Axon length maintenance and synapse integrity are regulated by c-AMP-dependent protein kinase A (PKA) during larval growth of the drosophila sensory neurons.

Axonal extension and synaptic targeting are usually completed during early development, but the axonal length and synaptic integrity need to be actively maintained during later developmental stages and the adult life. Failure in the axonal length maintenance and the subsequent axonal degeneration have been associated with neurological disorders, but currently little is known about the genetic factors controlling this process. Here, we show that regulated intracellular levels of cAMP-dependent protein kinase A (PKA) are critical for the axon maintenance during the transition from the early to the later larval stages in the Drosophila class IV dendritic arborization (da) sensory neurons. Our data indicate that when the intracellular levels of PKA are increased via genetic manipulations, these peripheral neurons initially form synapses with wild-type appearance, at their predicted ventral nerve cord (VNC) target sites (in the first and second instar larval stages), but that their synapses disintegrate, and the axons retract and become fragmented in the subsequent larval stages (third larval stage). The affected axonal endings at the disintegrated synaptic sites still express the characteristic presynaptic and cytoskeletal markers such as Bruchpilot and Fascin, indicating that the synapse had been initially properly formed, but that it later lost its integrity. Finally, the phenotype is significantly more prominent in the axons of the neurons whose cell bodies are located in the posterior body segments. We propose that the reason for this is the fact that during the larval development the posterior neurons face a much greater challenge while trying to keep up with the fast-paced growth of the larval body, and that PKA is critical for this process. Our data reveal PKA as a novel factor in the axonal length and synapse integrity maintenance in sensory neurons. These results could be of help in understanding neurological disorders characterized by destabilized synapses.

Impairments in dendrite morphogenesis as etiology for neurodevelopmental disorders and implications for therapeutic treatments.

Dendrite morphology is pivotal for neural circuitry functioning. While the causative relationship between small-scale dendrite morphological abnormalities (shape, density of dendritic spines) and neurodevelopmental disorders is well established, such relationship remains elusive for larger-scale dendrite morphological impairments (size, shape, branching pattern of dendritic trees). Here, we summarize published data on dendrite morphological irregularities in human patients and animal models for neurodevelopmental disorders, with focus on autism and schizophrenia. We next discuss high-risk genes for these disorders and their role in dendrite morphogenesis. We finally overview recent developments in therapeutic attempts and we discuss how they relate to dendrite morphology. We find that both autism and schizophrenia are accompanied by dendritic arbor morphological irregularities, and that majority of their high-risk genes regulate dendrite morphogenesis. Thus, we present a compelling argument that, along with smaller-scale morphological impairments in dendrites (spines and synapse), irregularities in larger-scale dendrite morphology (arbor shape, size) may be an important part of neurodevelopmental disorders' etiology. We suggest that this should not be ignored when developing future therapeutic treatments.

Importance of gene dosage in controlling dendritic arbor formation during development.

Proper dendrite morphology is crucial for normal nervous system functioning. While a number of genes have been implicated in dendrite morphogenesis in both invertebrates and mammals, it remains unclear how developing dendrites respond to changes in gene dosage and what type of patterns their responses may follow. To understand this, I review here evidence from the recent literature, focusing on the genetic studies performed in the Drosophila larval dendritic arborization class IV neuron, an excellent cell type to understand dendrite morphogenesis. I summarize how class IV arbors change morphology in response to developmental fluctuations in the expression levels of 47 genes, studied by means of genetic manipulations such as loss-of-function and gain-of-function, and for which sufficient information is available. I find that arbors can respond to changing gene dosage in several distinct ways, each characterized by a singular dose-response curve. Interestingly, in 72% of cases arbors are sensitive, and thus adjust their morphology, in response to both decreases and increases in the expression of a given gene, indicating that dendrite morphogenesis is a process particularly sensitive to gene dosage. By summarizing the parallels between Drosophila and mammals, I show that many Drosophila dendrite morphogenesis genes have orthologs in mammals, and that some of these are associated with mammalian dendrite outgrowth and human neurodevelopmental disorders. One notable disease-related molecule is kinase Dyrk1A, thought to be a causative factor in Down syndrome. Both increases and decreases in Dyrk1A gene dosage lead to impaired dendrite morphogenesis, which may contribute to Down syndrome pathoetiology.

Developmental shaping of dendritic arbors in Drosophila relies on tightly regulated intra-neuronal activity of protein kinase A (PKA).

Dendrites develop morphologies characterized by multiple levels of complexity that involve neuron type specific dendritic length and particular spatial distribution. How this is developmentally regulated and in particular which signaling molecules are crucial in the process is still not understood. Using Drosophila class IV dendritic arborization (da) neurons we test in vivo the effects of cell-autonomous dose-dependent changes in the activity levels of the cAMP-dependent Protein Kinase A (PKA) on the formation of complex dendritic arbors. We find that genetic manipulations of the PKA activity levels affect profoundly the arbor complexity with strongest impact on distal branches. Both decreasing and increasing PKA activity result in a reduced complexity of the arbors, as reflected in decreased dendritic length and number of branching points, suggesting an inverted U-shape response to PKA. The phenotypes are accompanied by changes in organelle distribution: Golgi outposts and early endosomes in distal dendritic branches are reduced in PKA mutants. By using Rab5 dominant negative we find that PKA interacts genetically with the early endosomal pathway. We test if the possible relationship between PKA and organelles may be the result of phosphorylation of the microtubule motor dynein components or Rab5. We find that Drosophila cytoplasmic dynein components are direct PKA phosphorylation targets in vitro, but not in vivo, thus pointing to a different putative in vivo target. Our data argue that tightly controlled dose-dependent intra-neuronal PKA activity levels are critical in determining the dendritic arbor complexity, one of the possible ways being through the regulation of organelle distribution.

Cytokine signaling through the JAK/STAT pathway is required for long-term memory in Drosophila.

Cytokine signaling through the JAK/STAT pathway regulates multiple cellular responses, including cell survival, differentiation, and motility. Although significant attention has been focused on the role of cytokines during inflammation and immunity, it has become clear that they are also implicated in normal brain function. However, because of the large number of different genes encoding cytokines and their receptors in mammals, the precise role of cytokines in brain physiology has been difficult to decipher. Here, we took advantage of Drosophila's being a genetically simpler model system to address the function of cytokines in memory formation. Expression analysis showed that the cytokine Upd is enriched in the Drosophila memory center, the mushroom bodies. Using tissue- and adult-specific expression of RNAi and dominant-negative proteins, we show that not only is Upd specifically required in the mushroom bodies for olfactory aversive long-term memory but the Upd receptor Dome, as well as the Drosophila JAK and STAT homologs Hop and Stat92E, are also required, while being dispensable for less stable memory forms.

Knockdown of spalt function by RNAi causes de-repression of Hox genes and homeotic transformations in the crustacean Artemia franciscana.

Hox genes play a central role in the specification of distinct segmental identities in the body of arthropods. The specificity of Hox genes depends on their restricted expression domains, their interaction with specific cofactors and selectivity for particular target genes. spalt genes are associated with the function of Hox genes in diverse species, but the nature of this association varies: in some cases, spalt collaborates with Hox genes to specify segmental identities, in others, it regulates Hox gene expression or acts as their target. Here we study the role of spalt in the branchiopod crustacean Artemia franciscana. We find that Artemia spalt is expressed in the pre-segmental 'growth zone' and in stripes in each of the trunk (thoracic, genital and post-genital) segments that emerge from this zone. Using RNA interference (RNAi), we show that knocking down the expression of spalt has pleiotropic effects, which include thoracic to genital (T-->G), genital to thoracic (G-->T) and post-genital to thoracic (PG-->T) homeotic transformations. These transformations are associated with a stochastic de-repression of Hox genes in the corresponding segments of RNAi-treated animals (AbdB for T-->G and Ubx/AbdA for G-->T and PG-->T transformations). We discuss a possible role of spalt in the maintenance of Hox gene repression in Artemia and in other animals.

Expression of hunchback during trunk segmentation in the branchiopod crustacean Artemia franciscana.

Comparative studies have shown that some aspects of segmentation are widely conserved among arthropods. Yet, it is still unclear whether the molecular prepatterns that are required for segmentation in Drosophila are likely to be similarly conserved in other arthropod groups. Homologues of the Drosophila gap genes, like hunchback, show regionally restricted expression patterns during the early phases of segmentation in diverse insects, but their expression patterns in other arthropod groups are not yet known. Here, we report the cloning of a hunchback orthologue from the crustacean Artemia franciscana and its expression during the formation of trunk segments. Artemia hunchback is expressed in a series of segmental stripes that correspond to individual thoracic/trunk, genital, and postgenital segments. However, this expression is not associated with the segmenting ectoderm but is restricted to mesodermal cells that associate with the ectoderm in a regular metameric pattern. All cells in the early segmental mesoderm appear to express hunchback. Later, mesodermal expression fades, and a complex expression pattern appears in the central nervous system (CNS), which is comparable to hunchback expression in the CNS of insects. No regionally restricted expression, reminiscent of gap gene expression, is observed during trunk segmentation. These patterns suggest that the expression patterns of hunchback in the mesoderm and in the CNS are likely to be ancient and conserved among crustaceans and insects. In contrast, we find no evidence for a conserved role of hunchback in axial patterning in the trunk ectoderm.

Ancestral role of caudal genes in axis elongation and segmentation.

caudal (cad/Cdx) genes are essential for the formation of posterior structures in Drosophila, Caenorhabditis elegans, and vertebrates. In contrast to Drosophila, the majority of arthropods generate their segments sequentially from a posteriorly located growth zone, a process known as short-germ development. caudal homologues are expressed in the growth zone of diverse short-germ arthropods, but until now their functional role in these animals had not been studied. Here, we use RNA interference to examine the function of caudal genes in two short-germ arthropods, the crustacean Artemia franciscana and the beetle Tribolium castaneum. We show that, in both species, caudal is required for the formation of most body segments. In animals with reduced levels of caudal expression, axis elongation stops, resulting in severe truncations that remove most trunk segments. We also show that caudal function is required for the early phases of segmentation and Hox gene expression. The observed phenotypes suggest that in arthropods caudal had an ancestral role in axis elongation and segmentation, and was required for the formation of most body segments. Similarities to the function of vertebrate Cdx genes in the presomitic mesoderm, from which somites are generated, indicate that this role may also predate the origin of the Bilateria.

Posterior patterning genes and the identification of a unique body region in the brine shrimp Artemia franciscana.

All arthropods share the same basic set of Hox genes, although the expression of these genes differs among divergent groups. In the brine shrimp Artemia franciscana, their expression is limited to the head, thoracic/trunk and genital segments, but is excluded from more posterior parts of the body which consist of six post-genital segments and the telson (bearing the anus). Nothing is currently known about the genes that specify the identity of these posterior structures. We examine the expression patterns of four candidate genes, Abdominal-B, caudal/Cdx, even-skipped/Evx and spalt, the homologues of which are known to play an important role in the specification of posterior structures in other animals. Abdominal-B is expressed in the genital segments of Artemia, but not in the post-genital segments at any developmental stage. The expression of caudal, even-skipped and spalt in the larval growth-zone suggests they may play a role in the generation of body segments (perhaps comparable with the role of gap and segmentation genes in insects), but not a direct role in defining the identity of post-genital segments. The expression of caudal at later stages suggests a role in the specification of anal structures. A PCR screen designed to isolate Hox genes expressed specifically in the posterior part of the body failed to identify any new Hox genes. We conclude that the post-genital segments of Artemia are not defined by any of the genes known to play a role in the specification of posterior segments in other arthropods. We argue that these segments constitute a unique body region that bears no obvious homology to previously characterised domains of Hox gene activity.