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Localized rbp4 expression in the yolk syncytial layer plays a role in yolk cell extension and early liver development.

Li Z, Korzh V, Gong Z - BMC Dev. Biol. (2007)

Bottom Line: Knockdown of Rbp4 in the YSL resulted in shortened yolk extension as well as the formation of two liver buds, which could be due to impaired migration of liver progenitor cells. rbp4 appears also to regulate the extracellular matrix protein Fibronectin1 (Fn1) specifically in the ventro-lateral yolk, indicating a role of Fn1 in liver progenitor migration.The characteristic expression pattern of rbp4 suggests that the YSL is patterned despite its syncytial nature.YSL-expressed Rbp4 plays a role in formation of both yolk extension and liver bud, the latter may also require migration of liver progenitor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore. g0203805@nus.edu.sg

ABSTRACT

Background: The number of genes characterized in liver development is steadily increasing, but the origin of liver precursor cells and the molecular control of liver formation remain poorly understood. Existing theories about formation of zebrafish visceral organs emphasize either their budding from the endodermal rod or formation of independent anlage followed by their later fusion, but none of these is completely satisfactory in explaining liver organogenesis in zebrafish.

Results: Expression of a gene encoding the retinol binding protein 4 (Rbp4) was analyzed in zebrafish. rbp4, which is expressed mainly in the liver in adults, was shown to be expressed in the yolk syncytial layer (YSL) during early embryogenesis. At 12-16 hpf rbp4 expression was restricted to the ventro-lateral YSL and later expanded to cover the posterior YSL. We demonstrated that rbp4 expression was negatively regulated by Nodal and Hedgehog (Hh) signalling and positively controlled by retinoic acid (RA). Knockdown of Rbp4 in the YSL resulted in shortened yolk extension as well as the formation of two liver buds, which could be due to impaired migration of liver progenitor cells. rbp4 appears also to regulate the extracellular matrix protein Fibronectin1 (Fn1) specifically in the ventro-lateral yolk, indicating a role of Fn1 in liver progenitor migration. Since exocrine pancreas, endocrine pancreas, intestine and heart developed normally in Rbp4 morphants, we suggest that rbp4 expression in the YSL is required only for liver development.

Conclusion: The characteristic expression pattern of rbp4 suggests that the YSL is patterned despite its syncytial nature. YSL-expressed Rbp4 plays a role in formation of both yolk extension and liver bud, the latter may also require migration of liver progenitor cells.

Show MeSH
Analyses of liver and pancreas development in Rbp4 morphants. (A-D) Dorsal view of transferrin expression at 48 hpf in a control embryo (A) and in 48 hpf embryos injected with increasing dosage of Spl MO as indicated (B-D). The midline is indicated by a horizontal point/dash line. (E, F) Cross sections of the control embryo in (A) and morphant in (C) respectively. The section plane is indicated in (A, C) by the vertical dash line. Dashed circles in (E, F) represent gut and the vertical poin/dash lines indicate the midline. (G, H) Lateral view of shh expression in 24 hpf control embryo (G) and morphant (H). (I, J) Dorsal view of GFP expression in the principle islet of pancreas in 48 hpf control Tg(ins:gfp) embryo (I) and morphant (J). (K-N) Control and morphant stained using two-color WISH with fluorescein-labeled somatostatin 2(red) and Dig-labeled elaA (blue). Panels (K, M) shows dorsal views of 3 dpf control and morphant respectively. Panels (L, N) are cross section at the planes as indicated in (K, M). Abbreviations: elaA, elastaseA; sst2, somatostatin 2; shh, sonic hedgehog; tf, transferrin.
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Figure 4: Analyses of liver and pancreas development in Rbp4 morphants. (A-D) Dorsal view of transferrin expression at 48 hpf in a control embryo (A) and in 48 hpf embryos injected with increasing dosage of Spl MO as indicated (B-D). The midline is indicated by a horizontal point/dash line. (E, F) Cross sections of the control embryo in (A) and morphant in (C) respectively. The section plane is indicated in (A, C) by the vertical dash line. Dashed circles in (E, F) represent gut and the vertical poin/dash lines indicate the midline. (G, H) Lateral view of shh expression in 24 hpf control embryo (G) and morphant (H). (I, J) Dorsal view of GFP expression in the principle islet of pancreas in 48 hpf control Tg(ins:gfp) embryo (I) and morphant (J). (K-N) Control and morphant stained using two-color WISH with fluorescein-labeled somatostatin 2(red) and Dig-labeled elaA (blue). Panels (K, M) shows dorsal views of 3 dpf control and morphant respectively. Panels (L, N) are cross section at the planes as indicated in (K, M). Abbreviations: elaA, elastaseA; sst2, somatostatin 2; shh, sonic hedgehog; tf, transferrin.

Mentions: To study the developmental changes in the morphants, several markers expressed in the YSL and endodermal organs were employed. The liver marker transferrin [38] indicated two liver buds on both sides of the body axis in embryos injected with either ATG-MO or Spl-MO injection, but not in Mis-MO injected embryos (Table 2). Compared with 27.6% of ATG-MO morphants with two liver buds, a much higher percentage (72.1%) of Spl-MO morphants showed this phenotype (Table 2). The two liver buds were either connected with each other or separated (Figure 4A–D). This phenotype also appeared in a dose-dependant manner; the high dose of Spl-MO caused an increase in a number of morphants with separated liver buds (data not shown). Duplication of liver or other unpaired visceral organs such as pancreas, heart and interrenal gland have been previously observed in mutants with defective midline formation [14,39-41]. In order to examine whether the phenomenon of duplicated liver in Rbp4 morphants was also due to midline defects, several midline markers were evaluated in the morphants at 18 and 24 hpf. shh (Figure 4G, H),no tail and twhh were expressed normally suggesting that formation of the notochord and floor plate was not affected. Therefore the duplication of the liver bud in Rbp4 morphants is not related to defects in midline formation. To further analyze liver morphology, cross-section of 48 hpf morphants was performed. From anterior to posterior, the two liver buds contain several layers of cells similar to that in control (Figure 4E, F). Thus it seems that the two liver buds undergo at least initial stages of hepatogenesis. At the same time, these sections also showed that the gut was normal. In contrast to the YSL-injection, injection of morpholino at 1-cell stage resulted in a lower percentage of the duplicated liver phenotype (5.8%, n = 52). In addition, defects in the brain and tail were observed (data not shown), indicating Rbp4 may have other functions during development. While this study focused on a role of Rbp4 during liver development, other functions of Rbp4 will be analyzed in future.


Localized rbp4 expression in the yolk syncytial layer plays a role in yolk cell extension and early liver development.

Li Z, Korzh V, Gong Z - BMC Dev. Biol. (2007)

Analyses of liver and pancreas development in Rbp4 morphants. (A-D) Dorsal view of transferrin expression at 48 hpf in a control embryo (A) and in 48 hpf embryos injected with increasing dosage of Spl MO as indicated (B-D). The midline is indicated by a horizontal point/dash line. (E, F) Cross sections of the control embryo in (A) and morphant in (C) respectively. The section plane is indicated in (A, C) by the vertical dash line. Dashed circles in (E, F) represent gut and the vertical poin/dash lines indicate the midline. (G, H) Lateral view of shh expression in 24 hpf control embryo (G) and morphant (H). (I, J) Dorsal view of GFP expression in the principle islet of pancreas in 48 hpf control Tg(ins:gfp) embryo (I) and morphant (J). (K-N) Control and morphant stained using two-color WISH with fluorescein-labeled somatostatin 2(red) and Dig-labeled elaA (blue). Panels (K, M) shows dorsal views of 3 dpf control and morphant respectively. Panels (L, N) are cross section at the planes as indicated in (K, M). Abbreviations: elaA, elastaseA; sst2, somatostatin 2; shh, sonic hedgehog; tf, transferrin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Analyses of liver and pancreas development in Rbp4 morphants. (A-D) Dorsal view of transferrin expression at 48 hpf in a control embryo (A) and in 48 hpf embryos injected with increasing dosage of Spl MO as indicated (B-D). The midline is indicated by a horizontal point/dash line. (E, F) Cross sections of the control embryo in (A) and morphant in (C) respectively. The section plane is indicated in (A, C) by the vertical dash line. Dashed circles in (E, F) represent gut and the vertical poin/dash lines indicate the midline. (G, H) Lateral view of shh expression in 24 hpf control embryo (G) and morphant (H). (I, J) Dorsal view of GFP expression in the principle islet of pancreas in 48 hpf control Tg(ins:gfp) embryo (I) and morphant (J). (K-N) Control and morphant stained using two-color WISH with fluorescein-labeled somatostatin 2(red) and Dig-labeled elaA (blue). Panels (K, M) shows dorsal views of 3 dpf control and morphant respectively. Panels (L, N) are cross section at the planes as indicated in (K, M). Abbreviations: elaA, elastaseA; sst2, somatostatin 2; shh, sonic hedgehog; tf, transferrin.
Mentions: To study the developmental changes in the morphants, several markers expressed in the YSL and endodermal organs were employed. The liver marker transferrin [38] indicated two liver buds on both sides of the body axis in embryos injected with either ATG-MO or Spl-MO injection, but not in Mis-MO injected embryos (Table 2). Compared with 27.6% of ATG-MO morphants with two liver buds, a much higher percentage (72.1%) of Spl-MO morphants showed this phenotype (Table 2). The two liver buds were either connected with each other or separated (Figure 4A–D). This phenotype also appeared in a dose-dependant manner; the high dose of Spl-MO caused an increase in a number of morphants with separated liver buds (data not shown). Duplication of liver or other unpaired visceral organs such as pancreas, heart and interrenal gland have been previously observed in mutants with defective midline formation [14,39-41]. In order to examine whether the phenomenon of duplicated liver in Rbp4 morphants was also due to midline defects, several midline markers were evaluated in the morphants at 18 and 24 hpf. shh (Figure 4G, H),no tail and twhh were expressed normally suggesting that formation of the notochord and floor plate was not affected. Therefore the duplication of the liver bud in Rbp4 morphants is not related to defects in midline formation. To further analyze liver morphology, cross-section of 48 hpf morphants was performed. From anterior to posterior, the two liver buds contain several layers of cells similar to that in control (Figure 4E, F). Thus it seems that the two liver buds undergo at least initial stages of hepatogenesis. At the same time, these sections also showed that the gut was normal. In contrast to the YSL-injection, injection of morpholino at 1-cell stage resulted in a lower percentage of the duplicated liver phenotype (5.8%, n = 52). In addition, defects in the brain and tail were observed (data not shown), indicating Rbp4 may have other functions during development. While this study focused on a role of Rbp4 during liver development, other functions of Rbp4 will be analyzed in future.

Bottom Line: Knockdown of Rbp4 in the YSL resulted in shortened yolk extension as well as the formation of two liver buds, which could be due to impaired migration of liver progenitor cells. rbp4 appears also to regulate the extracellular matrix protein Fibronectin1 (Fn1) specifically in the ventro-lateral yolk, indicating a role of Fn1 in liver progenitor migration.The characteristic expression pattern of rbp4 suggests that the YSL is patterned despite its syncytial nature.YSL-expressed Rbp4 plays a role in formation of both yolk extension and liver bud, the latter may also require migration of liver progenitor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore. g0203805@nus.edu.sg

ABSTRACT

Background: The number of genes characterized in liver development is steadily increasing, but the origin of liver precursor cells and the molecular control of liver formation remain poorly understood. Existing theories about formation of zebrafish visceral organs emphasize either their budding from the endodermal rod or formation of independent anlage followed by their later fusion, but none of these is completely satisfactory in explaining liver organogenesis in zebrafish.

Results: Expression of a gene encoding the retinol binding protein 4 (Rbp4) was analyzed in zebrafish. rbp4, which is expressed mainly in the liver in adults, was shown to be expressed in the yolk syncytial layer (YSL) during early embryogenesis. At 12-16 hpf rbp4 expression was restricted to the ventro-lateral YSL and later expanded to cover the posterior YSL. We demonstrated that rbp4 expression was negatively regulated by Nodal and Hedgehog (Hh) signalling and positively controlled by retinoic acid (RA). Knockdown of Rbp4 in the YSL resulted in shortened yolk extension as well as the formation of two liver buds, which could be due to impaired migration of liver progenitor cells. rbp4 appears also to regulate the extracellular matrix protein Fibronectin1 (Fn1) specifically in the ventro-lateral yolk, indicating a role of Fn1 in liver progenitor migration. Since exocrine pancreas, endocrine pancreas, intestine and heart developed normally in Rbp4 morphants, we suggest that rbp4 expression in the YSL is required only for liver development.

Conclusion: The characteristic expression pattern of rbp4 suggests that the YSL is patterned despite its syncytial nature. YSL-expressed Rbp4 plays a role in formation of both yolk extension and liver bud, the latter may also require migration of liver progenitor cells.

Show MeSH