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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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In situ hybridization of Tc-HexA1 and Hexokinase activity during the first 24 hours of beetle embryogenesis.(A–E) In situ hybridization and respective nuclear DAPI stainings (A’–E’). In all panels head is to the left and dorsal side up. (F) Hex activity during the first 24 hours of embryonic development. (A,A’) Eggs during the first four hours after egg lay (AEL), when rapid cleavages occur display ubiquitous Tc-HexA1 mRNA. (B,B’) Eggs between four and eight hours (4–8 hours) also show ubiquitous Tc-HexA1 expression. (C,C’) During gastrulation between 8–12 hours Tc-HexA1 expression largely decreases, remaining low between 12–16 hours in D,D’. (E,E’) During germ band elongation (16–20 hours) Tc-HexA1 expression is upregulated and occurs only in the embryonic region (emb in D), being absent in the serosa (ser). (F) Specific Hexokinase activity (U/mg protein). High activity is detected in egg extracts from 0–4 hours and after 16 hours, which correlates to Tc-HexA1 mRNA expression pattern. pp - posterior pit, emb - embryonic tissue, ser - serosa.
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pone-0065125-g004: In situ hybridization of Tc-HexA1 and Hexokinase activity during the first 24 hours of beetle embryogenesis.(A–E) In situ hybridization and respective nuclear DAPI stainings (A’–E’). In all panels head is to the left and dorsal side up. (F) Hex activity during the first 24 hours of embryonic development. (A,A’) Eggs during the first four hours after egg lay (AEL), when rapid cleavages occur display ubiquitous Tc-HexA1 mRNA. (B,B’) Eggs between four and eight hours (4–8 hours) also show ubiquitous Tc-HexA1 expression. (C,C’) During gastrulation between 8–12 hours Tc-HexA1 expression largely decreases, remaining low between 12–16 hours in D,D’. (E,E’) During germ band elongation (16–20 hours) Tc-HexA1 expression is upregulated and occurs only in the embryonic region (emb in D), being absent in the serosa (ser). (F) Specific Hexokinase activity (U/mg protein). High activity is detected in egg extracts from 0–4 hours and after 16 hours, which correlates to Tc-HexA1 mRNA expression pattern. pp - posterior pit, emb - embryonic tissue, ser - serosa.

Mentions: Since Tc-HexA1 appears to be expressed during early hours of embryogenesis we investigated its mRNA localization by in situ hybridization. Tc-HexA1 is detected ubiquitously during the first four hours of embryonic development probably due to maternal mRNA deposition (Figure 4A). At that stage only a few nuclei can be observed by nuclear DAPI stainings (Figure 4A’). During the next four hours of embryonic development extensive cell division takes place and Tc-HexA1 expression is still observed (Figure 4B, B’). Soon after, Tc-HexA1 expression starts to diminish and the lowest levels are observed shortly before gastrulation (8–12 hours), when the posterior pit (pp) can be observed (Figure 4C,C’). During gastrulation and beginning of germ band extension mRNA levels remain low (Figure 4D) and the embryonic (emb) cells at the ventral side can be distinguished from the serosa cells, the latter with large nuclei (Figure 4D’). During germ band extension (16–20 hours) Tc-HexA1 expression is upregulated and identified only at the embryonic region; expression in the polyploid serosa (ser) cells is absent (Figure 4E, E’). Taken together, the spatial analysis of Tc-HexA1 expression suggest a temporal control at early stages (0–12 hours) and a spatial control shortly after (16–20 hours) with embryonic cells expressing this enzyme and extra-embryonic cells lacking it (Figure 4D,E).


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)

In situ hybridization of Tc-HexA1 and Hexokinase activity during the first 24 hours of beetle embryogenesis.(A–E) In situ hybridization and respective nuclear DAPI stainings (A’–E’). In all panels head is to the left and dorsal side up. (F) Hex activity during the first 24 hours of embryonic development. (A,A’) Eggs during the first four hours after egg lay (AEL), when rapid cleavages occur display ubiquitous Tc-HexA1 mRNA. (B,B’) Eggs between four and eight hours (4–8 hours) also show ubiquitous Tc-HexA1 expression. (C,C’) During gastrulation between 8–12 hours Tc-HexA1 expression largely decreases, remaining low between 12–16 hours in D,D’. (E,E’) During germ band elongation (16–20 hours) Tc-HexA1 expression is upregulated and occurs only in the embryonic region (emb in D), being absent in the serosa (ser). (F) Specific Hexokinase activity (U/mg protein). High activity is detected in egg extracts from 0–4 hours and after 16 hours, which correlates to Tc-HexA1 mRNA expression pattern. pp - posterior pit, emb - embryonic tissue, ser - serosa.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672164&req=5

pone-0065125-g004: In situ hybridization of Tc-HexA1 and Hexokinase activity during the first 24 hours of beetle embryogenesis.(A–E) In situ hybridization and respective nuclear DAPI stainings (A’–E’). In all panels head is to the left and dorsal side up. (F) Hex activity during the first 24 hours of embryonic development. (A,A’) Eggs during the first four hours after egg lay (AEL), when rapid cleavages occur display ubiquitous Tc-HexA1 mRNA. (B,B’) Eggs between four and eight hours (4–8 hours) also show ubiquitous Tc-HexA1 expression. (C,C’) During gastrulation between 8–12 hours Tc-HexA1 expression largely decreases, remaining low between 12–16 hours in D,D’. (E,E’) During germ band elongation (16–20 hours) Tc-HexA1 expression is upregulated and occurs only in the embryonic region (emb in D), being absent in the serosa (ser). (F) Specific Hexokinase activity (U/mg protein). High activity is detected in egg extracts from 0–4 hours and after 16 hours, which correlates to Tc-HexA1 mRNA expression pattern. pp - posterior pit, emb - embryonic tissue, ser - serosa.
Mentions: Since Tc-HexA1 appears to be expressed during early hours of embryogenesis we investigated its mRNA localization by in situ hybridization. Tc-HexA1 is detected ubiquitously during the first four hours of embryonic development probably due to maternal mRNA deposition (Figure 4A). At that stage only a few nuclei can be observed by nuclear DAPI stainings (Figure 4A’). During the next four hours of embryonic development extensive cell division takes place and Tc-HexA1 expression is still observed (Figure 4B, B’). Soon after, Tc-HexA1 expression starts to diminish and the lowest levels are observed shortly before gastrulation (8–12 hours), when the posterior pit (pp) can be observed (Figure 4C,C’). During gastrulation and beginning of germ band extension mRNA levels remain low (Figure 4D) and the embryonic (emb) cells at the ventral side can be distinguished from the serosa cells, the latter with large nuclei (Figure 4D’). During germ band extension (16–20 hours) Tc-HexA1 expression is upregulated and identified only at the embryonic region; expression in the polyploid serosa (ser) cells is absent (Figure 4E, E’). Taken together, the spatial analysis of Tc-HexA1 expression suggest a temporal control at early stages (0–12 hours) and a spatial control shortly after (16–20 hours) with embryonic cells expressing this enzyme and extra-embryonic cells lacking it (Figure 4D,E).

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