Segmentally homologous neurons acquire two different terminal neuropeptidergic fates in the Drosophila nervous system

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In this study, we identify the means by which segmentally homologous neurons acquire different neuropeptide fates in Drosophila. Ventral abdominal (Va)-neurons in the A1 segment of the ventral nerve cord express DH31 and AstA neuropeptides (neuropeptidergic fate I) by virtue of Ubx activity, whereas the A2-A4 Va-neurons express the Capa neuropeptide (neuropeptidergic fate II) under the influence of abdA. These different fates are attained through segment-specific programs of neural subtype specification undergone by segmentally homologous neurons. This is an attractive alternative by which Hox genes can shape Drosophila segmental neural architecture (more sophisticated than the previously identified binary “to live” or “not to live” mechanism). These data refine our knowledge of the mechanisms involved in diversifying neuronal identity within the central nervous systemThis study was supported by grant number: BFU2013-43858-

Specification of the Drosophila Orcokinin A neurons by combinatorial coding

This is a post-peer-review, pre-copyedit version of an article published in Cell and Tissue Research. The final authenticated version is available online at: http://dx.doi.org10.1007/s00441-022-03721-xThe central nervous system contains a daunting number of different cell types. Understanding how each cell acquires its fate remains a major challenge for neurobiology. The developing embryonic ventral nerve cord (VNC) of Drosophila melanogaster has been a powerful model system for unraveling the basic principles of cell fate specification. This pertains specifically to neuropeptide neurons, which typically are stereotypically generated in discrete subsets, allowing for unambiguous single-cell resolution in different genetic contexts. Here, we study the specification of the OrcoA-LA neurons, characterized by the expression of the neuropeptide Orcokinin A and located laterally in the A1-A5 abdominal segments of the VNC. We identified the progenitor neuroblast (NB; NB5-3) and the temporal window (castor/grainyhead) that generate the OrcoA-LA neurons. We also describe the role of the Ubx, abd-A, and Abd-B Hox genes in the segment-specific generation of these neurons. Additionally, our results indicate that the OrcoA-LA neurons are “Notch Off” cells, and neither programmed cell death nor the BMP pathway appears to be involved in their specification. Finally, we performed a targeted genetic screen of 485 genes known to be expressed in the CNS and identified nab, vg, and tsh as crucial determinists for OrcoA-LA neurons. This work provides a new neuropeptidergic model that will allow for addressing new questions related to neuronal specification mechanisms in the futureThis work was supported by a grant from the MINECO (BFU2016-78327-P) to J.B-S and The University of Queensland, Australia, to S

Dachshund acts with Abdominal-B to trigger programmed cell death in the Drosophila central nervous system at the frontiers of Abd-B expression

A striking feature of the nervous system pertains to the appearance of different neural cell subtypes at different axial levels. Studies in the Drosophila central nervous system reveal that one mechanism underlying such segmental differences pertains to the segment-specific removal of cells by programmed cell death (PCD). One group of genes involved in segment-specific PCD is the Hox homeotic genes. However, while segment-specific PCD is highly precise, Hox gene expression is evident in gradients, raising the issue of how the Hox gene function is precisely gated to trigger PCD in specific segments at the outer limits of Hox expression. The Drosophila Va neurons are initially generated in all nerve cord segments but removed by PCD in posterior segments. Va PCD is triggered by the posteriorly expressed Hox gene Abdominal-B (Abd-B). However, Va PCD is highly reproducible despite exceedingly weak Abd-B expression in the anterior frontiers of its expression. Here, we found that the transcriptional cofactor Dachshund supports Abd-B-mediated PCD in its anterior domain. In vivo bimolecular fluorescence complementation analysis lends support to the idea that the Dachshund/Abd-B interplay may involve physical interactions. These findings provide an example of how combinatorial codes of transcription factors ensure precision in Hox-mediated PCD in specific segments at the outer limits of Hox expressionMinisterio de Ciencia y Educación, Grant/Award Number: PID2019-110952GB-I0

Selective role of the DNA helicase Mcm5 in BMP retrograde signaling during Drosophila neuronal differentiation

The MCM2-7 complex is a highly conserved hetero-hexameric protein complex, critical for DNA unwinding at the replicative fork during DNA replication. Overexpression or mutation in MCM2-7 genes is linked to and may drive several cancer types in humans. In mice, mutations in MCM2-7 genes result in growth retardation and mortality. All six MCM2-7 genes are also expressed in the developing mouse CNS, but their role in the CNS is not clear. Here, we use the central nervous system (CNS) of Drosophila melanogaster to begin addressing the role of the MCM complex during development, focusing on the specification of a well-studied neuropeptide expressing neuron: the Tv4/FMRFa neuron. In a search for genes involved in the specification of the Tv4/FMRFa neuron we identified Mcm5 and find that it plays a highly specific role in the specification of the Tv4/FMRFa neuron. We find that other components of the MCM2-7 complex phenocopies Mcm5, indicating that the role of Mcm5 in neuronal subtype specification involves the MCM2-7 complex. Surprisingly, we find no evidence of reduced progenitor proliferation, and instead find that Mcm5 is required for the expression of the type I BMP receptor Tkv, which is critical for the FMRFa expression. These results suggest that the MCM2-7 complex may play roles during CNS development outside of its well-established role during DNA replicatio

TFG 2013/2014

Amb aquesta publicació, EINA, Centre universitari de Disseny i Art adscrit a la Universitat Autònoma de Barcelona, dóna a conèixer el recull dels Treballs de Fi de Grau presentats durant el curs 2013-2014. Voldríem que un recull com aquest donés una idea més precisa de la tasca que es realitza a EINA per tal de formar nous dissenyadors amb capacitat de respondre professionalment i intel·lectualment a les necessitats i exigències de la nostra societat. El treball formatiu s’orienta a oferir resultats que responguin tant a paràmetres de rigor acadèmic i capacitat d’anàlisi del context com a l’experimentació i la creació de nous llenguatges, tot fomentant el potencial innovador del disseny.Con esta publicación, EINA, Centro universitario de diseño y arte adscrito a la Universidad Autónoma de Barcelona, da a conocer la recopilación de los Trabajos de Fin de Grado presentados durante el curso 2013-2014. Querríamos que una recopilación como ésta diera una idea más precisa del trabajo que se realiza en EINA para formar nuevos diseñadores con capacidad de responder profesional e intelectualmente a las necesidades y exigencias de nuestra sociedad. El trabajo formativo se orienta a ofrecer resultados que respondan tanto a parámetros de rigor académico y capacidad de análisis, como a la experimentación y la creación de nuevos lenguajes, al tiempo que se fomenta el potencial innovador del diseño.With this publication, EINA, University School of Design and Art, affiliated to the Autonomous University of Barcelona, brings to the public eye the Final Degree Projects presented during the 2013-2014 academic year. Our hope is that this volume might offer a more precise idea of the task performed by EINA in training new designers, able to speak both professionally and intellectually to the needs and demands of our society. The educational task is oriented towards results that might respond to the parameters of academic rigour and the capacity for contextual analysis, as well as to considerations of experimentation and the creation of new languages, all the while reinforcing design’s innovative potential

Análisis de nuevos mecanismos de especificación neuropeptidérgica en Drosophila melanogaster

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 20-09-2019Esta tesis tiene embargado el acceso al texto completo hasta el 20-03-2021Durante el desarrollo del sistema nervioso central (SNC) de Drosophila melanogaster se genera un numero abrumador de subtipos celulares. De hecho, cada una de las casi 400 células que componen cada hemisegmento de la cuerda nerviosa ventral (CNV) es prácticamente única. Invertebrados y vertebrados parecen compartir los principios comunes de la especificación neuronal, en los cuales las cascadas de factores de transcripción establecen patrones temporales de división en los progenitores, cuyos resultados son modificados posteriormente por las señales espaciales para generar diversidad. Finalmente, códigos combinatorios de factores selectores terminales refinan aún más las características únicas de cada célula. En esta tesis doctoral se emplean técnicas de genética clásica y de transcriptómica con el objetivo de ahondar en el conocimiento sobre la especificación neuronal. Por un lado, se han caracterizado los mecanismos básicos de desarrollo de las neuronas del sistema de la Orcokinina A estableciéndolo como un interesante modelo para el estudio de la adquisición de destinos terminales neuropeptidérgicos. Se trata de un grupo de 10 neuronas localizadas en los segmentos abdominales A1-A5 de la CNV de D. melanogaster. Estas células nacen a partir del neuroblasto (NB) 5-3 en una ventana temporal castor-grainyhead (cas-grh). Su patrón de expresión a lo largo de la CNV está determinado por los genes homeóticos Ultrabithorax (Ubx) y abdominal A (abdA). Asimismo, requieren la ausencia de expresión de Krüppel (Kr) y la inactivación de la ruta de Notch para su correcta especificación. Además, gracias a una eficiente búsqueda genética dirigida se han identificado más de 60 factores que podrían estar implicados en su cascada selectora terminal. De ellos, se han estudiado tres en mayor profundidad: Nab, Teashirt (Tsh) y Vestigial (Vg), llegando a establecer relaciones jerárquicas de activación entre ellos. Por otro lado, y utilizando las células FMRFamidérgicas Tv4 como modelo, en esta tesis doctoral se ha descrito el papel de Mcm5 como factor implicado en la especificación neuronal, siendo éste el primer trabajo que otorga esta función a una helicasa replicativa. Los estudios de expresión génica diferencial llevados a cabo a partir de la secuenciación de ARN sugieren que este factor ejerce su función a través de la activación de la ruta de TFG-β/BMPAn overwhelming number of cellular subtypes is generated during the central nervous system (CNS) of Drosophila melanogaster development. Each of the nearly 400 cells that compounds a single hemisegment of the ventral nerve cord (VNC) is almost unique. In this regard, it seems that invertebrates and vertebrates share the most basic values of neuronal specification. Different transcription factors cues establish division temporal patterns in progenitors, and the results are subsequently modified by spatial signals to generate diversity. Finally, combinatorial cascades of terminal selectors further refine the most unique characteristics of each cell. Techniques of classical genetics and transcriptomics are used in this thesis in order to deepening understand the neuronal specification rules. On the one hand, the basic mechanisms of development have been characterized in the Orcokinin A system, turning it into an interesting neuropeptidergic model for new terminal cell fate specification studies. Orcokinin A system consists of 10 neurons located in the VNC of D. melanogaster, specifically along the abdominal segments A1-A5. These cells are born from the neuroblast (NB) 5-3 in a temporal mixed window castorgrainyhead (cas-grh). The homeotic genes Ultrabithorax (Ubx) and abdominal A (abdA) determine this expression pattern throughout the VNC. These cells also require the absence of Krüppel (Kr) expression and the Notch pathway inactivation for their correct specification. In addition, thanks to a great efficient targeted screening, more than 60 transcription factors that could be involved in the terminal selector cascade have been identified. Among these, three of them have been deeper studied: Nab, Teashirt (Tsh) and Vestigial (Vg), establishing also hierarchical relations of activation between them. On the other hand, the role of Mcm5 in the specification of neuronal cell fates has been described using the FMRFamidergic Tv4 cells as a model. This study is the first work that confers this function to a replicative helicase. The differential gene expression analysis carried out from RNA sequencing assays suggest that Mcm5 is performing through the activation of the TFG-β/BMP pathway

Neuronal Cell Fate Specification by the Convergence of Different Spatiotemporal Cues on a Common Terminal Selector Cascade.

Specification of the myriad of unique neuronal subtypes found in the nervous system depends upon spatiotemporal cues and terminal selector gene cascades, often acting in sequential combinatorial codes to determine final cell fate. However, a specific neuronal cell subtype can often be generated in different parts of the nervous system and at different stages, indicating that different spatiotemporal cues can converge on the same terminal selectors to thereby generate a similar cell fate. However, the regulatory mechanisms underlying such convergence are poorly understood. The Nplp1 neuropeptide neurons in the Drosophila ventral nerve cord can be subdivided into the thoracic-ventral Tv1 neurons and the dorsal-medial dAp neurons. The activation of Nplp1 in Tv1 and dAp neurons depends upon the same terminal selector cascade: col>ap/eya>dimm>Nplp1. However, Tv1 and dAp neurons are generated by different neural progenitors (neuroblasts) with different spatiotemporal appearance. Here, we find that the same terminal selector cascade is triggered by Kr/pdm>grn in dAp neurons, but by Antp/hth/exd/lbe/cas in Tv1 neurons. Hence, two different spatiotemporal combinations can funnel into a common downstream terminal selector cascade to determine a highly related cell fate

<i>grain</i> is critical for dAp specification.

<p>(A, B) Eya and Nplp1 expression in VNCs at stage AFT. In <i>grn</i> mutants, Eya and Nplp1 expression in dAp cells is almost completely lost (long dotted bracket). (C-F) Expression of Dimm, Col and βgal (<i>ap</i>^<i>rK568</i>) in control and <i>grn</i> mutants. In <i>grn</i> mutants all three markers are strongly downregulated, specifically in dAp cells (long dotted brackets). In contrast, expression in Tv1 cells is unperturbed. (G) Quantification of Nplp1 and Eya expressing dAp cells in control and <i>grn</i> mutant VNCs (n > 10 VNCs; asterisks denote significant difference in <i>grn</i> mutants compared to control; <i>p</i> < 0.05, Student´s two-tailed <i>t</i> test; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002450#pbio.1002450.s001" target="_blank">S1 Data</a>). Genotypes: (A) <i>OregonR</i>. (B) <i>grn</i>^<i>7L12</i><i>/grn</i>^<i>SPJ9</i>. (C, E) <i>ap</i>^<i>rK568</i><i>/+</i>. (D, F) <i>ap</i>^<i>rK568</i><i>/+; grn</i>^<i>SPJ9</i><i>/grn</i>^<i>7L12</i>.</p

Cross-rescue reveals that <i>Kr</i> and <i>pdm</i> act upstream of <i>grn</i> and <i>col</i>.

<p>(A–D) Eya and Nplp1 expression in cross-rescue of <i>Kr</i> and <i>pdm</i> mutants with either <i>UAS-col</i> or <i>UAS-grn</i> misexpressed from <i>pros-Gal4</i>, at stage AFT. (A) While the number of Eya expressing dAp neurons in <i>Kr</i> mutants is not restored by misexpression of <i>grn</i>, (B) in <i>pdm</i> mutants misexpression of <i>grn</i> results in partial rescue in numbers of dAp neurons expressing both Eya and Nplp1. (C) Cross-rescue of <i>Kr</i> with <i>col</i> can fully rescue the mutant phenotype and results in ectopic numbers of dAp neurons expressing both Eya or Nplp1. (D) Cross-rescue of <i>pdm</i> with <i>col</i> can fully rescue the <i>pdm</i> mutant phenotype with respect to Eya and Nplp1 positive dAp neurons, and results in ectopic Eya and Nplp1 expression. (E, F) Quantification of Eya and Nplp1 positive dAp neurons from the different rescue experiments (n = 9 VNCs for <i>Kr; pros>grn</i>, n = 8 VNCs for <i>pdm; pros>col</i>. For all others n >10 VNCs; asterisk denotes <i>p</i> < 0.05, Student´s two-tailed <i>t</i> test; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002450#pbio.1002450.s001" target="_blank">S1 Data</a>). Genotypes: (A) <i>Kr</i>^<i>1</i>, <i>Kr</i>^<i>CD</i><i>; pros-Gal4</i>/ <i>UAS-grn</i>. (B) <i>Df(2L)ED773; pros-Gal4</i>/<i>UAS-grn</i>. (C) <i>Kr</i>^<i>1</i>, <i>Kr</i>^<i>CD</i><i>; pros-Gal4</i>/<i>UAS-col</i>. (D) <i>Df(2L)ED773; pros-Gal4</i>/<i>UAS-col</i>.</p

Early temporal genes are critical for dAp specification.

<p>(A–F) Expression of Eya and Nplp1 in control and temporal mutants, at St16 or AFT. (B) In <i>hb</i> mutants (boxed area), we observe two dorsal Ap cells (yellow dotted circles), which both express Eya, Col, and Dimm. Quantification of Nplp1 positive dAp cells in <i>hb</i> mutants fails since <i>hb</i> mutants do not develop into stage AFT at which Nplp1 is expressed. (C and D) Both <i>Kr</i> and <i>pdm</i> mutants show decreased numbers of Eya and Nplp1 expressing dAp cells (long dotted brackets). (E and F) Eya and Nplp1 expression in <i>cas</i> and <i>grh</i> mutants is not affected. (G–I) Dimm and Col expression shows a loss of both factors in the dAp cells in <i>Kr</i> and <i>pdm</i> mutants (long dotted brackets). (J) Cross-rescue of <i>Kr</i> mutants by <i>UAS-pdm</i> from <i>pros-Gal4</i> does not rescue Eya expression in dAp cells, but can partially rescue Nplp1 expression. (K) Col and Nplp1 expression in dAp cells of control and <i>Kr</i> mutants expressing <i>pdm</i> from <i>pros-Gal4</i> shows that <i>pdm</i> can restore the Col and Nplp1 expression in <i>Kr</i> mutants. (L) Quantification of Eya and Nplp1 positive dAp neurons in temporal mutants (n > 10; asterisk denotes <i>p</i> < 0.05; Student´s two-tailed <i>t</i> test; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002450#pbio.1002450.s001" target="_blank">S1 Data</a>). (M) Genetic model of the dAp specification cascade, showing that the early temporal genes <i>Kr</i> and <i>pdm</i> act to specify the dorsal Ap cell fate. (N) Quantification of Eya and Nplp1 positive dAp neurons in (n > 10 VNC; asterisk denotes <i>p</i> < 0.05; Student´s two-tailed <i>t</i> test; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002450#pbio.1002450.s001" target="_blank">S1 Data</a>). Numbers of Nplp1 positive dAp neurons in <i>Kr</i> mutants expressing <i>pdm</i> are significantly increased compared to <i>Kr</i> mutants. Genotypes: (A) <i>OregonR</i>. (B) <i>hb</i>^<i>P1</i>, <i>hb</i>^<i>FB</i>. (C, H) <i>Kr</i>^<i>1</i>, <i>Kr</i>^<i>CD</i>. (D, I) <i>Df(2L)ED773</i>. (E) <i>cas</i>^<i>Δ1</i><i>/ cas</i>^<i>Δ1</i>. (F) <i>grh</i>^<i>IM</i><i>/grh</i>^<i>IM</i>. (G) <i>OregonR</i>. (J, and K) <i>Kr</i>^<i>1</i>, <i>Kr</i>^<i>CD</i>; <i>pros-Gal4/UAS-nub</i>.</p