Héctor Herranz

MicroRNAs and gene regulatory networks: managing the impact of noise in biological systems

Biological systems are continuously challenged by an environment that is variable. Yet, a key feature of developmental and physiological processes is their remarkable stability. This review considers how microRNAs contribute to gene regulatory networks that confer robustness.

Generation of medial and lateral dorsal body domains by the pannier gene of Drosophila

The homeobox gene extradenticle (exd) acts as a cofactor of the homeotic genes in the specification of larval patterns during embryogenesis. To study its role in adult patterns, we have generated clones of mutant exd- cells and examined their effect on the different body parts. In some regions, exd- clones exhibit homeotic transformations similar to those produced by known homeotic mutations such as Ultrabithorax (Ubx), labial (lab), spineless-aristapedia (ssa) or Antennapedia (Antp). In other regions, the lack of exd causes novel homeotic transformations producing ectopic eyes and legs. Moreover, exd is also required for functions normally not associated with homeosis, such as the maintenance of the dorsoventral pattern, the specification of subpatterns in adult appendages or the arrangement of bristles in the mesonotum and genitalia. Our findings indicate that exd is critically involved in adult morphogenesis, not only in the homeotic function but also in several other developmental processes.Previously published experiments have shown that the endogenous Dfd gene can be ectopically activated by its own (heat-shock-driven) product in a subset of cells of different segments. This results in the differentiation of maxillary structures like cirri and mouth hooks in places where they normally do not appear, and represents a phenomenon of autocatalysis of homeotic gene function that differs from the normal activation process. We show that this out-of-context activation occurs in cells belonging to the anterior compartments of the three thoracic and the A1 to A8 abdominal segments and that it requires the normal function of the polarity genes wingless (wg) and engrailed (en). The wg product, in addition to that of Dfd, appears to be sufficient to activate the endogenous Dfd gene in many embryonic cells. We have studied the effect of several homeotic genes on Dfd activation and phenotypic expression: Scr, Antp, Ubx and Abd-B repress Dfd both transcriptionally and at the phenotypic level, if their products are in sufficient amounts. The endogenous abd-A gene does not have a noticeable effect, but when it is replaced by an hsp70-abd-A gene, which produces a high and uniform level of expression, the phenotypic expression of Dfd is suppressed. Our results also suggest that the differentiation of cirri is induced by Dfd-expressing cells in non-expressing neighboring cells, and that this interaction occurs across the parasegmental border.During evolution, many animal groups have developed specialised outgrowths of the body wall, limbs or appendages. The type of appendage depends on the identity of the segment where they appear, indicating that the Hox genes contribute to appendage specification. Moreover, work carried out principally in Drosophila has identified the gene products and the mechanisms involved in pattern formation in the appendages. In this essay, we compare the morphogenetic processes in the appendages and the body wall; the function of the Hox genes and the response to the signalling molecules involved in local patterning. We speculate that, although the basic mechanisms are similar, there are significant differences in the manner the body trunk and appendages respond to them.[ES] La pared celular es un elemento morfogenetico esencial que determina la forma final de las celulas y que las protege contra la lisis. En S. pombe esta esta constituida por ? y s-glucano y manoproteinas y tanto la sintesis como remodelacion de su estructura requiere de diferentes enzimas estrictamente reguladas. En S. pombe existe poca informacion de como se lleva a cabo la incorporacion del material de membrana y sobre la regulacion de las enzimas implicadas en la sintesis y remodelacion de la pared celular por los mecanismos de transporte vesicular. Para abordar el estudio de como el trafico vesicular mediado por clatrina afecta a la morfogenesis de S. pombe y en particular cual es su papel en la regulacion de la sintesis de la pared celular se ha analizado el papel tanto de la clatrina, mediante el analisis de diferentes mutantes de la cadena ligera de la clatrina, como el del adaptador AP-2, que interviene en el proceso de endocitosis mediada por clatrina. Se ha demostrado que la delecion de la cadena ligera de la clatrina resulta letal para las celulas de S. pombe y que esta letalidad se rescata al incubar las celulas en un medio suplementado con sorbitol. En este caso aunque las celulas pueden sobrevivir poseen graves defectos morfologicos, en crecimiento, en trafico vesicular, en desarrollo sexual, etc. Se ha podido comprobar que la ausencia de Clc1p afecta drasticamente a la estabilidad de Chc1p hecho que hace que, a diferencia de otros organismos, la supervivencia de S. pombe sea mas dependiente de la presencia clatrina. Ademas se ha demostrado que la letalidad causada por la ausencia de Clc1p se debe principalmente a defectos graves en la sintesis de la pared celular que afectan directamente a la sintesis del glucano. Los resultados obtenidos muestran que una reduccion en la cantidad de clatrina causa un leve impacto en el transporte vesicular en general y en otros procesos y elementos biologicos, pero afecta gravemente a la secrecion de enzimas de sintesis/remodelacion de la pared celular, como las s(1,3)glucan sintasa y endoglucanasas. En cuanto al complejo adaptador AP-2 se ha comprobado, que a diferencia de lo que se conoce hasta el momento en otros organismos unicelulares, este forma un complejo con la clatrina y se ha demostrado que tiene un papel en la endocitosis general de S. pombe. Asi mismo se ha descubierto que AP-2 puede estar interviniendo en la sintesis de la pared celular ya que su ausencia afecta a la actividad s-glucan sintasa y hace que S. pombe sea hiper-sensible a compuestos que afectan a la integridad de la pared celular.We characterized a novel protein of the Ras family, p19 (H-RasIDX). The c-H-ras proto-oncogene undergoes alternative splicing of the exon termed IDX. We show that the alternative p19 mRNA is stable and as abundant as p21 (p21 H-Ras4A) mRNA in all of the human tissues and cell lines tested. IDX is spliced into stable mRNA in different mammalian species, which present a high degree of nucleotide conservation. Both the endogenous and the transiently expressed p19 protein are detected in COS-1 and HeLa cells and show nuclear diffuse and speckled patterns as well as cytoplasmic localization. In yeast two-hybrid assays, p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multiprotein complexes in different signaling pathways. This observation suggests that p19 and p21 play differential and complementary roles in the cell.Resumen del trabajo presentado al Congreso Nacional de Biotecnologia, celebrado en Murcia del 18 al 21 de junio de 2017.A. G. G. thanks Ramon Areces Foundation for a grant. J. C. thanks NIH-CA24487 for financial support.Ministerio de Educacion y Ciencia and grant S-0505/MAT-0283 from Comunidad Autonoma de Madrid to M.S. and by an Institutional grant from Fundacion Ramon Areces to the Centro de Biologia Molecular “Severo Ochoa”We report a genetic and molecular study of UbxMX6 and Ubx195rx1, two mutations in the Ultrabithorax (Ubx) locus which appear to have a strong effect on the activity of the homologous Ubx gene. These mutations show the characteristic embryonic and adult phenotypes of Ubx null alleles, and also fail to produce any detectable Ubx product. Yet, genetic and phenotypic analyses involving a large number of trans heterozygous combinations of UbxMX6 and Ubx195rx1 with different classes of Ubx mutations, indicate that they hyperactivate the homologous gene. This effect is induced on wildtype or mutant forms of Ubx, provided that the pairing in the bithorax region is normal, i.e. these mutations have a strong positive effect on transvection. We also show that, unlike all the other known cases of transvection in Ubx, this is not zeste-dependent. Southern analyses indicate that UbxMX6 is a 3.4 kb deletion, and Ubx195rx1 is an approximately 11 kb insertion of foreign DNA, both in the promoter region. We speculate that the region altered in the mutations may have a wildtype function to ensure cis-autonomy of the regulation of Ubx transcription.Resumen del trabajo presentado al Congreso Nacional de Biotecnologia, celebrado en Murcia del 18 al 21 de junio de 2017.The pannier (pnr) gene of Drosophila encodes a zinc-finger transcription factor of the GATA family and is involved in several developmental processes during embryonic and imaginal development. We report some novel aspects of the regulation and function of pnr during embryogenesis. Previous work has shown that pnr is activated by decapentaplegic (dpp) in early development, but we find that after stage 10, the roles are reversed and pnr becomes an upstream regulator of dpp. This function of pnr is necessary for the activation of the Dpp pathway in the epidermal cells implicated in dorsal closure and is not mediated by the JNK pathway, which is also necessary for Dpp activity in these cells. In addition, we show that pnr behaves as a selector-like gene in generating morphological diversity in the dorsoventral body axis. It is responsible for maintaining a subdivision of the dorsal half of the embryo into two distinct, dorsomedial and dorsolateral, regions, and also specifies the identity of the dorsomedial region. These results, together with prior work on its function in adults, suggest that pnr is a major factor in the genetic subdivision of the body of Drosophila.10th International Symposium on Reproductive Physiology of Fish (10th ISRPF), Expanding the khowledge base of reproductive success: from genes to the environment, 25-30 May 2014, Olhao, Portugal.-- 1 pageBy using a hsp70-Ubx fusion gene, we have ectopically expressed a Ubx product in the embryonic head primordia and studied the developmental effects on the larval head. We find that after high and persistent levels of Ubx product, the head is replaced by three (C1, C2 and C3) abdominal-like denticle belts. The C2 and C3 belts are the homeotic transformations of parasegments 1 and 2, respectively, while the C1 belt probably derives from the transformation and subsequent fusion of the most anterior procephalic primordia. On the basis of their response to the Ubx product and other arguments, we propose that the larval head is made of two genetically distinct components; one is the procephalon and the anterior region of the mandibular lobe, and the other is part of the parasegmental trunk and includes parasegments 1 and 2. Our results also indicate that most or all the larval head structures derive from precursor cells of ventral origin.The Iroquois (Iro) family of genes are found in nematodes, insects and vertebrates. They usually occur in one or two genomic clusters of three genes each and encode transcriptional controllers that possess a characteristic homeodomain. The Iro genes function early in development to specify the identity of diverse territories of the body, such as the dorsal head and dorsal mesothorax of Drosophila and the neural plate of Xenopus. In some aspects they act in the same way as classical selector genes, but they display specific properties that place them into a category of their own. Later in development in both Drosophila and vertebrates, the Iro genes function again to subdivide those territories into smaller domains.The pannier (pnr) gene encodes a GATA transcription factor and acts in several developmental processes in Drosophila, including embryonic dorsal closure, specification of cardiac cells and bristle determination. We show that pnr is expressed in the mediodorsal parts of thoracic and abdominal segments of embryos, larvae and adult flies. Its activity confers cells with specific adhesion properties that make them immiscible with non-expressing cells. Thus there are two genetic domains in the dorsal region of each segment: a medial (MED) region where pnr is expressed and a lateral (LAT) region where it is not. The homeobox gene iroquois (iro) is expressed in the LAT region. These regions are not formed by separate polyclones of cells, but are defined topographically. We show that ectopic pnr in the wing induces MED thoracic development, indicating that pnr specifies the identity of the MED regions. Correspondingly, when pnr is removed from clones of cells in the MED domain, they sort out and apparently adopt the LAT fate. We propose that (1) the subdivision into MED and LAT regions is a general feature of the Drosophila body plan and (2) pnr is the principal gene responsible for this subdivision. We argue that pnr acts like a classical selector gene but differs in that its expression is not propagated through cell divisions.We have developed a specific polyclonal antibody that recognizes the protein products of the abdominal-A (abd-A) gene, a member of the bithorax complex of Drosophila. The normal expression domain extends from parasegments 7 to 13, in good correspondence with previous genetic and molecular results. However, while the anterior border of expression is precisely demarcated by a parasegmental boundary, the posterior border does not coincide with a lineage boundary. Within the normal domain, the expression of abd-A shows intrametameric modulation; the amount of product is higher in posterior compartments and in the most anterior cells of the anterior compartments and then gradually decreases. We have examined the effect on abd-A expression of a number of mutations, some mapping within and others outside the abd-A transcription unit. Those mapping to the transcription unit eliminate or severely reduce the amount of abd-A antigen, while those mapping outside produce an abnormal distribution of abd-A protein. Finally, we show that the abd-A gene is down-regulated in part of the Abdominal-B (Abd-B) domain, precisely in those regions where the Abd-B gene is expressed at high levels.Resumen del trabajo presentado al Yeast Genetics Meeting, celebrado en Stanford, California (USA) del 22 al 26 de agosto de 2018.The effect of the anti-tumoral drug lauryl gallate on the infectivity of the African swine fever virus among other DNA (Herpes simplex and Vaccinia) and RNA (Influenza, Porcine transmissible gastroenteritis and Sindbis) viruses, involved in animal and human diseases, is analyzed. Viral production was strongly inhibited in different cell lines at non-toxic concentrations of the drug (1-10 μM), reducing the titres from 3 to more than 5 log. units depending on the multiplicity of infection. In our model system (African swine fever virus in Vero cells), the addition of the drug 1 h before virus adsorption, completely abolished virus productivity in a one-step growth virus cycle. Interestingly, no inhibitory effect was observed when lauryl gallate was added after 5 to 8 hpi. Both cellular and viral DNA synthesis and late viral transcription were inhibited by the drug, but, however, the early viral protein synthesis and the virus-mediated increasing of p53 remained unaffected. Activation of the apoptotic effector caspase-3 was not detected after lauryl gallate treatment of Vero cells, and, furthermore, the presence of the drug abrogated the activation of this protease induced by the virus infection. The overall results likely indicate that a cellular factor/function might be the target of the antiviral action of alkyl gallates.Tesis Doctoral presentada por Eduardo Rodenas Martinez en el Centro Andaluz de Biologia del Desarrollo, centro mixto CSIC-UPO.Resumen del trabajo presentado al Yeast Genetics Meeting, celebrado en Stanford, California (USA) del 22 al 26 de agosto de 2018.

Self‐refinement of Notch activity through the transmembrane protein Crumbs: modulation of γ‐Secretase activity

Cell interactions mediated by Notch family receptors have been implicated in the specification of tissue boundaries. Tightly localized activation of Notch is crucial for the formation of sharp boundaries. In the Drosophila wing imaginal disc, the Notch receptor is expressed in all cells. However, Notch activity is limited to a narrow stripe of cells along the dorsal–ventral compartment boundary, where it induces the expression of target genes. How a widely expressed protein becomes tightly regulated at the dorsal–ventral boundary in the Drosophila wing is not completely understood. Here, we show that the transmembrane protein Crumbs is involved in a feedback mechanism used by Notch to refine its own activation domain at the Drosophila wing margin. Crumbs reduces the activity of the γ‐Secretase complex, which mediates the proteolytic intracellular processing of Notch. These results indicate a novel molecular mechanism of the regulation of Notch signal, and also that defects in Crumbs might be involved in similar abnormal γ‐Secretase complex activity observed in Alzheimers disease.

Mutual Repression by Bantam miRNA and Capicua Links the EGFR/MAPK and Hippo Pathways in Growth Control

BACKGROUND The epidermal growth factor receptor (EGFR) and Hippo signaling pathways control cell proliferation and apoptosis to promote tissue growth during development. Misregulation of these pathways is implicated in cancer. Our understanding of the mechanisms that integrate the activity of these pathways remains fragmentary. This study identifies bantam microRNA as a common target of these pathways and suggests a mechanistic link between them. RESULTS The EGFR pathway acts through bantam to control tissue growth. bantam expression is regulated by the EGFR pathway, acting via repression of the transcriptional repressor Capicua. Thus EGFR signaling induces bantam expression by alleviating the effects of a repressor. bantam in turn acts in a negative feedback loop to limit Capicua expression. CONCLUSIONS bantam appears to be a transcriptional target of both the EGFR and Hippo growth control pathways. Feedback regulation by bantam on Capicua provides a means to link signal propagation by the EGFR pathway to activity of the Hippo pathway and may play an important role in integration of these two pathways in growth control.

The functions of pannier during Drosophila embryogenesis

The homeobox gene extradenticle (exd) acts as a cofactor of the homeotic genes in the specification of larval patterns during embryogenesis. To study its role in adult patterns, we have generated clones of mutant exd- cells and examined their effect on the different body parts. In some regions, exd- clones exhibit homeotic transformations similar to those produced by known homeotic mutations such as Ultrabithorax (Ubx), labial (lab), spineless-aristapedia (ssa) or Antennapedia (Antp). In other regions, the lack of exd causes novel homeotic transformations producing ectopic eyes and legs. Moreover, exd is also required for functions normally not associated with homeosis, such as the maintenance of the dorsoventral pattern, the specification of subpatterns in adult appendages or the arrangement of bristles in the mesonotum and genitalia. Our findings indicate that exd is critically involved in adult morphogenesis, not only in the homeotic function but also in several other developmental processes.Previously published experiments have shown that the endogenous Dfd gene can be ectopically activated by its own (heat-shock-driven) product in a subset of cells of different segments. This results in the differentiation of maxillary structures like cirri and mouth hooks in places where they normally do not appear, and represents a phenomenon of autocatalysis of homeotic gene function that differs from the normal activation process. We show that this out-of-context activation occurs in cells belonging to the anterior compartments of the three thoracic and the A1 to A8 abdominal segments and that it requires the normal function of the polarity genes wingless (wg) and engrailed (en). The wg product, in addition to that of Dfd, appears to be sufficient to activate the endogenous Dfd gene in many embryonic cells. We have studied the effect of several homeotic genes on Dfd activation and phenotypic expression: Scr, Antp, Ubx and Abd-B repress Dfd both transcriptionally and at the phenotypic level, if their products are in sufficient amounts. The endogenous abd-A gene does not have a noticeable effect, but when it is replaced by an hsp70-abd-A gene, which produces a high and uniform level of expression, the phenotypic expression of Dfd is suppressed. Our results also suggest that the differentiation of cirri is induced by Dfd-expressing cells in non-expressing neighboring cells, and that this interaction occurs across the parasegmental border.During evolution, many animal groups have developed specialised outgrowths of the body wall, limbs or appendages. The type of appendage depends on the identity of the segment where they appear, indicating that the Hox genes contribute to appendage specification. Moreover, work carried out principally in Drosophila has identified the gene products and the mechanisms involved in pattern formation in the appendages. In this essay, we compare the morphogenetic processes in the appendages and the body wall; the function of the Hox genes and the response to the signalling molecules involved in local patterning. We speculate that, although the basic mechanisms are similar, there are significant differences in the manner the body trunk and appendages respond to them.[ES] La pared celular es un elemento morfogenetico esencial que determina la forma final de las celulas y que las protege contra la lisis. En S. pombe esta esta constituida por ? y s-glucano y manoproteinas y tanto la sintesis como remodelacion de su estructura requiere de diferentes enzimas estrictamente reguladas. En S. pombe existe poca informacion de como se lleva a cabo la incorporacion del material de membrana y sobre la regulacion de las enzimas implicadas en la sintesis y remodelacion de la pared celular por los mecanismos de transporte vesicular. Para abordar el estudio de como el trafico vesicular mediado por clatrina afecta a la morfogenesis de S. pombe y en particular cual es su papel en la regulacion de la sintesis de la pared celular se ha analizado el papel tanto de la clatrina, mediante el analisis de diferentes mutantes de la cadena ligera de la clatrina, como el del adaptador AP-2, que interviene en el proceso de endocitosis mediada por clatrina. Se ha demostrado que la delecion de la cadena ligera de la clatrina resulta letal para las celulas de S. pombe y que esta letalidad se rescata al incubar las celulas en un medio suplementado con sorbitol. En este caso aunque las celulas pueden sobrevivir poseen graves defectos morfologicos, en crecimiento, en trafico vesicular, en desarrollo sexual, etc. Se ha podido comprobar que la ausencia de Clc1p afecta drasticamente a la estabilidad de Chc1p hecho que hace que, a diferencia de otros organismos, la supervivencia de S. pombe sea mas dependiente de la presencia clatrina. Ademas se ha demostrado que la letalidad causada por la ausencia de Clc1p se debe principalmente a defectos graves en la sintesis de la pared celular que afectan directamente a la sintesis del glucano. Los resultados obtenidos muestran que una reduccion en la cantidad de clatrina causa un leve impacto en el transporte vesicular en general y en otros procesos y elementos biologicos, pero afecta gravemente a la secrecion de enzimas de sintesis/remodelacion de la pared celular, como las s(1,3)glucan sintasa y endoglucanasas. En cuanto al complejo adaptador AP-2 se ha comprobado, que a diferencia de lo que se conoce hasta el momento en otros organismos unicelulares, este forma un complejo con la clatrina y se ha demostrado que tiene un papel en la endocitosis general de S. pombe. Asi mismo se ha descubierto que AP-2 puede estar interviniendo en la sintesis de la pared celular ya que su ausencia afecta a la actividad s-glucan sintasa y hace que S. pombe sea hiper-sensible a compuestos que afectan a la integridad de la pared celular.We characterized a novel protein of the Ras family, p19 (H-RasIDX). The c-H-ras proto-oncogene undergoes alternative splicing of the exon termed IDX. We show that the alternative p19 mRNA is stable and as abundant as p21 (p21 H-Ras4A) mRNA in all of the human tissues and cell lines tested. IDX is spliced into stable mRNA in different mammalian species, which present a high degree of nucleotide conservation. Both the endogenous and the transiently expressed p19 protein are detected in COS-1 and HeLa cells and show nuclear diffuse and speckled patterns as well as cytoplasmic localization. In yeast two-hybrid assays, p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multiprotein complexes in different signaling pathways. This observation suggests that p19 and p21 play differential and complementary roles in the cell.Resumen del trabajo presentado al Congreso Nacional de Biotecnologia, celebrado en Murcia del 18 al 21 de junio de 2017.A. G. G. thanks Ramon Areces Foundation for a grant. J. C. thanks NIH-CA24487 for financial support.Ministerio de Educacion y Ciencia and grant S-0505/MAT-0283 from Comunidad Autonoma de Madrid to M.S. and by an Institutional grant from Fundacion Ramon Areces to the Centro de Biologia Molecular “Severo Ochoa”We report a genetic and molecular study of UbxMX6 and Ubx195rx1, two mutations in the Ultrabithorax (Ubx) locus which appear to have a strong effect on the activity of the homologous Ubx gene. These mutations show the characteristic embryonic and adult phenotypes of Ubx null alleles, and also fail to produce any detectable Ubx product. Yet, genetic and phenotypic analyses involving a large number of trans heterozygous combinations of UbxMX6 and Ubx195rx1 with different classes of Ubx mutations, indicate that they hyperactivate the homologous gene. This effect is induced on wildtype or mutant forms of Ubx, provided that the pairing in the bithorax region is normal, i.e. these mutations have a strong positive effect on transvection. We also show that, unlike all the other known cases of transvection in Ubx, this is not zeste-dependent. Southern analyses indicate that UbxMX6 is a 3.4 kb deletion, and Ubx195rx1 is an approximately 11 kb insertion of foreign DNA, both in the promoter region. We speculate that the region altered in the mutations may have a wildtype function to ensure cis-autonomy of the regulation of Ubx transcription.Resumen del trabajo presentado al Congreso Nacional de Biotecnologia, celebrado en Murcia del 18 al 21 de junio de 2017.The pannier (pnr) gene of Drosophila encodes a zinc-finger transcription factor of the GATA family and is involved in several developmental processes during embryonic and imaginal development. We report some novel aspects of the regulation and function of pnr during embryogenesis. Previous work has shown that pnr is activated by decapentaplegic (dpp) in early development, but we find that after stage 10, the roles are reversed and pnr becomes an upstream regulator of dpp. This function of pnr is necessary for the activation of the Dpp pathway in the epidermal cells implicated in dorsal closure and is not mediated by the JNK pathway, which is also necessary for Dpp activity in these cells. In addition, we show that pnr behaves as a selector-like gene in generating morphological diversity in the dorsoventral body axis. It is responsible for maintaining a subdivision of the dorsal half of the embryo into two distinct, dorsomedial and dorsolateral, regions, and also specifies the identity of the dorsomedial region. These results, together with prior work on its function in adults, suggest that pnr is a major factor in the genetic subdivision of the body of Drosophila.10th International Symposium on Reproductive Physiology of Fish (10th ISRPF), Expanding the khowledge base of reproductive success: from genes to the environment, 25-30 May 2014, Olhao, Portugal.-- 1 pageBy using a hsp70-Ubx fusion gene, we have ectopically expressed a Ubx product in the embryonic head primordia and studied the developmental effects on the larval head. We find that after high and persistent levels of Ubx product, the head is replaced by three (C1, C2 and C3) abdominal-like denticle belts. The C2 and C3 belts are the homeotic transformations of parasegments 1 and 2, respectively, while the C1 belt probably derives from the transformation and subsequent fusion of the most anterior procephalic primordia. On the basis of their response to the Ubx product and other arguments, we propose that the larval head is made of two genetically distinct components; one is the procephalon and the anterior region of the mandibular lobe, and the other is part of the parasegmental trunk and includes parasegments 1 and 2. Our results also indicate that most or all the larval head structures derive from precursor cells of ventral origin.The Iroquois (Iro) family of genes are found in nematodes, insects and vertebrates. They usually occur in one or two genomic clusters of three genes each and encode transcriptional controllers that possess a characteristic homeodomain. The Iro genes function early in development to specify the identity of diverse territories of the body, such as the dorsal head and dorsal mesothorax of Drosophila and the neural plate of Xenopus. In some aspects they act in the same way as classical selector genes, but they display specific properties that place them into a category of their own. Later in development in both Drosophila and vertebrates, the Iro genes function again to subdivide those territories into smaller domains.The pannier (pnr) gene encodes a GATA transcription factor and acts in several developmental processes in Drosophila, including embryonic dorsal closure, specification of cardiac cells and bristle determination. We show that pnr is expressed in the mediodorsal parts of thoracic and abdominal segments of embryos, larvae and adult flies. Its activity confers cells with specific adhesion properties that make them immiscible with non-expressing cells. Thus there are two genetic domains in the dorsal region of each segment: a medial (MED) region where pnr is expressed and a lateral (LAT) region where it is not. The homeobox gene iroquois (iro) is expressed in the LAT region. These regions are not formed by separate polyclones of cells, but are defined topographically. We show that ectopic pnr in the wing induces MED thoracic development, indicating that pnr specifies the identity of the MED regions. Correspondingly, when pnr is removed from clones of cells in the MED domain, they sort out and apparently adopt the LAT fate. We propose that (1) the subdivision into MED and LAT regions is a general feature of the Drosophila body plan and (2) pnr is the principal gene responsible for this subdivision. We argue that pnr acts like a classical selector gene but differs in that its expression is not propagated through cell divisions.We have developed a specific polyclonal antibody that recognizes the protein products of the abdominal-A (abd-A) gene, a member of the bithorax complex of Drosophila. The normal expression domain extends from parasegments 7 to 13, in good correspondence with previous genetic and molecular results. However, while the anterior border of expression is precisely demarcated by a parasegmental boundary, the posterior border does not coincide with a lineage boundary. Within the normal domain, the expression of abd-A shows intrametameric modulation; the amount of product is higher in posterior compartments and in the most anterior cells of the anterior compartments and then gradually decreases. We have examined the effect on abd-A expression of a number of mutations, some mapping within and others outside the abd-A transcription unit. Those mapping to the transcription unit eliminate or severely reduce the amount of abd-A antigen, while those mapping outside produce an abnormal distribution of abd-A protein. Finally, we show that the abd-A gene is down-regulated in part of the Abdominal-B (Abd-B) domain, precisely in those regions where the Abd-B gene is expressed at high levels.Resumen del trabajo presentado al Yeast Genetics Meeting, celebrado en Stanford, California (USA) del 22 al 26 de agosto de 2018.The effect of the anti-tumoral drug lauryl gallate on the infectivity of the African swine fever virus among other DNA (Herpes simplex and Vaccinia) and RNA (Influenza, Porcine transmissible gastroenteritis and Sindbis) viruses, involved in animal and human diseases, is analyzed. Viral production was strongly inhibited in different cell lines at non-toxic concentrations of the drug (1-10 μM), reducing the titres from 3 to more than 5 log. units depending on the multiplicity of infection. In our model system (African swine fever virus in Vero cells), the addition of the drug 1 h before virus adsorption, completely abolished virus productivity in a one-step growth virus cycle. Interestingly, no inhibitory effect was observed when lauryl gallate was added after 5 to 8 hpi. Both cellular and viral DNA synthesis and late viral transcription were inhibited by the drug, but, however, the early viral protein synthesis and the virus-mediated increasing of p53 remained unaffected. Activation of the apoptotic effector caspase-3 was not detected after lauryl gallate treatment of Vero cells, and, furthermore, the presence of the drug abrogated the activation of this protease induced by the virus infection. The overall results likely indicate that a cellular factor/function might be the target of the antiviral action of alkyl gallates.Tesis Doctoral presentada por Eduardo Rodenas Martinez en el Centro Andaluz de Biologia del Desarrollo, centro mixto CSIC-UPO.Resumen del trabajo presentado al Yeast Genetics Meeting, celebrado en Stanford, California (USA) del 22 al 26 de agosto de 2018.

Oncogenic cooperation between SOCS family proteins and EGFR identified using a Drosophila epithelial transformation model

MicroRNAs (miRNAs) are emerging as cooperating factors that promote the activity of oncogenes in tumor formation and disease progression. This poses the challenge of identifying the miRNA targets responsible for these interactions. In this study, we identify the growth regulatory miRNA bantam and its target, Socs36E, as cooperating factors in EGFR-driven tumorigenesis and metastasis in a Drosophila model of epithelial transformation. bantam promotes growth by limiting expression of Socs36E, which functions as a negative growth regulator. Socs36E has only a modest effect on growth on its own, but behaves as a tumor suppressor in combination with EGFR activation. The human ortholog of SOCS36E, SOCS5, behaves as a candidate tumor suppressor in cellular transformation in cooperation with EGFR/RAS pathway activation.

Cell Competition Drives the Formation of Metastatic Tumors in a Drosophila Model of Epithelial Tumor Formation

Cell competition is a homeostatic process in which proliferating cells compete for survival. Elimination of otherwise normal healthy cells through competition is important during development and has recently been shown to contribute to maintaining tissue health during organismal aging. The mechanisms that allow for ongoing cell competition during adult life could, in principle, contribute to tumorigenesis. However, direct evidence supporting this hypothesis has been lacking. Here, we provide evidence that cell competition drives tumor formation in a Drosophila model of epithelial cancer. Cells expressing EGFR together with the conserved microRNA miR-8 acquire the properties of supercompetitors. Neoplastic transformation and metastasis depend on the ability of these cells to induce apoptosis and engulf nearby cells. miR-8 expression causes genome instability by downregulating expression of the Septin family protein Peanut. Cytokinesis failure due to downregulation of Peanut is required for tumorigenesis. This study provides evidence that the cellular mechanisms that drive cell competition during normal tissue growth can be co-opted to drive tumor formation and metastasis. Analogous mechanisms for cytokinesis failure may lead to polyploid intermediates in tumorigenesis in mammalian cancer models.

The miRNA machinery targets Mei-P26 and regulates Myc protein levels in the Drosophila wing

MicroRNAs (miRNAs) have been implicated in cell‐cycle regulation and in some cases shown to have a role in tissue growth control. Depletion of miRNAs was found to have an effect on tissue growth rates in the wing primordium of Drosophila, a highly proliferative epithelium. Dicer‐1 (Dcr‐1) is a double‐stranded RNAseIII essential for miRNA biogenesis. Adult cells lacking dcr‐1, or with reduced dcr‐1 activity, were smaller than normal cells and gave rise to smaller wings. dcr‐1 mutant cells showed evidence of being susceptible to competition by faster growing cells in vivo and the miRNA machinery was shown to promote G1–S transition. We present evidence that Dcr‐1 acts by regulating the TRIM‐NHL protein Mei‐P26, which in turn regulates dMyc protein levels. Mei‐P26 is a direct target of miRNAs, including the growth‐promoting bantam miRNA. Thus, regulation of tissue growth by the miRNA pathway involves a double repression mechanism to control dMyc protein levels in a highly proliferative and growing epithelium.

Scarface, a secreted serine protease-like protein, regulates polarized localization of laminin A at the basement membrane of the Drosophila embryo

Cell–matrix interactions brought about by the activity of integrins and laminins maintain the polarized architecture of epithelia and mediate morphogenetic interactions between apposing tissues. Although the polarized localization of laminins at the basement membrane is a crucial step in these processes, little is known about how this polarized distribution is achieved. Here, in Drosophila, we analyse the role of the secreted serine protease‐like protein Scarface in germ‐band retraction and dorsal closure—morphogenetic processes that rely on the activity of integrins and laminins. We present evidence that scarface is regulated by c‐Jun amino‐terminal kinase and that scarface mutant embryos show defects in these morphogenetic processes. Anomalous accumulation of laminin A on the apical surface of epithelial cells was observed in these embryos before a loss of epithelial polarity was induced. We propose that Scarface has a key role in regulating the polarized localization of laminin A in this developmental context.

Robustness and Stability of the Gene Regulatory Network Involved in DV Boundary Formation in the Drosophila Wing

Gene regulatory networks have been conserved during evolution. The Drosophila wing and the vertebrate hindbrain share the gene network involved in the establishment of the boundary between dorsal and ventral compartments in the wing and adjacent rhombomeres in the hindbrain. A positive feedback-loop between boundary and non-boundary cells and mediated by the activities of Notch and Wingless/Wnt-1 leads to the establishment of a Notch dependent organizer at the boundary. By means of a Systems Biology approach that combines mathematical modeling and both in silico and in vivo experiments in the Drosophila wing primordium, we modeled and tested this regulatory network and present evidence that a novel property, namely refractoriness to the Wingless signaling molecule, is required in boundary cells for the formation of a stable dorsal-ventral boundary. This new property has been validated in vivo, promotes mutually exclusive domains of Notch and Wingless activities and confers stability to the dorsal-ventral boundary. A robustness analysis of the regulatory network complements our results and ensures its biological plausibility.