Kathleen A. Klukas

Chicken embryo lens cultures mimic differentiation in the lens

Embryonic chicken lenses, which had been disrupted by trypsin, were grown in culture. These cultures mimic lens development as it occurred in vivo, forming lens-like structures known as lentoids. Using a variety of techniques including electron microscopic analysis, autoradiography, immunofluorescence, and polyacrylamide gel electrophoresis, it was shown that the lentoid cells had many characteristics in common with the differentiated cells of the intact lens, the elongated fiber cells. These characteristics included a shut off of DNA synthesis, a loss of cell organelles, an increase in cell volume, an increase in delta-crystallin protein, and the development of extensive intercellular junctions. The cultures began as a simple epithelial monolayer but then underwent extensive morphogenesis as they differentiated. This morphogenesis involved three distinctive morphological types which appeared in sequence as an epithelial monolayer of polygonal shaped cells with pavement packing, elongated cells oriented end to end, and the multilayered, multicellular lentoids. These distinct morphological stages of differentiation in culture mimic morphogenesis as it occurs in the lens.

Improvements for the anatomical characterization of insect neurons in whole mount : the use of cyanine-derived fluorophores and laser scanning confocal microscopy

The optical sectioning capability of the laser scanning confocal microscope was utilized to image dye-filled neurons within whole-mounted insect ganglia. Specific pterothoracic interneurons, in the mothManduca sexta, were retrogradely filled with Neurobiotin and subsequently visualized with a monoclonal anti-biotin conjugated with one of the following fluorophores: fluorescein, and the newly developed cyanines, Cy3.18 (Cy3) and Cy5.18 (Cy5). Overall, the Cy5 fluorophore was best suited for imaging insect neurons within ganglia. This new methodology allowed us to identify and characterize morphologically a collection of descending multisegmental interneurons with large or small diameter somata. A variety of larger molecular weight (10000 daltons) tracers was also used to examine the possibility of nonselective filling of neurons with Neurobiotin, possibly through gap junctions. We also investigated the usefulness of Cy3 and Cy5 as fluorophores for transmitter immunostaining of neurons in whole mount. Neurons immunoreactive for serotonin and the neuropeptides, FMR Famide and SCPb, were imaged in the brain and the pterothoracic ganglion. The central projections of some of these immunoreactive neurons were imaged in their entirety.

Arrangement of MP26 in lens junctional membranes: Analysis with proteases and antibodies

SummaryThe major membrane protein of the bovine lens fiber cell is a 26-kilodalton (kD) protein (MP26), which appears to be a component of the extensive junctional specializations found in these cells. To examine the arrangement of MP26 within the junctional membranes, various proteases were incubated with fiber cell membranes that had been isolated with or without urea and/or detergents. These membranes were analyzed with electron microscopy and SDS-PAGE to determine whether the junctional specializations or the proteins were altered by proteolysis. Microscopy revealed no obvious structural changes. Electrophoresis showed that chymotrypsin, papain, and trypsin degraded MP26 to 21–22 kD species. A variety of protease treatments, including overnight digestions, failed to generate additional proteolysis. Regions on MP26 which were sensitive to these three proteases overlapped. Smaller peptides were cleaved from MP26 with V8 protease and carboxypptidases A and B. Protein domains cleaved by these proteases also overlapped with regions sensitive to chymotrypsin, papain, and trypsin. Specific inhibition of the carboxypeptidases suggested that cleavage obtained with these preparations was not likely due to contaminating endoproteases. Since antibodies are not thought to readily penetrate the 2–3 nm extracellular gap in the fiber cell junctions, antibodies to MP26 were used to analyze the location of the protease-sensitive domains. Antisera were applied to control (26 kD) and proteolyzed (22 kD) membranes, with binding being evaluated by means of ELISA reactions on intact membranes. Antibody labeling was also done following SDS-PAGE and transfer to derivatized paper. Both assays showed a significant decrease in binding following proteolysis, with the 22 kD product showing no reaction with the anti-MP26 sera. These investigations suggest that MP26 is arranged with approximately fourfifths of the primary sequence “protected” by the lipid bilayer and the narrow extracellular gap. One-fifth of the molecule, including the C-terminus, appears to be exposed on the cytoplasmic side of the membrane.

Cellular localization of bursicon using antisera against partial peptide sequences of this insect cuticle‐sclerotizing neurohormone

Bursicon is the final neurohormone released at the end of the molting cycle. It triggers the sclerotization (tanning) of the insect cuticle. Until now, its existence has been verified only by bioassays. In an attempt to identify this important neurohormone, bursicon was purified from homogenates of 2,850 nerve cords of the cockroach Periplaneta americana by using high performance liquid chromatography technology and two‐dimensional gel electrophoresis. Bursicon bioactivity was found in four distinct protein spots at approximately 30 kDa between pH 5.3 and 5.9. The protein of one of these spots at pH 5.7 was subsequently microsequenced, and five partial amino acid sequences were retrieved. Evidence is presented that two of these sequences are derived from bursicon. Antibodies raised against the two sequences labeled bursicon‐containing neurons in the central nervous systems of P. americana. One of these antisera labeled bursicon‐containing neurons in the crickets Teleogryllus commodus and Gryllus bimaculatus, and the moth Manduca sexta. A cluster of four bilaterally paired neurons in the brain of Drososphila melanogaster was also labeled. In addition, this antiserum detected three spots corresponding to bursicon in Western blots of two‐dimensional gels. The 12‐amino acid sequence detected by this antiserum, thus, seems to be conserved even among species that are distantly related. J. Comp. Neurol. 452:163–177, 2002.

Steroid regulation of octopamine expression during metamorphic development of the moth Manduca sexta.

Octopamine (OA), a biogenic amine similar to norepinephrine, has profound and well‐documented actions on the nervous systems of invertebrates. In the insect, Manduca sexta, we examined the developmental plasticity of OA synthesis, studied its endocrine regulation, and observed previously undescribed OA‐immunoreactive (ir) neurons. We found that levels of tyramine beta‐hydroxylase (TβH), an essential enzyme for the biosynthesis of OA, increase during metamorphosis. Based on the established and influential roles of the steroid hormone 20‐hydroxyecdysone (20‐HE) during development, we tested the hypothesis that increases in TβH levels and OA immunoreactivity are regulated by the rise in 20‐HE occurring during pupal‐adult development. We determined that the levels of TβH in the terminal abdominal ganglion (neuromeres 6–9) remain at a constant level during pupal development and the early stages of adult development. Beginning at ca. pupal stage 8, however, the levels of TβH begin to rise, reaching a maximum level by pupal stage 12. By removing the source of ecdysteroid hormone through ligation, and by subsequent replacement of 20‐HE via infusion, we found evidence indicating that the preadult rise of 20‐HE is both necessary and sufficient for the increased levels of TβH. During the course of our study, we also identified previously unreported OA‐ir neurons. In particular, adult‐specific OA‐ir lateral cells were found, as were relatively small OA‐ir dorsal median pairs that doubled in size during adult development. Abdominal ganglia not exposed to the preadult rise in 20‐HE possessed neither the OA‐ir lateral neurons nor the somatic growth of the smaller OA‐ir median neurons. These newly described OA‐ir neurons probably contribute to the steroid‐induced elevations of TβH observed at the end of metamorphosis. J. Comp. Neurol. 424:283–296, 2000.

Distribution and development of dopamine- and octopamine-synthesizing neurons in the medicinal leech

Although the medicinal leech is a well‐studied system in which many neurons and circuits have been identified with precision, descriptions of the distributions of some of the major biogenic amines, such as dopamine (DA) and octopamine (OA), have yet to be completed. In the European medicinal leech Hirudo medicinalis and the American medicinal leech Macrobdella decora,we have presented the first immunohistochemical study of DA neurons in the entire central nervous system, and of OA‐immunoreactive (ir) neurons in the head and tail brains. Dopaminergic neurons were identified using the glyoxylic acid method and antisera to DA and its rate‐limiting synthetic enzyme tyrosine hydroxylase (TH). Octopaminergic neurons were recognized using a highly specific antiserum raised against OA. An antibody raised against DA‐β‐hydroxylase (DβH), the mammalian enzyme that converts DA to norepinephrine (NE), was found to immunostain OA‐ir neurons. This antibody appears to cross‐react with the closely related invertebrate enzyme tyramine‐β‐hydroxylase, which converts tyramine to OA, suggesting that the OA‐ir cells are indeed octopaminergic, capable of synthesizing OA. Because the DβH antiserum selectively immunostained the OA‐ir neurons, but not the DA‐synthesizing cells, our results also indicate that the DA‐ir neurons synthesize DA and not NE as their end product. The expression of TH immunoreactivity was found to emerge relatively early in development, on embryonic day 9 (47–48% of development). In contrast, OA expression remained absent as late as embryonic day 20. Higher order processes of some of the dopaminergic and octopaminergic neurons in the adult brain were observed to project to a region previously described as a neurohemal complex. Several TH‐ir processes were also seen in the stomatogastric nerve ring, suggesting that DA may play a role in the regulation of biting behavior. By mapping the distributions and developmental expression pattern of DA and OA neurons in the leech, we aim to gain a better understanding of the functional roles of aminergic neurons and how they influence behavior.J. Comp. Neurol. 442:115–129, 2002.

Dopamine-synthesizing neurons include the putative H-cell homologue in the moth Manduca sexta

The catecholamine dopamine (DA) plays a fundamental role in the regulation of behavior and neurodevelopment across animal species. Uncovering the embryonic origins of neurons that express DA opens a path for a deeper understanding of how DA expression is regulated and, in turn, how DA regulates the activities of the nervous system. In a well‐established insect model, Manduca sexta, we identified the putative homologue of the embryonic grasshopper “H‐cell” using intracellular techniques, laser scanning confocal microscopy, and immunohistochemistry. In both species, this neuron possesses four axons and has central projections resembling the letter H. The H‐cell in grasshoppers is known to be derived from the midline precursor 3 cell (MP3) and to pioneer the pathways of the longitudinal connectives; in Drosophila, the H‐cell is also known to be derived from MP3. In the current study, we demonstrate that the Manduca H‐cell is immunoreactive to antibodies raised against DA and its rate‐limiting synthetic enzyme, tyrosine hydroxylase (TH). In larvae and adults, one DA/TH‐immunoreactive (‐ir) H‐cell per ganglion is present. In embryos, individual ganglia contain a single midline TH‐ir cell body positioned along side its putative sibling. Such observations are consistent with the known secondary transformation (in grasshoppers) of only one of the two MP3 progeny during early development. Although a hallmark feature of invertebrate neurons is the fairly stereotypical position of neuronal somata, we found that the H‐cell somata can “flip‐flop” by 180° between an anterior and posterior position. This variability appears to be random and is not restricted to any particular ganglion. Curiously, what is segment‐specific is the absence of the DA/TH‐ir H‐cell in the metathoracic (T3) ganglion as well as the unique structure of the H‐cell in the subesophageal ganglion. Because this is the first immunohistochemical study of DA neurons in Manduca, we have provided the distribution pattern and morphologies of dopaminergic neurons, in addition to the H‐cells, within the ventral nerve cord during development. J. Comp. Neurol. 430:501–517, 2001.

Novel mouse IgG-like immunoreactivity expressed by neurons in the moth Manduca sexta: Developmental regulation and colocalization with crustacean cardioactive peptide

Immunoglobulin‐related molecules have been shown to play important roles in cell‐cell recognition events during the development of both vertebrate and invertebrate nervous systems. In the moth, Manduca sexta, we report the presence of novel, mouse, immunoglobulin G (mIgG)‐like immunoreactivity in a discrete population of identified neurosecretory neurons (the NS‐Ls also known as the cell 27s) and interneurons (the IN‐704s). A number of polyclonal anti‐mIgG antibodies were used to immunostain these cells in wholemount. The mIgG‐like‐immunoreactive (IR) neurons were present during embryogenesis through the developing adult stages, but disappeared in the postemerged adult. Biochemical analysis of M. sexta ventral nerve cords revealed that the mIgG‐like antigen is a membrane‐associated 27‐kDa protein which is likely responsible for the mIgG‐like immunostaining observed.

Dopamine Signaling in the Bee

Dopamine (DA) is a signaling molecule derived from the amino acid tyrosine. It is an important neuromodulator, neurotransmitter and neurohormone in invertebrates as well as in vertebrates and numerous studies suggest roles for this amine in motor function, learning and memory, aggression, arousal and sleep, and in a number of other behaviors. A growing body of evidence suggests that DA plays a diversity of roles also in Apis mellifera. Three honey bee DA receptor genes have been cloned and characterized. In this chapter we focus on their likely involvement in the regulation of locomotor activity, ovary development, and olfactory learning and memory.

Hormone-dependent expression of fasciclin II during ganglionic migration and fusion in the ventral nerve cord of the moth Manduca sexta

The ventral nerve cord of holometabolous insects is reorganized during metamorphosis. A prominent feature of this reorganization is the migration of subsets of thoracic and abdominal larval ganglia to form fused compound ganglia. Studies in the hawkmoth Manduca sexta revealed that pulses of the steroid hormone 20‐hydroxyecdysone (20E) regulate ganglionic fusion, but little is known about the cellular mechanisms that make migration and fusion possible. To test the hypothesis that modulation of cell adhesion molecules is an essential component of ventral nerve cord reorganization, we used antibodies selective for either the transmembrane isoform of the cell adhesion receptor fasciclin II (TM‐MFas II) or the glycosyl phosphatidylinositol‐linked isoform (GPI‐MFas II) to study cell adhesion during ganglionic migration and fusion. Our observations show that expression of TM‐MFas II is regulated temporally and spatially. GPI‐MFas II was expressed on the surface of the segmental ganglia and the transverse nerve, but no evidence was obtained for regulation of GPI‐MFas II expression during metamorphosis of the ventral nerve cord. Manipulation of 20E titers revealed that TM‐MFas II expression on neurons in migrating ganglia is regulated by hormonal events previously shown to choreograph ganglionic migration and fusion. Injections of actinomycin D (an RNA synthesis inhibitor) or cycloheximide (a protein synthesis inhibitor) blocked ganglionic movement and the concomitant increase in TM‐MFas II, suggesting that 20E regulates transcription of TM‐MFas II. The few neurons that showed TM‐MFas II immunoreactivity independent of endocrine milieu were immunoreactive to an antiserum specific for eclosion hormone (EH), a neuropeptide regulator of molting. J. Comp. Neurol. 509:319–339, 2008.