Estudio de la regulación de la proliferación y la formación de patrón en el desarrollo del abdomen por los genes Hox Ultrabithorax y abdominal-A de Drosophila melanogaster

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-06-2017Esta tesis tiene embargado el acceso al texto completo hasta el 27-12-2018Los genes Hox son una familia de genes altamente conservados que especifican la identidad segmental a lo largo del eje antero-posterior en todos los organismos bilaterales. Esto los convierte en buenos candidatos para dirigir los mecanismos que regulan el tamaño, la forma y la diferenciación. Por ejemplo, los genes Hox deben controlar la diferente tasa de división celular que tiene lugar en diferentes órganos y coordinarla con los mecanismos que regulan la formación del patrón. Este trabajo se propone analizar el papel de los genes Hox Ultrabithorax y abdominal-A de Drosophila melanogaster en la coordinación de estos procesos. Hemos estudiado cómo se controlan los diferentes comportamientos proliferativos de dos grupos de células de la mosca con diferentes ritmos de crecimiento: el área presuntiva del disco imaginal de ala, que forma el tórax adulto, y los histoblastos, que dan origen al abdomen. Los discos imaginales proliferan principalmente durante el período larvario, mientras que los histoblastos lo hacen durante el período pupal, permaneciendo quiescentes durante la larva. Ultrabithorax se expresa y requiere en el tórax posterior y en el primer segmento abdominal, mientras que la expresión de abdominal-A es necesaria en el segundo segmento abdominal y posteriores. La expresión ectópica de abdominal-A en la región proximal del disco imaginal del ala transforma el tórax en abdomen, pero las células no se dividen como histoblastos sino como células de disco. De forma recíproca, la falta de Ultrabithorax en el primer segmento abdominal hace que las células se desarrollen como torácicas (o como una estructura intermedia entre el tórax y el abdomen), pero los histoblastos no se dividen como las células de disco, sino que permanecen quiescentes. Es decir, en ambos casos la tasa de división celular no corresponde a la estructura que estas células formarán. Los estudios de los genes expresados en el tórax o el abdomen (string, neuralized, wingless, eyegone) apoyan la idea de que en estas transformaciones homeóticas, de abdomen a tórax o viceversa, las células de disco imaginal o los histoblastos, al menos durante el período larvario, no adquieren el patrón característico del segmento que van a diferenciar. Los experimentos de rescate de un fenotipo mutante abdominal-A indican que este gen no se requiere durante el período larvario, sino durante el pupal, para especificar un segmento abdominal; por el contrario, la conversión del tórax al abdomen requiere la actividad de este gen en ambos períodos. Por último, nuestro estudio de la función de los genes Ultrabithorax y abdominal-A ha demostrado que ambos son necesarios para conferir características morfológicas especiales a los histoblastos (forma de membrana, morfología nuclear), y que son redundantes para una función esencial en las células epidérmicas politénicas que rodean los nidos de histoblastos. Nuestro trabajo profundiza en el estudio de los mecanismos por los cuales los genes Hox coordinan la dinámica de crecimiento y la formación de patrón, así como en sus requerimientos temporales para el desarrollo de estructuras adultas.Hox genes are a highly conserved gene family, which specify segment identity along the antero-posterior axis in all bilateral organisms. This makes them good candidates to direct the mechanisms that regulate size, shape and differentiation. As an example, the Hox genes must control the different rate of division that takes place in different organs and coordinate it with the mechanisms that regulate pattern formation. In this work we propose to analyze the role of the genes Hox Ultrabithorax and abdominal-A of Drosophila melanogaster in the coordination of these processes. We have studied how they control the different proliferative behavior of two groups of cells of the fly with different growth rates: the presumptive notum area of the imaginal wing disc, which forms the adult thorax, and the histoblasts, which give rise to the abdomen. Imaginal discs proliferate mainly during the larval period while the histoblasts do so during the period of pupation, remaining quiescent during the larva. Ultrabithorax is expressed and required in the posterior thorax and first abdominal segment whereas abdominal-A expression is needed in the second and posterior abdominal segments. The ectopic expression of abdominal-A in the proximal region of the imaginal wing disc transforms the thorax into the abdomen, but the cells do not divide as histoblasts but as disc cells. Conversely, the lack of Ultrabithorax in the first abdominal segment makes the cells to develop as thoracic (or as an intermediate structure between the thorax and abdomen), but the histoblasts do not divide like the disc cells, but remain quiescent. That is, the rate of cell division does not correspond to the structure that these cells will form. Studies of genes expressed in the thorax or abdomen (string, neuralized, wingless, eyegone) support the idea that, in these homeotic transformations from the abdomen to the thorax or vice versa, imaginal disc cells or histoblasts, at least during the larval period, do not acquire the characteristic pattern of the segment to which they will differentiate. Experiments of rescue of an abdominal-A mutant phenotype indicate that this gene is not required during the larval period, but during the pupal one, to specify an abdominal segment; by contrast, conversion of the thorax to the abdomen requires the activity of this gene in both periods. Finally, our study of the function of the Ultrabithorax and abdominal-A genes has shown that both are needed to confer special morphological characteristics to histoblasts (membrane form, nuclear morphology), and that are redundant for an essential function In the polytene epidermal cells surrounding the histoblasts of the abdomen. Our work delves into the study of the mechanisms by which Hox genes coordinate growth dynamics and pattern formation, as well as their temporal requirements for the development of adult structures

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-

A targeted genetic screen identifies crucial players in the specification of the Drosophila abdominal Capaergic neurons

The central nervous system contains a wide variety of neuronal subclasses generated by neural progenitors. The achievement of a unique neural fate is the consequence of a sequence of early and increasingly restricted regulatory events, which culminates in the expression of a specific genetic combinatorial code that confers individual characteristics to the differentiated cell. How the earlier regulatory events influence post-mitotic cell fate decisions is beginning to be understood in the Drosophila NB 5-6 lineage. However, it remains unknown to what extent these events operate in other lineages. To better understand this issue, we have used a very highly specific marker that identifies a small subset of abdominal cells expressing the Drosophila neuropeptide Capa: the ABCA neurons. Our data support the birth of the ABCA neurons from NB 5-3 in a cas temporal window in the abdominal segments A2–A4. Moreover, we show that the ABCA neuron has an ABCA-sibling cell which dies by apoptosis. Surprisingly, both cells are also generated in the abdominal segments A5–A7, although they undergo apoptosis before expressing Capa. In addition, we have performed a targeted genetic screen to identify players involved in ABCA specification. We have found that the ABCA fate requires zfh2, grain, Grunge and hedgehog genes. Finally, we show that the NB 5-3 generates other subtype of Capa-expressing cells (SECAs) in the third suboesophageal segment, which are born during a pdm/cas temporal window, and have different genetic requirements for their specification.This work was supported by a grant from the Spanish Ministerio de Ciencia e Innovación (BFU-2008-04683-C02-02 to L.T.)

Lineage-unrelated neurons generated in different temporal windows and expressing different combinatorial codes can converge in the activation of the same terminal differentiation gene

It is becoming increasingly clear that the activation of specific terminal differentiation genes during neural development is critically dependent upon the establishment of unique combinatorial transcription factor codes within distinct neural cell subtypes. However, it is still unclear to which extent these codes are shared by lineage-unrelated neurons expressing the same terminal differentiation genes. Additionally, it is not known if the activation of a specific terminal differentiation gene is restricted to cells born at a particular developmental time point. Here, we utilize the terminal differentiation gene FMRFa which is expressed by the Ap4 and SE2 neurons in the Drosophila ventral nerve cord, to explore these issues in depth. We find that the Ap4 and SE2 neurons are generated by different neural progenitors and use different combinatorial codes to activate FMRFa expression. Additionally, we find that the Ap4 and SE2 neurons are generated in different temporal gene expression windows. Extending the investigation to include a second Drosophila terminal differentiation gene, Leucokinin, we find similar results, suggesting that neurons generated by different progenitors might commonly use different transcription factor codes to activate the same terminal differentiation gene. Furthermore, these results imply that the activation of a particular terminal differentiation gene in temporally unrestricted.This work was supported by a grant from the Spanish Ministerio de Ciencia e Innovación (BFU-2008- 04683-C02-02 to L.T.)

Lineage-unrelated neurons generated in different temporal windows and expressing different combinatorial codes can converge in the activation of the same terminal differentiation gene

This work was supported by a grant from the Spanish Ministerio de Ciencia e Innovación (BFU-2008-04683-C02-02 to L.T.).It is becoming increasingly clear that the activation of specific terminal differentiation genes during neural development is critically dependent upon the establishment of unique combinatorial transcription factor codes within distinct neural cell subtypes. However, it is still unclear to which extent these codes are shared by lineage-unrelated neurons expressing the same terminal differentiation genes. Additionally, it is not known if the activation of a specific terminal differentiation gene is restricted to cells born at a particular developmental time point. Here, we utilize the terminal differentiation gene FMRFa which is expressed by the Ap4 and SE2 neurons in the Drosophila ventral nerve cord, to explore these issues in depth. We find that the Ap4 and SE2 neurons are generated by different neural progenitors and use different combinatorial codes to activate FMRFa expression. Additionally, we find that the Ap4 and SE2 neurons are generated in different temporal gene expression windows. Extending the investigation to include a second Drosophila terminal differentiation gene, Leucokinin, we find similar results, suggesting that neurons generated by different progenitors might commonly use different transcription factor codes to activate the same terminal differentiation gene. Furthermore, these results imply that the activation of a particular terminal differentiation gene in temporally unrestricted.Ministerio de Ciencia e Innovación (BFU-2008-04683-C02-02)Depto. de Biología CelularFac. de Ciencias BiológicasTRUEpu

Detection of Anti–Leishmania infantum Antibodies in Wild European and American Mink (Mustela lutreola and Neovison vison) from Northern Spain, 2014–20

The European mink (Mustela lutreola) is listed as a critically endangered species because of ongoing population reduction from habitat degradation and the effects of introduced species, such as American mink (Neovison vison). This small, fragmented population becomes vulnerable to many other threats, including diseases. Leishmaniosis is a zoonotic disease caused by the protozoan parasite Leishmania infantum found in the Mediterranean area, which affects many mammals, including wild small mammals. Furthermore, clinical disease caused by L. infantum has recently been described in other mustelids. To assess the exposure to Leishmania sp. infection in mink species in northern Spain, blood samples from 139 feral American mink and 42 native European mink from north Spain were evaluated for Leishmania sp. infection using enzyme-linked immunosorbent assays against Leishmania spp. antibodies, with 52.4% of American mink and 45.3% of European mink being found seropositive. This finding raises questions regarding how the disease may affect these species and the potential repercussions for conservation efforts. Despite a high seroprevalence being observed in wild mink of both species in this study, association with clinical or pathologic signs of disease has yet to be elucidated.Depto. de Medicina y Cirugía AnimalFac. de VeterinariaTRUEpu

Identification of Diuretic hormone 31 (DH31) and Allatostatin A (AstA) neuropeptides as Va-A1 terminal markers.

<p>(A) Model of the segmentally synonymous Va-neurons [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194281#pone.0194281.ref008" target="_blank">8</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194281#pone.0194281.ref009" target="_blank">9</a>]. In A1, the Va neuron fate is unknown. (B) Dimm expression marks Va-neurons in four abdominal segments (A1 to A4). (C) Overlap of Capa (red), Dimm (green), and Dac (blue) at Stg 18 h (after egg laying; AEL) in wild type. Capa is expressed in A2-A4 abdominal segments, but not in Va-A1. (D) Overlap of DH31 (red), Dimm (green), and Dac (blue) at Stg 18 in wild type. The DH31 neuropeptide is expressed in Va-A1 neurons. (E) Overlap of DH31 (red) and AstA (green) at Stg 18 h AEL in wild type, showing that AstA is also expressed in Va-A1 neurons. Genotype: <i>OregonR</i>.</p

Postmitotic <i>abdominal-A</i> ignores posterior prevalence in Va-neurons.

<p>(A) Expression of Dimm (green) and DH31 (red) shows that Va-Capa neurons are transformed into Va-DH31 neurons in <i>abdA</i> mutants. (B-C) Expression of Dimm (green) and Ubx (red) in control (B) and <i>abdA</i> mutants (C). Ubx expression is increased in A2-A4 Va-neurons. (D) Expression of AstA (red), Capa (blue), and Dimm (green) in a <i>cas>abdA</i> background. Va-DH31 fate was expanded anteriorly and Va-neurons in A5 and A6 do not undergo PCD. (E-G) Quantitation of genetic studies [<i>n</i> ≥8 VNC in all genotypes; asterisks indicate significant difference compared with controls (Student’s <i>t</i>-test, <i>P</i><0.001)] Genotypes: (A, C) <i>abdA</i>^<i>MX1</i>/<i>abdA</i>^<i>MX1</i>, (B) <i>OregonR</i>, (D) <i>cas-Gal4</i>/<i>UAS-abdA</i>.</p

<i>Antennapedia</i> shows phenotypic dominance over the co-expressed <i>Ultrabithorax</i> in Va-T3 neurons.

<p>(A) Expression of DH31 (red) and Dimm (green) showing that T3 Va-neurons expressed DH31 in <i>Antp</i> mutants. (B-C) Expression of Ubx (green) and Dimm (red) in <i>Antp</i> mutants (B) and controls (C) at Stg 15. Ubx is normally expressed at low level in T3 Va-neurons at Stg 15; this expression is unaffected in <i>Antp</i> mutants. (D-E) Expression of AstA (red), Capa (blue), and Dimm (green) in controls (D) and a <i>cas>Antp</i> background (E). Note that Capa expression (but not DH31/AstA) is downregulated when <i>Antp</i> is overexpressed. (F) and (G) Quantitation of these genetic studies [<i>n</i>≥11 VNC in all genotypes; asterisks indicate significant difference compared with control (Student’s <i>t</i>-test, <i>P</i><0.001)]. Genotypes: (A, C) <i>Antp</i>^{<i>NS-rvc12</i>}/<i>Antp</i>^<i>14</i>, (B, D) <i>OregonR</i>, (E) <i>cas-Gal4/UAS-Antp</i>.</p

<i>Ultrabithorax</i> induces DH31 expression.

<p>(A) Scheme summarizing Hox expression in Va-neurons (modified from[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194281#pone.0194281.ref008" target="_blank">8</a>]). Expression of Dimm (green) and DH31 (red) in <i>Ubx</i> mutants (B) and a <i>cas>Ubx</i> background (C). Dimm and Dac expression is lost in <i>Ubx</i> mutants, whereas <i>Ubx</i> misexpression results in anterior expansion of DH31, Dimm and Dac expression into Va-T2-T3 neurons. (D) Expression of AstA (red), Capa (blue), and Dimm (green) on a <i>cas>Ubx</i> background. Note posterior expansion of Va-Capa neurons into Va-A5 neurons. (E) Quantitation of these genetic studies [<i>n</i> ≥8 VNC in all genotypes; asterisks indicate significant difference compared with controls (Student’s <i>t</i>-test, <i>P</i> <0.001)]. Genotypes: (B) <i>Ubx</i>^<i>1</i>/<i>Ubx</i>^<i>1</i>, (C-D) <i>cas-Gal4/UAS-Ubx</i>, (E) <i>OregonR</i>, <i>Ubx</i>^<i>1</i>/<i>Ubx</i>^<i>1</i>, <i>cas-Gal4/UAS-Ubx</i>, <i>elav-Gal4/UAS-Ubx</i>.</p