Relevant Degree Programs
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - May 2019)
Terminal differentiation of neurons often requires retrograde signals from the target cells they innervate, which trigger neural subtype-specific gene expression upon target contact. Target-derived BMP signaling and transcription factors including the LIM-Homeodomain transcription factor Apterous are required for FMRFa neuropeptide gene expression in Drosophila Tv4 neurons. We modeled the integrative mechanism of these extrinsic and intrinsic inputs at a Tv4 neuron-specific FMRFa enhancer. We show that Tv4-specific FMRFa expression requires two separable cis-elements, a BMP-response element (BMP-RE) that binds Mad, and a homeodomain response element (HD-RE) that binds Apterous. Strikingly, we find that concatemers of these two short (~30bp) cis-elements each independently drive spatial and temporal expression appropriate for Tv4-specific FMRFa. Thus, specific and robust expression is generated from the synergy of two low-activity heterotypic cis-elements that encode the same output from distinct inputs. We further examined the timing mechanism of FMRFa initiation, which models predict would be solely based on target contact. In contrast, we find that the timed downregulation of the COUP-TF I/II nuclear receptor Seven up functions to de-repress HD-RE and BMP-RE activity immediately prior to target contact. Thus, we reveal that the active suppression of neurotransmitter identity, prior to target contact, is an innate component of the target-dependent mechanism for timed gene activation.Further examination of the FMRFa BMP-RE shows that the sequence of the Mad and Medea binding sites to be “perfectly wrong.” It displays sequence similarity to both the previously characterized BMP Silencing Element (SE) and Activating Elements (AE) but with base substitutions that should abrogate Smad binding. Biochemical and reporter construct analysis demonstrate that the FMRFa BMP-RE is an atypical activating element that is specifically attenuated in its ability to interact with the co-Smad Medea. In vivo reporter assays show that this attenuation is required to drive appropriate expression in the CNS. These findings represent only the third verified type of BMP-RE found in Drosophila leading us to call this class an AE2 element.
Over the past years, numerous studies have advanced our understanding of the generation and function of the sex-specific neuronal populations that control sex-specific behaviours. Prior to the work presented in this thesis, no female-specific subsets of neurons had been identified in Drosophila; thus, all models and studies of sex-specific neurons have had a male bias.This thesis describes the first identification and characterization of a female-specific neuronal population in the central nervous system of Drosophila, the Ilp7-motoneurons. These neurons innervate the oviduct and are required for egg-laying. We further identified cellular and genetic mechanisms that direct the dimorphic generation of these female-specific neurons. Programmed cell death of post-mitotic nascent Ilp7-motoneurons in males accounts for their female-specific generation in a process regulated by a non-canonical and dosage-sensitive pro-apoptotic role for the male fruitless isoform (fruM). Thus, we find that analysis of female-specific neuron generation unveils novel mechanisms of dimorphic nervous system construction.Our characterization of Ilp7-motoneurons led to a collaboration with Eric Lai (Sloan Kettering, USA), to study the neuronal basis of the female sterility phenotype of the ∆mir mutant, a deficiency in the bidirectional mir-iab-4 and mir-iab-8 miRNA locus of the Bithorax-Complex. We find that female sterility arises from derepression of mir-iab-4/8 targets, Ultrabithorax and homothorax, in fru-expressing neuronal populations of the posterior abdominal segments of the ventral nerve cord. This results in numerous phenotypes that each likely contribute to sterility. ∆mir females have reduced Ilp7-motor innervation of the oviduct. ∆mir virgin females are constitutively unreceptive to males; however, if mated, they fail to increase egg production. Our data suggests a novel mechanism that may explain this phenotype; after mating, sex peptide from the male seminal fluid is retained in the female reproductive tract, rather than being transferred to the hemolymph, where it is believed to effect the increase in egg production. Ongoing work aims to identify the neuronal populations that are disrupted in ∆mir mutants. Taken together, this thesis provides novel insight and models to further our understandingof female-specific neuronal differentiation, a field that has long been under-represented in theliterature.
Appropriate integration of neurons into functioning networks is the ultimate goal ofneuronal differentiation. My thesis examined the mechanism and timing of neuronaldifferentiation in Drosophila melanogaster in relation to the functional requirements of adeveloping neuronal network.Specifically, my thesis aimed to address the role of extrinsic signaling in inducing andmaintaining the expression of genes important to the function of neurons within their network.CCAP-neurons were chosen as a model because: 1) GAL4 drivers are available for cell-specificgenetic manipulation. 2) The critical role of CCAP-neurons in the behavior, ecdysis, provides foran easily assayed phenotype if these neurons fail to function properly. 3) Four peptide hormonesare selectively expressed in differentiated CCAP-neurons that are essential for the normalfunction of CCAP-neurons in ecdysis; this provides a direct link between gene expression andbehavior. 4) Ecdysis is reiterated at multiple developmental steps, thus the CCAP-neuronalpopulation functions throughout development. Together, these factors allow my work to relateneuronal subtype-specific differentiation to the regulation of gene expression and then directly tobehavior.Larval Drosophila CCAP-neurons comprise ~46 neurons [~36 interneurons (CCAP-INs)and 10 efferent-neurons (CCAP-ENs)] that express a number of terminal differentiation genes(TDGs; such as neuropeptides). To begin, we delineated mechanisms underlying the expressionof four TDGs, the peptide hormones CCAP, MIP, Bursicon-α and Bursicon-β, which togethermediate the functional output of those neurons. Importantly, my studies found that a specificsubset of CCAP-neurons, the CCAP-ENs, is both necessary and sufficient for ecdysis, and thattheir function in ecdysis is mediated by extrinsic BMP-dependent peptide hormone expression.Additionally, we found that the change in the ecdysis behavioral sequence from larval to pupalecdysis is supported by the recruitment of a ‘late’ subset of CCAP-neurons that are born in theembryo but undergo extrinsic ecdysone-triggered, temporally-tuned differentiation immediatelyprior to pupal ecdysis.
The diversification of cellular subtype during development is directed by combinatorially acting transcription factors and signaling pathways that act to regulate subtype specific gene expression profiles in post-mitotic cells. These key transcription factors and signaling pathways operate in a transcriptional network, which act to establish cellular subtype identity over the course of a developing cellular lineage. Lineage progression towards ever increasing cellular diversity is often viewed as a ratchet mechanism of irreversible steps resulting in the specification and then terminal differentiation of cell subtype identities. From this viewpoint, terminally differentiated cells have long been considered as irreversibly locked into their identity. In a landmark article, Blau and Baltimore (Blau and Baltimore, 1991) postulated that a cell’s identity, or differentiated status, requires persistent active regulation, rather than lapsing into a passive ‘locked-in’ state. While little genetic evidence was available at the time, sufficient evidence has since accumulated to propose that the terminally differentiated state, or identity, of a cell subtype indeed requires active maintenance. Currently, however, we have only the most rudimentary understanding of the regulatory mechanisms that maintain neuronal identity. This thesis presents a systematic effort to characterize the role of the transcriptional networks that differentiate neuronal identity in the mature neurons of the adult nervous system. Using the Drosophila Tv cluster neurons I show the persistent requirement of 1) target derived signals and 2) networks of transcription factors for the maintenance of the cellular subtype specific expression profiles of terminal differentiation genes, genes that define these neuron’s function and identity. This work establishes one of the most comprehensive transcriptional models for maintenance of cell identity to date. It also provides novel mechanistic insights showing that cellular differentiation is a persistent process that requires active maintenance, rather than being passively ‘locked-in’ or unalterable. As such, the work of this thesis provides critical insight that provides a strong foundation for further efforts to determine how neuronal identity is maintained.
Master's Student Supervision (2010 - 2018)
Differences in the number, morphology and function of neurons between the sexes underlie sexually dimorphic behaviours and physiology. In Drosophila, neuronal sexual dimorphism is determined by the sex determination cascade, most of which occurs downstream of Transformer (Tra). Tra is only expressed in females where it splices the sex determination effectors, fruitless (fru) and doublesex (dsx), into female-specific isoforms (non-coding fruF and coding dsxF transcripts). In males, Tra is not expressed, which leads to default splicing of fru and dsx into male-specific isoforms (coding fruM and dsxM transcripts). Sex-specific isoforms of Fru and Dsx direct most, if not all, sex differences during development of the nervous system. The development of the male nervous system has been well-studied, whereas the mechanisms that give rise to female-specific dimorphisms have been less researched. We used a subset of insulin-like peptide 7 (Ilp7)-expressing neurons as a model for studying the development of neuronal sex differences. These neurons express fru but not dsx, and innervate the reproductive organs. Using a genetic approach, we found novel roles for tra and fru in generating a female-specific ventral subset of Ilp7 neurons (FS-Ilp7 neurons). We found FruM-dependent male-specific programmed cell death (PCD) of FS-Ilp7 neurons underlies their female-specific generation. Furthermore, we found that FruM is necessary for serotonergic differentiation and for proper axonal targeting of Ilp7 neurons in males. In females, we show that forcing male-specific splicing of fru is insufficient to trigger PCD, because, unexpectedly, Tra prevents FruM-dependent PCD in two ways to ensure FS-Ilp7 neuronal survival; not only does Tra act canonically in fru splicing, but it also acts non-canonically in parallel or downstream of fru splicing to block fruM-dependent PCD. We conclude that FruM controls both neuronal numbers via PCD and arborization in post-embryonic Ilp7 neurons, and that Tra plays a novel failsafe function in females to establish and then reinforce the decision to generate female-specific neurons.
The lifelong identity and function of a neuron is dictated by the set of effector genes it expresses in its terminally differentiated state. Neuron-specific effector gene expression is established and maintained by networks of transcription factors. How elaborate cascades of transcription factors establish neuronal identities during development has been studied extensively; however, how transcription factor networks are maintained in mature neurons to maintain effector gene expression remains poorly understood. I used the well-characterized transcription factor networks in Drosophila Tv1 and Tv4 neurons to further understand how transcription factor networks are maintained in mature neurons. I focused on the transcription factors Apterous and Dimmed, and investigated the cis- and trans- regulatory transcriptional mechanisms that govern initiation and maintenance of their expression. I previously identified a 756 bp genomic region (dimm.K1) upstream of dimmed that is sufficient to initiate and maintain reporter expression in Tv1 and Tv4 neurons. Here I characterized the trans-regulatory inputs of dimm.K1 reporter expression. I found that different transcriptional inputs are required for initiation vs. maintenance of dimm.K1 reporter expression. Further, inputs required for maintenance differ not only between cell subtypes (Tv1 vs. Tv4) but also between individual cells of the same subtype (Tv1 neurons in thoracic segments 1 and 3). I also compared apterous initiation and maintenance. My previous work had identified a 3.845 kb genomic region (ap.K1) upstream of apterous that is sufficient to initiate, but not maintain, reporter expression in Tv1 and Tv4 neurons. In this thesis I show that the addition of a region with putative TRE activity to the ap.K1 initiation element is sufficient to add maintenance of reporter expression in Tv1 and Tv4 neurons. Overall, I show that a variety of mechanisms can maintain the expression of transcription factors in mature neurons, and that these are different and more varied than the mechanisms of initiation. Furthermore, for any single transcription factor, the initiation mechanisms that are shared by different cell subtypes can diverge into diverse cell-specific maintenance mechanisms.