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Title: Specification of Drosophila neuropeptidergic neurons by the splicing component brr2}
Article:
Abstract:
During embryonic development, a number of genetic cues act to generate neuronal diversity. While intrinsic transcriptional cascades are well-known to control neuronal sub-type cell fate, the target cells can also provide critical input to specific neuronal cell fates. Such signals, denoted retrograde signals, are known to provide critical survival cues for neurons, but have also been found to trigger terminal differentiation of neurons. One salient example of such target-derived instructive signals pertains to the specification of the Drosophila FMRFamide neuropeptide neurons, the Tv4 neurons of the ventral nerve cord. Tv4 neurons receive a BMP signal from their target cells, which acts as the final trigger to activate the FMRFa gene. A recent FMRFa-eGFP genetic screen identified several genes involved in Tv4 specification, two of which encode components of the U5 subunit of the spliceosome: brr2( l( 3) 72Ab) and Prp8. In this study, we focus on the role of RNA processing during target-derived signaling. We found that brr2 and Prp8 play crucial roles in controlling the expression of the FMRFa neuropeptide specifically in six neurons of the VNC( Tv4 neurons). Detailed analysis of brr2 revealed that this control is executed by two independent mechanisms, both of which are required for the activation of the BMP retrograde signaling pathway in Tv4 neurons:( 1) Proper axonal pathfinding to the target tissue in order to receive the BMP ligand.( 2) Proper RNA splicing of two genes in the BMP pathway: the thickveins( tkv) gene, encoding a BMP receptor subunit, and the Medea gene, encoding a co-Smad. These results reveal involvement of specific RNA processing in diversifying neuronal identity within the central nervous system.
Introduction:
While transcriptional networks and signalling pathways have been a primary focus during the neural specification processes, relatively little is known about how post-transcriptional modulations control the identity of individual cells. Splicing mechanisms have been widely studied and are known to play an essential role, not only during general mRNA processing, but also as a a regulatory mechanism for generating cellular diversity during development[1–4]. Alternative splicing plays a major part in the generation of protein variation, through mRNA isoform generation, and can also act as a regulatory element, by establishing splicing patterns of batteries of genes specific to certain cell types, tissues or even organisms[5]. Salient examples of alternate splicing patterns stem from Drosophila, where sex determination is regulated by dimorphic splicing of pre-mRNAs for Sex lethal, transformer, doublesex and fruitless[6]. In addition, splicing factors can specify identity in a tissue, such as the splicing factor embryonic lethal abnormal vision( elav), which promotes the production of the Ngr180 neuroglia-specific isoform in the nervous system, as opposed to the ubiquitous Ngr167 isoform[7]. However, to what extent splicing determines specific cell identities is not well understood. The Drosophila Ap cluster is an excellent model for studying mechanisms involved in neural specification in the central nervous system( CNS). The Ap cluster is a well-studied subset of four neurons located laterally in each of the thoracic hemisegments( T1-T3), readily identifiable by the expression of the factors Ap and Eyes absent( Eya). All four Ap neurons are part of the last-born cells of the NB5-6T neuroblast, within which extensive regulatory cascades have been shown to determine the specific fate of each of the neurons of this cluster. These include the Tv1 and Tv4 neuropeptidergic neurons( expressing Nplp1 and FMRFamide, respectively), and also the Tv2 and Tv3 neurons[8–19]. Intriguingly, the Tv4 neuron requires a target-derived retrograde signal, mediated by BMP ligand Glass bottom boat( Gbb) activation of BMP signaling, to terminally differentiate and activate the FMRFa gene[11, 20]. This makes FMRFa expression in Tv4 neurons an excellent model for addressing the molecular genetic aspects of retrograde instructive signals during nervous system development. A recent genetic screen looking for genes involved in Tv4 specification, using an FMRFa-eGFP transgene, identified the genes l( 3) 72Ab( FlyBase denomination; herein referred to as brr2) and Prp8[21]. brr2 encodes a small ribonucleoprotein( snRNP) type DExD/H Box ATPase with helicase activity, while Prp8 encodes a PROCT domain protein, both of which associate with the snRNAs U5 complex and are principal components of the spliceosome[22]. Apart from their well-established role in splicing, most notably in yeast, very little is known about Brr2 and Prp8 function. The human ortholog of brr2, BRR2 has been described to be involved in retinitis pigmentosa disease( RP)[23, 24], but no role in cell specification. Here, we address the function of brr2 and Prp8, focusing on brr2 and its putative role in RNA processing in the acquisition of Tv4 neuronal fate. Our results conclude that brr2 plays a key and highly specific role controlling the specification of the Tv4 neuron identity. This control is fulfilled by two mechanisms that lead to the activation of the BMP retrograde signaling pathway:( 1) correct axonal pathfinding to the target tissue, essential to receive the BMP ligand and,( 2) specific RNA processing of the transcripts encoding the BMP receptor subunit Thickveins( Tkv) and Medea, part of the Smad complex, both essential for TGF-β receptor signaling pathway activity. These data demonstrate that specific RNA processing plays a key role in ensuring proper TGFβ/BMP target-derived signalling, and extends our knowledge of the post-transcriptional mechanisms involved in diversifying neuronal identity.
Discussion:
In this study, we describe a cell-specific role of Brr2 in controlling the specification of the neuropeptidergic identity of the Tv4 neuron in the embryonic Drosophila Central Nervous System( CNS). This control is achieved through its role in three different steps necessary for the Tv4 neuron specification:( 1) the axon pathfinding;( 2) Dimm expression; and( 3) activation of the BMP signalling. The role of Brr2 in the spliceosome complex has been widely studied and it is known to be essential for proper RNA processing[22, 40]. Brr2 has also been identified as a fundamental component of the alternative splicing process in Drosophila[17], and in addition, brr2 expression is ubiquitous in Drosophila[36, 37]. These findings prompted us to anticipate a widely defective splicing pattern. However, our RNA-Seq analysis revealed that a limited number of genes were affected. Furthermore, our genetic analysis show that while brr2 is necessary for proFMRFa and Nplp1 expression in the Ap cluster, expression of these neuropeptides in other cells in the VNC( SE2 cells for proFMRFa and dAp for Nplp1) was not affected. In addition, after a detailed analysis of pMad expression along the VNC of brr2 mutants, we are not able to find differences with respect to the wild type pMad expression. This set of results demonstrates that the roles of ubiquitous components of the spliceosome can be highly cell-type selective. Why Brr2 activity is restricted to promoting BMP signalling only in the Tv4 neuron of the Ap cluster, but not in other areas of the VNC, remains unclear. tkv and Medea are key components of the BMP signalling pathway and act in a number of neurons projecting out of the CNS. However, the number of pMad-positive neurons is not affected in brr2 mutants. Protein-protein interaction has been described between Eya and Brr2 in a co-immunoprecipitation analysis in Drosophila[41]. The same study also described a similar interaction between Prp8 and Ap. It is tempting to speculate that these interactions could allow specific recruitment/activity of the spliceosome complex in Ap cluster cells, granting efficiency and specificity to this component of the spliceosome. The association between transcription and splicing is frequent and increases expression efficiency[42, 43]. Examples of these phenomena can be found in Drosophila Mef2 transcription factor family, where integration of both processes is involved in muscle fibre specification[2]. Moreover, cell-specific splicing events have been previously reported in the Drosophila nervous system, for instance with regards to splicing of the Dscam gene[44–46]. Although the role of brr2 in the Tv4 neuron has been delimitated with high resolution in the case of the post-transcriptional effects upon tkv and Medea, the underlying mechanisms in the axonal pathfinding defects and the lack of Dimm expression observed in Tv4 neurons remains unresolved. Regarding the pathfinding phenotype, the RNA-Seq analysis in this study revealed significant changes in 8 genes and splicing defects in 57 genes related to axon guidance. The specific role, if any, of each of these genes in the Ap cluster is unknown. Nevertheless, our study has identified clear candidate genes which, together with the well-described axonal pathfinding of Tv4, provide an excellent starting point for studying axonal guidance. Using specific mutant backgrounds and co-expression of different splicing variants identified in this study, it would be possible to shed light into the function of those genes, not only at the expression level but with regards to different isoforms, as have been previously described for the axon pathfinding gene lola[47, 48]. With respect to Dimm, our RNA-Seq analysis did not identify any defects in known genes which could explain the lack of its expression, which suggest that further analysis is required to identify more genes related to Nplp1 and proFMRFa neuropeptide expression. The low efficiency of the rescue mediated via expression of active forms of tkv and sax( apterous-Gal4>saxA, tkvA) suggests that Brr2 could be regulating additional components downstream of pMad. The nuclear localization of pMad in a subset of cells where proFMRFa was not restored suggests that the splicing defects in Medea are not interfering with its function, at least with respect to its translocation to the nucleus. Indeed, these results match those from in vitro studies wherein this mRNA isoform encodes a Medea protein with apparently normal DNA-binding activity[39]. Nevertheless, its behavior in vivo is unknown and we cannot rule out that changes in the Medea splicing may affect the function of the Med/pMad complex in other ways, maybe related to cofactor binding as well as DNA-binding. The specification of Tv4 neurons requires an elaborate gene cascade, which converges to activate the expression of the FMRFa neuropeptide gene[8, 10–14, 32]. One of the necessary components in this cascade is retrograde BMP signalling, and a number of events are necessary to activate the BMP signalling in the Tv4 neuron. Here, we demonstrate how the brr2 gene regulates this process at three different points: Control of pathfinding of the Tv4 axon, which is necessary for BMP ligand reception and regulation of proper splicing of two crucial players of the BMP signalling pathway, tkv and Medea. In summary, here we report post-transcriptional mechanisms that sculpt neural architecture at a cell-specific level, controlling precise aspects of cell development that together define precise and discrete cell identities. This increases our understanding of how alternative splicing is utilized during neural development. Future studies will be needed to determine if similar cell-specific splicing mechanisms is utilized in other lineages contributing to neural diversity, and emerging new technologies and bioinformatics for RNA studies will facilitate this work.
The nervous system displays daunting cellular diversity , largely generated through complex regulatory input operating on stem cells and their neural lineages during development . Most of the reported mechanisms acting to generate neural diversity pertain to transcriptional regulation . In contrast , little is known regarding the post-transcriptional mechanisms involved . Here , we use a specific group of neurons , Apterous neurons , in the ventral nerve cord of Drosophila melanogaster as our model , to analyze the function of two essential components of the spliceosome; Brr2 and Prp8 . Apterous neurons require a BMP retrograde signal for terminal differentiation , and we find that brr2 and Prp8 play crucial roles during this process . brr2 is critical for two independent events; axon pathfinding and BMP signaling , both of which are required for the activation of the retrograde signaling pathway necessary for Apterous neurons . These results identify a post-transcriptional mechanism as key for specifying neuronal identity , by ensuring the execution of a retrograde signal .