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Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum.

Fraga A, Ribeiro L, Lobato M, Santos V, Silva JR, Gomes H, da Cunha Moraes JL, de Souza Menezes J, de Oliveira CJ, Campos E, da Fonseca RN - PLoS ONE (2013)

Bottom Line: In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis.Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality.Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos.

View Article: PubMed Central - PubMed

Affiliation: Laboratório Integrado de Bioquímica Hatisaburo Masuda (LIBHM), Núcleo de Pesquisas Ecológicas e Sócioambientais de Macaé (NUPEM), Universidade Federal do Rio de Janeiro (UFRJCampus Macaé), Rio de Janeiro, Brazil.

ABSTRACT
Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.

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Tc-GSK-3 is expressed in the embryonic tissue throughout embryogenesis.(A–D) Tc-GSK-3 expression and respective nuclear DAPI stainings (A’–D’). (A,A’) In the first four hours after egg laying (AEL), Tc-GSK-3 is ubiquitously expressed. Note the few nuclei at the periphery. In the next four hours (4–8 hours) a similar ubiquitous expression pattern is observed (data not shown). (B–B’) Between 8–12 hours AEL Tc-GSK-3 expression is mainly concentrated at the embryonic cells (emb) and not in the extraembryonic polyploid serosal (ser) cells. (C–C’) This pattern of strong expression of Tc-GSK-3 in the embryonic cells remains between 12–16 hours AEL, when serosal cells surround the embryonic ones. (D–D’) Between 16–20 hours AEL Tc-GSK-3 expression is still largely confined to the embryo (emb), which is undergoing germ band elongation. At 20–24 hours AEL a similar expression profile is observed (data not shown).
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pone-0065125-g007: Tc-GSK-3 is expressed in the embryonic tissue throughout embryogenesis.(A–D) Tc-GSK-3 expression and respective nuclear DAPI stainings (A’–D’). (A,A’) In the first four hours after egg laying (AEL), Tc-GSK-3 is ubiquitously expressed. Note the few nuclei at the periphery. In the next four hours (4–8 hours) a similar ubiquitous expression pattern is observed (data not shown). (B–B’) Between 8–12 hours AEL Tc-GSK-3 expression is mainly concentrated at the embryonic cells (emb) and not in the extraembryonic polyploid serosal (ser) cells. (C–C’) This pattern of strong expression of Tc-GSK-3 in the embryonic cells remains between 12–16 hours AEL, when serosal cells surround the embryonic ones. (D–D’) Between 16–20 hours AEL Tc-GSK-3 expression is still largely confined to the embryo (emb), which is undergoing germ band elongation. At 20–24 hours AEL a similar expression profile is observed (data not shown).

Mentions: One of the key enzymes involved in glycogen regulation and degradation is the glycogen synthase kinase (GSK-3), which phosphorylates Glycogen Synthase (GS) responsible for glycogen synthesis. Phosphorylation of GS by GSK-3 decreases its activity. In addition to its metabolic role, GSK-3 also acts as a key downstream component of the Wnt pathway (reviewed in [48]). To investigate the role of GSK-3 during T. castaneum embryogenesis we performed in situ hybridization using embryos collected every four hours as previously described for Tc-HexA1. First, Tc-GSK-3 seems to be maternally provided since its mRNA is detected along the whole egg during the first four hours of development (Figure 7A,A’). In the interval between 4 and 8 hours and between 8–12 hours, Tc-GSK-3 is detected only at the germ rudiment, a ventral-posterior part of the egg, constituted by embryo and the amnion (Figure 7B,B’-emb, data not shown), and not in the extra-embryonic serosa (Figure 7B, ser, see also [49]).


Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum.

Fraga A, Ribeiro L, Lobato M, Santos V, Silva JR, Gomes H, da Cunha Moraes JL, de Souza Menezes J, de Oliveira CJ, Campos E, da Fonseca RN - PLoS ONE (2013)

Tc-GSK-3 is expressed in the embryonic tissue throughout embryogenesis.(A–D) Tc-GSK-3 expression and respective nuclear DAPI stainings (A’–D’). (A,A’) In the first four hours after egg laying (AEL), Tc-GSK-3 is ubiquitously expressed. Note the few nuclei at the periphery. In the next four hours (4–8 hours) a similar ubiquitous expression pattern is observed (data not shown). (B–B’) Between 8–12 hours AEL Tc-GSK-3 expression is mainly concentrated at the embryonic cells (emb) and not in the extraembryonic polyploid serosal (ser) cells. (C–C’) This pattern of strong expression of Tc-GSK-3 in the embryonic cells remains between 12–16 hours AEL, when serosal cells surround the embryonic ones. (D–D’) Between 16–20 hours AEL Tc-GSK-3 expression is still largely confined to the embryo (emb), which is undergoing germ band elongation. At 20–24 hours AEL a similar expression profile is observed (data not shown).
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Related In: Results  -  Collection

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pone-0065125-g007: Tc-GSK-3 is expressed in the embryonic tissue throughout embryogenesis.(A–D) Tc-GSK-3 expression and respective nuclear DAPI stainings (A’–D’). (A,A’) In the first four hours after egg laying (AEL), Tc-GSK-3 is ubiquitously expressed. Note the few nuclei at the periphery. In the next four hours (4–8 hours) a similar ubiquitous expression pattern is observed (data not shown). (B–B’) Between 8–12 hours AEL Tc-GSK-3 expression is mainly concentrated at the embryonic cells (emb) and not in the extraembryonic polyploid serosal (ser) cells. (C–C’) This pattern of strong expression of Tc-GSK-3 in the embryonic cells remains between 12–16 hours AEL, when serosal cells surround the embryonic ones. (D–D’) Between 16–20 hours AEL Tc-GSK-3 expression is still largely confined to the embryo (emb), which is undergoing germ band elongation. At 20–24 hours AEL a similar expression profile is observed (data not shown).
Mentions: One of the key enzymes involved in glycogen regulation and degradation is the glycogen synthase kinase (GSK-3), which phosphorylates Glycogen Synthase (GS) responsible for glycogen synthesis. Phosphorylation of GS by GSK-3 decreases its activity. In addition to its metabolic role, GSK-3 also acts as a key downstream component of the Wnt pathway (reviewed in [48]). To investigate the role of GSK-3 during T. castaneum embryogenesis we performed in situ hybridization using embryos collected every four hours as previously described for Tc-HexA1. First, Tc-GSK-3 seems to be maternally provided since its mRNA is detected along the whole egg during the first four hours of development (Figure 7A,A’). In the interval between 4 and 8 hours and between 8–12 hours, Tc-GSK-3 is detected only at the germ rudiment, a ventral-posterior part of the egg, constituted by embryo and the amnion (Figure 7B,B’-emb, data not shown), and not in the extra-embryonic serosa (Figure 7B, ser, see also [49]).

Bottom Line: In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis.Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality.Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos.

View Article: PubMed Central - PubMed

Affiliation: Laboratório Integrado de Bioquímica Hatisaburo Masuda (LIBHM), Núcleo de Pesquisas Ecológicas e Sócioambientais de Macaé (NUPEM), Universidade Federal do Rio de Janeiro (UFRJCampus Macaé), Rio de Janeiro, Brazil.

ABSTRACT
Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.

Show MeSH
Related in: MedlinePlus