprisingly, our analyses revealed that neuroblasts in lgl mutant brains undergo asymmetric division to produce progenitor cells, which then revert back into neuroblasts. In lgl mutant brains, Numb remained localized in the cortex of mitotic neuroblasts and failed to segregate exclusively into the progenitor cell following completion of asymmetric division. These results led us to propose that elevated aPKC function in the cortex of mitotic neuroblasts reduces the function of Numb in the future progenitor cells. We identified that the acyl-CoA binding domain containing 3 protein (ACBD3) binding region is essential for asymmetric segregation of Numb in mitotic neuroblasts and suppression of the supernumerary neuroblast phenotype induced by increased aPKC function. The ACBD3 binding region of Numb harbors two aPKC phosphorylation sites, serines 48 and 52. Surprisingly, while the phosphorylation status at these two sites directly impinged on asymmetric segregation of Numb in mitotic neuroblasts, both the phosphomimetic and non-phosphorylatable forms of Numb suppressed formation of excess neuroblasts triggered by increased cortical aPKC function. Thus, we propose that precise regulation of cortical aPKC kinase activity distinguishes the sibling cell identity in part by ensuring asymmetric partitioning of Numb into the future progenitor cell where Numb maintains restricted potential independently of regulation by aPKC.     

Lethal giant larvae controls the localization of notch-signaling regulators numb, neuralized, and Sanpodo in Drosophila sensory-organ precursor cells

Asymmetric distribution of fate determinants is a fundamental mechanism underlying the acquisition of distinct cell fates during asymmetric division. In Drosophila neuroblasts, the apical DmPar6/DaPKC complex inhibits Lethal giant larvae (Lgl) to promote the basal localization of fate determinants. In contrast, in the sensory precursor (pI) cells that divide asymmetrically with a planar polarity, Lgl inhibits Notch signaling in the anterior pI daughter cell, pIIb, by a yet-unknown mechanism. We show here that Lgl promotes the cortical recruitment of Partner of Numb (Pon) and regulates the asymmetric distribution of the fate determinants Numb and Neuralized during the pI cell division. Analysis of Pon-GFP and Histone2B-mRFP distribution in two-color movies confirmed that Lgl regulates Pon localization. Moreover, posterior DaPKC restricts Lgl function to the anterior cortex at mitosis. Thus, Lgl functions similarly in neuroblasts and in pI cells. We also show that Lgl promotes the acquisition of the pIIb cell fate by inhibiting the plasma membrane localization of Sanpodo and thereby preventing the activation of Notch signaling in the anterior pI daughter cell. Thus, Lgl regulates cell fate by controlling Pon cortical localization, asymmetric localization of Numb and Neuralized, and plasma-membrane localization of Sandopo.     

Neurogenesis in the developing crab brain: Postembryonic generation of neurons persists beyond metamorphosis

A considerable amount of information is available about the structure and function of the central nervous system in adult crustaceans. However, little effort has been directed toward understanding embryonic and larval neurogenesis in these animals. In the present study we recorded neurogenesis in the brain of laboratory-reared larvae of the spider crab Hyas araneus. Proliferating cells were detected immunocytochemically after in vivo labeling with 5-bromo-2′-deoxyuridine. This method has already been used to study the proliferation of neuroblasts in the ventral nerve cord of spider crab larvae. In the brain, a set of mitotically highly active neuroblasts was found in newly hatched zoea 1 larvae. These neuroblasts are individually identifiable due to their position and therefore a schematic map of the cerebral neuroblasts could be established. The number of active neuroblasts is high from hatching throughout the molt to the zoea 2. This proliferative action then decreases dramatically and has ceased at the time of first metamorphosis toward the megalopa larva. However, many ganglion mother cells born by unequal division of neuroblasts then go through their final division throughout the subsequent megalopa stage. In the brain, all mitotic activity has ceased at the time of second metamorphosis with the exception of a cluster of labeled nuclei within the olfactory lobe cells. In this cluster, the generation of neurons persists beyond the second metamorphosis into the crab 1 stage. Meanwhile, the neuropil volume of the olfactory lobes increases 10-fold from hatching to the crab 1. These results are discussed with regard to reports on neuronal proliferation during adult life in insects and rodents. © 1996 John Wiley & Sons, Inc.     

Segment-specific generation of Drosophila Capability neuropeptide neurons by multi-faceted Hox cues

In the Drosophila ventral nerve cord, the three pairs of Capability neuropeptide-expressing Va neurons are exclusively found in the second, third and fourth abdominal segments (A2–A4). To address the underlying mechanisms behind such segment-specific cell specification, we followed the developmental specification of these neurons. We find that Va neurons are initially generated in all ventral nerve cord segments and progress along a common differentiation path. However, their terminal differentiation only manifests itself in A2–A4, due to two distinct mechanisms: segment-specific programmed cell death (PCD) in posterior segments, and differentiation to an alternative identity in segments anterior to A2. Genetic analyses reveal that the Hox homeotic genes are involved in the segment-specific appearance of Va neurons. In posterior segments, the Hox gene Abdominal-B exerts a pro-apoptotic role on Va neurons, which involves the function of several RHG genes. Strikingly, this role of Abd-B is completely opposite to its role in the segment-specific apoptosis of other classes of neuropeptide neurons, the dMP2 and MP1 neurons, where Abd-B acts in an anti-apoptotic manner. In segments A2–A4 we find that abdominal A is important for the terminal differentiation of Va cell fate. In the A1 segment, Ultrabithorax acts to specify an alternate Va neuron fate. In contrast, in thoracic segments, Antennapedia suppresses the Va cell fate. Thus, Hox genes act in a multi-faceted manner to control the segment-specific appearance of the Va neuropeptide neurons in the ventral nerve cord.     

Serotonin immunoreactive neurons in the central nervous system of an insect (Periplaneta americana)

Serotonin-like immunoreactivity was mapped in the central nervous system (CNS) of the cockroach, Periplaneta americana. Immunoreactive staining occurred in every ganglion of the CNS. The largest numbers of immunoreactive somata were detected in the optic lobes and the brain, and lowest numbers in the first and second thoracic ganglia. Dense stained fibers, ramifications, and varicosities were found in all ganglia, and numerous axon like processes occurred in all interganglionic connectives. Immunoreactive processes were not, however, detected in most of the peripherally projecting nerve roots. Processes were found only in roots of the suboesophageal ganglion and the tritocerebral lobes of the brain. A comparison of the map for serotonin immunoreactivity with one generated for the pentapeptide transmitter proctolin suggests that the two systems overlap only in the suboesophageal ganglion and the tritocerebrum. The amine and peptide may co-occur in neurons in these regions. The serotonin immunoreactive system appeared significantly different from the octopaminergic system of the ventral nerve cord. Seventy-two potentially identifiable immunoreactive cells were located in the cockroach CNS. Some of these may be suitable for physiological study of the functional role of serotonin.     

Mapping of serotonin-immunoreactive neurons in the larval nervous system of the flies Calliphora erythrocephala and Sarcophaga bullata

Summary Serotonin-immunoreactive (5-HTi) neurons were mapped in the larval central nervous system (CNS) of the dipterous flies Calliphora erythrocephala and Sarcophaga bullata. Immunocytochemistry was performed on cryostat sections, paraffin sections, and on the entire CNS (whole mounts).The CNS of larvae displays 96–98 5-HTi cell bodies. The location of the cell bodies within the segmental cerebral and ventral ganglia is consistent among individuals. The pattern of immunoreactive fibers in tracts and within neuropil regions of the CNS was resolved in detail. Some 5-HTi neurons in the CNS possess axons that run through peripheral nerves (antenno-labro-frontal nerves).The suboesophagealand thoracico-abdominal ganglia of the adult blowflies were studied for a comparison with the larval ventral ganglia. In the thoracico-abdominal ganglia of adults the same number of 5-HTi cell bodies was found as in the larvae except in the metathoracic ganglion, which in the adult contains two cell bodies less than in the larva. The immunoreactive processes within the neuropil of the adult thoracico-abdominal ganglia form more elaborate patterns than those of the larvae, but the basic organization of major fiber tracts was similar in larval and adult ganglia. Some aspects of postembryonic development are discussed in relation to the transformation of the distribution of 5-HTi neurons and their processes into the adult pattern.