Stefan Thor

A LIM-homeodomain combinatorial code for motor-neuron pathway selection

Different classes of vertebrate motor neuron that innervate distinct muscle targets express unique combinations of LIM-homeodomain transcription factors, suggesting that a combinatorial code of LIM-homeodomain proteins may underlie the control of motor-neuron pathway selection. Studies of LIM-homeodomain genes in mouse, Drosophila melanogaster and Caenorhabditis elegans have revealed functions of these genes in neuronal survival, axon guidance, neurotransmitter expression and neuronal function, but, to our knowledge, none of these studies have addressed the issue of a functional code. Here we study two members of this gene family in Drosophila, namely lim3, the homologue of the vertebrate Lhx3 and Lhx4 genes, and islet, thehomologue of the vertebrate Isl1 and Isl2 genes. We show that Drosophila lim3 is expressed by a specific subset of islet-expressing motor neurons and that mutating or misexpressing lim3 switches motor-neuron projections predictably. Our results provide evidence that lim3 and islet constitute a combinatorial code that generates distinct motor-neuron identities.

Genetic control of Drosophila nerve cord development.

The Drosophila ventral nerve cord has been a central model system for studying the molecular genetic mechanisms that control CNS development. Studies show that the generation of neural diversity is a multistep process initiated by the patterning and segmentation of the neuroectoderm. These events act together with the process of lateral inhibition to generate precursor cells (neuroblasts) with specific identities, distinguished by the expression of unique combinations of regulatory genes. The expression of these genes in a given neuroblast restricts the fate of its progeny, by activating specific combinations of downstream genes. These genes in turn specify the identity of any given postmitotic cell, which is evident by its cellular morphology and choice of neurotransmitter.

Segment-specific prevention of pioneer neuron apoptosis by cell-autonomous, postmitotic Hox gene activity

In vertebrates, neurons often undergo apoptosis after differentiating and extending their axons. By contrast, in the developing nervous system of invertebrate embryos apoptosis typically occurs soon after cells are generated. Here, we show that the Drosophila dMP2 and MP1 pioneer neurons undergo segment-specific apoptosis at late embryonic stages, long after they have extended their axons and have performed their pioneering role in guiding follower axons. This segmental specificity is achieved by differential expression of the Hox gene Abdominal B, which in posterior segments prevents pioneer neuron death postmitotically and cell-autonomously by repressing the RHG-motif cell death activators reaper and grim. Our results identify the first clear case of a cell-autonomous and anti-apoptotic role for a Hox gene in vivo. In addition, they provide a novel mechanism linking Hox positional information to differences in neuronal architecture along the anteroposterior axis by the selective elimination of mature neurons.

Curcumin Promotes A-beta Fibrillation and Reduces Neurotoxicity in Transgenic Drosophila

The pathology of Alzheimers disease (AD) is characterized by the presence of extracellular deposits of misfolded and aggregated amyloid-β (Aβ) peptide and intraneuronal accumulation of tangles comprised of hyperphosphorylated Tau protein. For several years, the natural compound curcumin has been proposed to be a candidate for enhanced clearance of toxic Aβ amyloid. In this study we have studied the potency of feeding curcumin as a drug candidate to alleviate Aβ toxicity in transgenic Drosophila. The longevity as well as the locomotor activity of five different AD model genotypes, measured relative to a control line, showed up to 75% improved lifespan and activity for curcumin fed flies. In contrast to the majority of studies of curcumin effects on amyloid we did not observe any decrease in the amount of Aβ deposition following curcumin treatment. Conformation-dependent spectra from p-FTAA, a luminescent conjugated oligothiophene bound to Aβ deposits in different Drosophila genotypes over time, indicated accelerated pre-fibrillar to fibril conversion of Aβ1–42 in curcumin treated flies. This finding was supported by in vitro fibrillation assays of recombinant Aβ1–42. Our study shows that curcumin promotes amyloid fibril conversion by reducing the pre-fibrillar/oligomeric species of Aβ, resulting in a reduced neurotoxicity in Drosophila.

Regulators Acting in Combinatorial Codes Also Act Independently in Single Differentiating Neurons

In the Drosophila ventral nerve cord, a small number of neurons express the LIM-homeodomain gene apterous (ap). These ap neurons can be subdivided based upon axon pathfinding and their expression of neuropeptidergic markers. ap, the zinc finger gene squeeze, the bHLH gene dimmed, and the BMP pathway are all required for proper specification of these cells. Here, using several ap neuron terminal differentiation markers, we have resolved how each of these factors contributes to ap neuron diversity. We find that these factors interact genetically and biochemically in subtype-specific combinatorial codes to determine certain defining aspects of ap neuron subtype identity. However, we also find that ap, dimmed, and squeeze additionally act independently of one another to specify certain other defining aspects of ap neuron subtype identity. Therefore, within single neurons, we show that single regulators acting in numerous molecular contexts differentially specify multiple subtype-specific traits.

Postmitotic specification of Drosophila insulinergic neurons from pioneer neurons.

Insulin and related peptides play important and conserved functions in growth and metabolism. Although Drosophila has proved useful for the genetic analysis of insulin functions, little is known about the transcription factors and cell lineages involved in insulin production. Within the embryonic central nervous system, the MP2 neuroblast divides once to generate a dMP2 neuron that initially functions as a pioneer, guiding the axons of other later-born embryonic neurons. Later during development, dMP2 neurons in anterior segments undergo apoptosis but their posterior counterparts persist. We show here that surviving posterior dMP2 neurons no longer function in axonal scaffolding but differentiate into neuroendocrine cells that express insulin-like peptide 7 (Ilp7) and innervate the hindgut. We find that the postmitotic transition from pioneer to insulin-producing neuron is a multistep process requiring retrograde bone morphogenetic protein (BMP) signalling and four transcription factors: Abdominal-B, Hb9, Fork Head, and Dimmed. These five inputs contribute in a partially overlapping manner to combinatorial codes for dMP2 apoptosis, survival, and insulinergic differentiation. Ectopic reconstitution of this code is sufficient to activate Ilp7 expression in other postmitotic neurons. These studies reveal striking similarities between the transcription factors regulating insulin expression in insect neurons and mammalian pancreatic beta-cells.

Motor neuron specification in worms, flies and mice: conserved and ‘lost’ mechanisms

Motor neuron differentiation has been studied intensively in both invertebrates and vertebrates in recent years. These studies have led to the identification of several key regulatory genes acting to generate motor neurons and to specify their subclass identities. By comparing findings from Caenorhabditis elegans, Drosophila and vertebrate model systems, it is apparent that both evolutionarily conserved and non-conserved mechanisms are used.

Specification of Drosophila motoneuron identity by the combinatorial action of POU and LIM-HD factors

In both vertebrates and invertebrates, members of the LIM-homeodomain (LIM-HD) family of transcription factors act in combinatorial codes to specify motoneuron subclass identities. In the developing Drosophila embryo, the LIM-HD factors Islet (Tailup) and Lim3, specify the set of motoneuron subclasses that innervate ventral muscle targets. However, as several subclasses express both Islet and Lim3, this combinatorial code alone cannot explain how these motoneuron groups are further differentiated. To identify additional factors that may act to refine this LIM-HD code, we have analyzed the expression of POU genes in the Drosophila embryonic nerve cord. We find that the class III POU protein, Drifter (Ventral veinless), is co-expressed with Islet and Lim3 specifically in the ISNb motoneuron subclass. Loss-of-function and misexpression studies demonstrate that the LIM-HD combinatorial code requires Drifter to confer target specificity between the ISNb and TN motoneuron subclasses. To begin to elucidate molecules downstream of the LIM-HD code, we examined the involvement of the Beaten path (Beat) family of immunoglobulin-containing cell-adhesion molecules. We find that beat Ic genetically interacts with islet and Lim3 in the TN motoneuron subclass and can also rescue the TN fasciculation defects observed in islet and Lim3 mutants. These results suggest that in the TN motoneuron context, Islet and Lim3 may specify axon target selection through the actions of IgSF call-adhesion molecules.

Segment-specific neuronal subtype specification by the integration of anteroposterior and temporal cues.

To address the question of how neuronal diversity is achieved throughout the CNS, this study provides evidence of modulation of neural progenitor cell “output” along the body axis by integration of local anteroposterior and temporal cues.

Independent roles of the dachshund and eyes absent genes in BMP signaling, axon pathfinding and neuronal specification.

In the Drosophila nerve cord, a subset of neurons expresses the neuropeptide FMRFamide related (Fmrf). Fmrf expression is controlled by a combinatorial code of intrinsic factors and an extrinsic BMP signal. However, this previously identified code does not fully explain the regulation of Fmrf. We have found that the Dachshund (Dac) and Eyes Absent (Eya) transcription co-factors participate in this combinatorial code. Previous studies have revealed an intimate link between Dac and Eya during eye development. Here, by analyzing their function in neurons with multiple phenotypic markers, we demonstrate that they play independent roles in neuronal specification, even within single cells. dac is required for high-level Fmrf expression, and acts potently together with apterous and BMP signaling to trigger Fmrf expression ectopically, even in motoneurons. By contrast, eya regulates Fmrf expression by controlling both axon pathfinding and BMP signaling, but cannot trigger Fmrf ectopically. Thus, we show that dac and eya perform entirely different functions in a single cell type to ultimately regulate a single phenotypic outcome.