Segmental and Regional Differences in Neuronal Expression of the Leech Hox Genes Lox1 and Lox2 During Embryogenesis

Using double immunofluorescence experiments, we described the expression of the leech Hox genes, Lox1 and Lox2 by central neurons that stained for either serotonin or the leech-specific neuronal marker, Laz1-1. The goal is to determine whether the segmental boundaries of Lox1 and Lox2 expression in identified neurons coincide with segmental and regional differences in the differentiation of these cells. A number of neurons described here have been previously identified. The anteromedial serotonergic neurons are restricted to rostral ganglion 1 (R1) to midbody ganglion 3 (M3), but only express Lox1 in M2 and M3. The posteromedial serotonergic neurons which are situated in all segments as bilateral pairs early in development, but later become unpaired starting at M3, expressed Lox1 only in M2 and M3, and Lox2 in M8 to M21, in all paired and unpaired stages. The Retzius neurons, which stain for serotonin, express Lox2 in M7 to M21 where they exhibit different morphologies from their segmental homologs of the sex ganglia in M5 and M6. The Laz1-1 immunoreactive (Laz1-1⁺) heart accessory-like neurons express Lox1 in M4 and Lox2 in M7 to M17, but not in their segmental homologs of the heart accessory (HA) neurons located exclusively in M5 and M6. Also, Laz1-1⁺ neurons, which we named Lz3 expressed Lox1 in M4 to M8 where they are unpaired, but express Lox2 in M9 to M16 where they are bilaterally paired. Other Laz1-1 cells show more restricted and isolated Lox1 and Lox2 expression patterns. These results suggest a role of Lox1 and/or Lox2 in defining the anteroposterior boundaries of segmentally iterated neurons.     

Segmental diversification of an identified leech neuron correlates with the segmental domain in which it expresses lox2, a member of the hox gene family

The cellular colocalization of LOX2 protein and small cardioactive peptide (SCP)-like immunoreactivity was studied in the nerve cord of the glossiphoniid leech Helobdella triserialis. Of the six neurons that express SCP in the midbody segments 7 to 17, only one, the MPS neuron, expresses LOX2 protein. The medial paired SCP (MPS) neurons are segmentally repeated and can be divided into three contiguous segmental domains according to cell body size and the timing and level of SCP expression. MPS neurons located in the anterior and middle segmental domains express LOX2 protein. In the middle domain, large MPS neurons begin to accumulate SCP shortly after the end of embryonic development, whereas in the anterior domain the MPS neurons are smaller and begin to express SCP at a later stage. In the posterior domain the MPS neurons exhibit a third phenotype—they have large cell bodies, express low levels of SCP starting from the midjuvenile stage, and do not show detectable LOX2 expression. Lineage tracer injections showed that the MPS neurons arise from a stereotyped cell lineage and are descended from the O teloblast stem cell. In midbody ganglia 2 to 6 and 18 to 21, there are lineally homologous neurons that do not express either LOX2 protein or SCP. Thus, the boundaries of LOX2 expression coincide precisely with two of the segmental boundaries of MPS differentiation, suggesting that expression of Lox2 at the level of this single identified neuron governs some, but not all, aspects of the neuron's segmental diversification. © 1996 John Wiley & Sons, Inc.     

Transplantation of neurons reveals processing areas and rules for synaptic connectivity in the cricket nervous system

In order to assess the nature of spatial cues in determining the characteristic projection sites of sensory neurons in the CNS, we have transplanted sensory neurons of the cricket Acheta domesticus to ectopic locations. Thoracic campaniform sensilla (CS) function as proprioceptors and project to an intermediate layer of neuropil in thoracic ganglia while cercal CS transduce tactile information and project into a ventral layer in the terminal abdominal ganglion (TAG). When transplanted to ectopic locations, these afferents retain their modality-specific projection in the host ganglion and terminate in the layer of neuropil homologous to that of their ganglion of origin. Thus, thoracic CS neurons project to intermediate neuropil when transplanted to the abdomen and cercal CS neurons project to a ventral layer of neuropil when transplanted to the thorax. We conclude that CS can be separated into two classes based on their characteristic axonal projections within each segmental ganglion. We also found that the sensory neurons innervating tactile hairs project to ventral neuropil in any ganglion they encounter after transplantation. Ectopic sensory neurons can form functional synaptic connections with identified interneurons located within the host ganglia. The new contacts formed by these ectopic sensory neurons can be with normal targets, which arborize within the same layer of neuropil in each segmental ganglion, or with novel targets, which lack dendrites in the normal ganglion and are thus normally unavailable for synaptogenesis. These observations suggest that a limited set of molecular markers are utilized for cell–cell recognition in each segmentally homologous ganglion. Regenerating sensory neurons can recognize novel postsynaptic neurons if they have dendrites in the appropriate layer of neuropil. We suggest that spatial constraints produced by the segmentation and the modality-specific layering of the nervous system have a pivotal role in determining synaptic specificity. © 1993 John Wiley & Sons, Inc.     

<Emphasis Type="Italic">Hau-Pax6A</Emphasis> expression in the central nervous system of the leech embryo

The leech Helobdella sp. (Austin) has two genes of the Pax6 subfamily, one of which is characterized in detail. Hau-Pax6A was expressed during embryonic development in a pattern similar to other bilaterian animals. RNA was detected in cellular precursors of the central nervous system (CNS) and in peripheral cells including a population associated with the developing eye. The CNS of the mature leech is a ventral nerve cord composed of segmental ganglia, and embryonic Hau-Pax6A expression was primarily localized to the N teloblast lineage that generates the majority of ganglionic neurons. Expression began when the ganglion primordia were four cells in length and was initially restricted to a single cell, n_s.a, whose descendants will form the ganglion’s anterior edge. At later stages, the Hau-Pax6A expression pattern expanded to include additional CNS precursors, including some descendants of the O teloblast. Expression persisted through the early stages of ganglion morphogenesis but disappeared from the segmented body trunk at the time of neuronal differentiation. The timing and iterated pattern of Hau-Pax6A expression in the leech embryo suggests that this gene may play a role in the segmental patterning of CNS morphogenesis.     

Dynamics of expression of apoptosis-regulatory proteins Bid, Bcl-2, Bcl-X, Bax and Bak during development of murine nervous system

We have used immunohistochemistry and immunoblotting to examine the expression of Bid and four other Bcl-2 family proteins (Bcl-2, Bcl-X, Bax and Bak) in the developing and adult murine central nervous system (CNS). Bid protein is widespread in embryonic and postnatal brain, and its expression is maintained at a high level late into the adulthood. Bid is expressed both in the germ disc, early neural tube, proliferating stem cells of ventricular zones, and in postmitotic, differentiated neurons of the developing central and peripheral nervous system. As the differentiation proceeds, the neurons express higher levels of Bid than the stem cells of the paraventricular zone. Both in embryonic and postnatal life, Bid protein is present in the most vital regions of brain, such as the limbic system, basal ganglia, mesencephalic tectum, Purkinje cells in cerebellum, and the ventral columns of spinal cord. The p15 cleaved form of Bid was detectable in the brain specimens at fetal stages of development, consistent with caspase-mediated activation of this pro-apoptotic Bcl-2 family protein. Among the Bcl-2 family proteins only Bid and Bcl-XL continue to be expressed at high levels in the adult brain.     

Restricted neural epidermal growth factor-like like 2 (NELL2) expression during muscle and neuronal differentiation

We have identified a secreted glycoprotein, neural epidermal growth factor-like like 2 (NELL2), in a screen designed to isolate molecules regulating sensory neuron genesis and differentiation in the dorsal root ganglia (DRG). In investigating NELL2 expression during embryogenesis, we demonstrate here that NELL2 is highly regulated spatially and temporally, being only transiently expressed in discrete regions of the central (CNS) and peripheral nervous systems (PNS) and in a subset of mesoderm derived structures during their peak periods of development. In the CNS and PNS, NELL2 is maximally expressed as motor and sensory neurons differentiate. Interestingly, its expression is restricted to sublineages of the neural crest, being strongly expressed throughout the immature DRG, but excluded from sympathetic ganglia. Similarly during muscle development, NELL2 is specifically expressed by hypaxial muscle precursor cells in the differentiating somite and derivatives in the forelimbs and body wall, but not by epaxial muscle precursors. Furthermore, NELL2 is differentially regulated in the CNS and PNS; in the CNS, NELL2 is only expressed by nascent, post-mitotic neurons as they commence their differentiation, yet in the PNS, NELL2 is expressed by subsets of progenitor cells in addition to nascent neurons. Based on this restricted spatial and temporal expression pattern, functional studies are in progress to determine NELL2's role during neuronal differentiation in both the PNS and CNS.     

Restricted neural epidermal growth factor-like like 2 (NELL2) expression during muscle and neuronal differentiation

We have identified a secreted glycoprotein, neural epidermal growth factor-like like 2 (NELL2), in a screen designed to isolate molecules regulating sensory neuron genesis and differentiation in the dorsal root ganglia (DRG). In investigating NELL2 expression during embryogenesis, we demonstrate here that NELL2 is highly regulated spatially and temporally, being only transiently expressed in discrete regions of the central (CNS) and peripheral nervous systems (PNS) and in a subset of mesoderm derived structures during their peak periods of development. In the CNS and PNS, NELL2 is maximally expressed as motor and sensory neurons differentiate. Interestingly, its expression is restricted to sublineages of the neural crest, being strongly expressed throughout the immature DRG, but excluded from sympathetic ganglia. Similarly during muscle development, NELL2 is specifically expressed by hypaxial muscle precursor cells in the differentiating somite and derivatives in the forelimbs and body wall, but not by epaxial muscle precursors. Furthermore, NELL2 is differentially regulated in the CNS and PNS; in the CNS, NELL2 is only expressed by nascent, post-mitotic neurons as they commence their differentiation, yet in the PNS, NELL2 is expressed by subsets of progenitor cells in addition to nascent neurons. Based on this restricted spatial and temporal expression pattern, functional studies are in progress to determine NELL2's role during neuronal differentiation in both the PNS and CNS.     

Immunoreactivity to molluskan neuropeptides in the central and stomatogastric nervous systems of the earthworm, Lumbricus terrestris L.

Polyclonal antisera against two related command neuropeptides (CNP2 and CNP4) described in neurons of the terrestrial snail Helix were used in a study of the nervous system of the earthworm Lumbricus. The CNP-like peptides belong to the same neuropeptide subfamily and bear a C-terminal signature sequence Tyr-Pro-Arg-X. The distribution patterns of immunoreactive (IR) neurons were studied in the central nervous system (CNS), skin, and stomatogastric nervous system of the earthworm. IR neurons were found in all CNS ganglia, the patterns being similar for both antibodies used. Several clusters of IR cells were observed in the cerebral and subesophageal ganglia. In the ventral cord ganglia, the number of IR cells decreased in the rostro-caudal direction, and the IR cells sent their fibers mostly into the median fiber bundle. Segmental nerves contained no IR fibers. After injury of the worm body, the number of IR neurons in the CNS significantly increased. In the skin, IR sensory neurons were present in sensory buds. The stomatogastric ganglia only contained IR fibers. Numerous scattered IR neurons were found in the inner subepithelial layer of the esophagus and formed the enteric plexus in which the cell bodies displayed a segmentally repeated pattern. Possible involvement of CNP-like-IR neurons in central integratory processes, sensory processes, and the regulation of feeding is discussed.This work was supported by INTAS (grant 01-2117), CRDF (grant RB1-2321-MO-02), and the Russian Foundation for Basic Research (grants 05-04-48724 and 03-04-48179).     

Ontogenesis of the snail,Helix aspersa: Embryogenesis timetable and ontogenesis of GABA-like immunoreactive neurons in the central nervous system

Late stages of embryogenesis in the terrestrial snail Helix aspersa L. were studied and a developmental timetable was produced. The distribution of gamma-aminobutyric acid-like immunoreactive (GABA-ir) elements in the CNS of the snail was studied from embryos to adulthood in wholemounts. In adults, approximately 226 GABA-ir neurons were located in the buccal, cerebral and pedal ganglia. The population of GABA-ir cells included four pairs of buccal neurons, three neuronal clusters in the pedal ganglia, two clusters and six single neurons in the cerebral ganglia. GABA-ir fibers were observed in all ganglia and in some nerves. The first detected pair of GABA-ir cells in the embryos appeared in the buccal ganglia at about 63–64% of embryonic development. Five pairs of GABA-ir cell bodies were observed in the cerebral ganglia at about 64–65% of development. During the following 30% of development three more pairs of GABA-ir neurons were detected in the buccal ganglia and over fifteen cells were detected in each cerebral ganglion. At the stage of 70% of development, the first pair of GABA-ir neurons was found in the pedal ganglia. In the suboesophageal ganglion complex, GABA-ir fibers were first detected at about 90% of embryonic development. In the posthatching period, the quantity of GABA-ir neurons reached the adult status in four days in the cerebral ganglia, and in three weeks in the pedal ganglia. In juveniles, transient expression of GABA was found in the pedal ganglia (fourth cluster).     

FMRfamide-like immunoreactivity in the ventral nerve cord of the larval eastern spruce budworm,Choristoneura fumiferana (clemens) (Lepidoptera : Tortricidae)

Distribution of FMRFamide-like immunoreactivity was examined in the larval ventral nerve cord of the eastern spruce budworm, Choristoneura fumiferana (Lepidoptera : Tortricidae). Indirect immunofluorescent methods revealed the existence of 3 groups of FLI neurons in each ganglion. The neurons are distributed in a bilaterally symmetrical fashion at the anterodorsal, midlateral and posteroventral regions of the ganglia. There are 4 FMRFamide-like immunoreactive fiber tracts on the dorsal surface of the ganglia to which the anterodorsal FLI neurons project ipsilaterally, while the midlateral pair projects both ipsi-, and contralaterally. The last abdominal ganglion (AG8) has 4 additional pairs of FLI neurons; and axons from some of these extend into the median abdominal nerve, which suggests some role for this neuropeptide in the control of posterior structures of the larva.     

FMRFamide-like immunoreactivity in the isolated,in vivo cultured ventral ganglion of the fly,Sarcophaga bullata (Parker) (Diptera: Sarcophagidae)

The influence of peripheral connectivity on the survival and differentiation of Phe-Met-Arg-Phe-amide-like immunoreactive (FLI) neurons in the ventral ganglion (VG) of the fly Sarcophaga bullata (Diptera: Sarcophagidae) was examined. Isolated larval VG were cultured in vivo for 13 days. The ganglia had undergone metamorphosis and resembled in situ metamorphosed VG in morphology and in the number and location of FLI neurons. The 3 pairs of large thoracic FLI neurons survived and became translocated to the midventral position extending immunoreactive axons into the dorsal neuropil. The 5 pairs of small FLI neurons also appeared de novo in the abdominal ganglion. However, the dorsal neural sheath of the cultured VG was devoid of FMRFamide-like immunoreactivity that was so characteristic of adult VG, which suggests the importance of peripheral connectivity for the metamorphic modification of FLI neurons.     

adrift, a novel bnl-induced Drosophila gene, required for tracheal pathfinding into the CNS

Neurons and glial cells provide guidance cues for migrating neurons. We show here that migrating epithelial cells also contact specific neurons and glia during their pathfinding, and we describe the first gene required in the process. In wild-type Drosophila embryos, the ganglionic tracheal branch navigates a remarkably complex path along specific neural and glial substrata, switching substrata five times before reaching its ultimate target in the CNS. In adrift mutants, ganglionic branches migrate normally along the intersegmental nerve, but sporadically fail to switch to the segmental nerve and enter the CNS; they wind up meandering along the ventral epidermis instead. adrift encodes a novel nuclear protein with an evolutionarily conserved motif. The gene is required in the trachea and is expressed in the leading cells of migrating ganglionic branches where it is induced by the branchless FGF pathway. We propose that Adrift regulates expression of tracheal genes required for pathfinding on the segmental nerve, and FGF induction of adrift expression in migrating tracheal cells promotes the switch from the intersegmental to the segmental nerve.     

Glutamate-like immunoreactivity in the leech central nervous system

Summary Using a monoclonal antibody for glutamate the distribution was determined of glutamate-like immunoreactive neurons in the leech central nervous system (CNS). Glutamate-like immunoreactive neurons (GINs) were found to be localized to the anterior portion of the leech CNS: in the first segmental ganglion and in the subesophageal ganglion. Exactly five pairs of GINs consistently reacted with the glutamate antibody. Two medial pairs of GINs were located in the subesophageal ganglion and shared several morphological characteristics with two medial pairs of GINs in the first segmental ganglion. An additional lateral pair of GINs was also located in segmental ganglion 1. A pair of glutamate-like immunoreactive neurons, which are potential homologs of the lateral pair of GINs in segmental ganglion 1, were occasionally observed in more posterior segmental ganglia along with a selective group of neuronal processes. Thus only a small, localized population of neurons in the leech CNS appears to use glutamate as their neurotransmitter.     

Glutamate-like immunoreactivity in the leech central nervous system.

Using a monoclonal antibody for glutamate the distribution was determined of glutamate-like immunoreactive neurons in the leech central nervous system (CNS). Glutamate-like immunoreactive neurons (GINs) were found to be localized to the anterior portion of the leech CNS: in the first segmental ganglion and in the subesophageal ganglion. Exactly five pairs of GINs consistently reacted with the glutamate antibody. Two medial pairs of GINs were located in the subesophageal ganglion and shared several morphological characteristics with two medial pairs of GINs in the first segmental ganglion. An additional lateral pair of GINs was also located in segmental ganglion 1. A pair of glutamate-like immunoreactive neurons, which are potential homologs of the lateral pair of GINs in segmental ganglion 1, were occasionally observed in more posterior segmental ganglia along with a selective group of neuronal processes. Thus only a small, localized population of neurons in the leech CNS appears to use glutamate as their neurotransmitter.