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

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

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

Mecanismos de adquisición de identidad neuronal y control del número celular en la cuerda nerviosa ventral de Drosophila melanogaster

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de Lectura: 26-01-2023Esta tesis tiene embargado el acceso al texto completo hasta el 26-07-2024El sistema nervioso presenta una complejidad en número y tipos celulares abrumadora, que permite funciones tan complejas como la memoria o el aprendizaje. Entender cómo durante el desarrollo embrionario un número limitado de células madre puede dar lugar a dicha complejidad, es fundamental para entender situaciones patológicas y para el desarrollo de terapias de medicina regenerativa. Para profundizar en este proceso, utilizamos como modelo la cuerda nerviosa ventral (CNV) del embrión de Drosophila melanogaster, que presenta gran diversidad de identidades neuronales con un número muy reducido de células y multitud de herramientas que facilitan su estudio. El desarrollo de diversos tipos celulares con el número de células adecuado, requiere la acción de factores de transcripción, que permiten la expresión diferencial de genes. Concretamente, los factores Hox y los factores temporales son fundamentales en este proceso. Sin embargo, se desconoce cómo se integra la información aportada por estos dos tipos de factores. Por ello, estudiamos su función en la determinación de dos tipos celulares pertenecientes al mismo linaje: las neuronas serotoninérgicas y las neuronas corazoninérgicas. Comprobamos que los factores Hox Ultrabithorax y Abdominal-A determinan el destino corazoninérgico y reprimen el destino serotoninérgico, mientras que los factores temporales Hunchback y Kruppel promueven el destino serotoninérgico y reprimen el destino corazoninérgico. Demostramos, por tanto, una lógica regulatoria entre estos factores para discernir entre dos identidades celulares concretas. Por otro lado, el número de genes relacionados con el control del número y tipo de células durante el desarrollo del sistema nervioso es todavía limitado. Por ello, realizamos una búsqueda genética visualizando distintos tipos celulares. Encontramos que el factor de dominio POU Ventral veins lacking (Vvl) actúa en la especificación neuronal, reprimiendo el destino neurona corazoninérgica en las neuronas serotoninérgicas. Además, Vvl participa en las neuronas Va-Capa controlando su número mediante la inducción de muerte celular programada. En definitiva, demostramos una lógica regulatoria entre factores Hox y factores temporales para especificar identidades neuronales concretas, y descubrimos que el factor de dominio POU Vvl es capaz de participar tanto en la especificación neuronal como en el control del número de células durante el desarrollo de la CNV de D. melanogasterThe nervous system is composed of an overwhelming diversity of cell types and number of cells, which allows functions as complex as memory or learning. Understanding how a limited number of stem cells can give rise to such complexity during embryonic development is essential for understanding pathological situations and for the development of regenerative medicine therapies. To study this process, we use the ventral nerve cord (VNC) of Drosophila melanogaster embryo as a model, which has a great diversity of systems of neurons composed by a very small number of cells and a multitude of tools that facilitate the study. During the development of the nervous system, the action of transcription factors is essential to specify different types of neurons in the correct numbers. Specifically, the Hox and temporal factors are fundamental in this process. However, little is known about how the information provided by these two types of factors is integrated. For this reason, we studied the function of Hox and temporal factors in the determination of two cell types belonging to the same lineage: serotonergic neurons and corazoninergic neurons. We found that the Hox factors Ultrabithorax and Abdominal-A determine the corazoninergic fate and repress the serotonergic fate, while the temporal factors Hunchback and Kruppel promote the serotonergic fate and repress the corazoninergic fate. Therefore, we demonstrate a regulatory logic between these factors to distinguish between two specific cell identities. On the other hand, the number of genes related with the control of the number and type of cells during the development of the nervous system is still limited. For this reason, we carried out a genetic screen visualizing different cell types. We found that the POU domain factor Ventral veins lacking (Vvl) participates in neuronal specification, repressing corazoninergic fate in serotonergic neurons. Besides, Vvl acts on Va-Capa neurons controlling the number of cells by inducing programmed cell death. In conclusion, we demonstrate a regulatory logic between Hox and temporal factors to determine specific neuronal identities. We also demonstrate that the POU domain factor Vvl can participate in both neuronal specification and cell number control during development of the VNC of D. melanogaste