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Expression Analysis of the Hippo Cascade Indicates a Role in Pituitary Stem Cell Development.

Lodge EJ, Russell JP, Patist AL, Francis-West P, Andoniadou CL - Front Physiol (2016)

Bottom Line: We have also carried out immunolocalisation studies to determine when YAP1 and TAZ, the transcriptional effectors of the Hippo pathway, are active.We find that YAP1/TAZ are active in the stem/progenitor cell population throughout development and at postnatal stages, consistent with their role in promoting the stem cell state.Our results demonstrate for the first time the collective expression of major components of the Hippo pathway during normal embryonic and postnatal development of the pituitary gland.

View Article: PubMed Central - PubMed

Affiliation: Craniofacial Development and Stem Cell Biology, Dental Institute, King's College London London, UK.

ABSTRACT
The pituitary gland is a primary endocrine organ that controls major physiological processes. Abnormal development or homeostatic disruptions can lead to human disorders such as hypopituitarism or tumors. Multiple signaling pathways, including WNT, BMP, FGF, and SHH regulate pituitary development but the role of the Hippo-YAP1/TAZ cascade is currently unknown. In multiple tissues, the Hippo kinase cascade underlies neoplasias; it influences organ size through the regulation of proliferation and apoptosis, and has roles in determining stem cell potential. We have used a sensitive mRNA in situ hybridization method (RNAscope) to determine the expression patterns of the Hippo pathway components during mouse pituitary development. We have also carried out immunolocalisation studies to determine when YAP1 and TAZ, the transcriptional effectors of the Hippo pathway, are active. We find that YAP1/TAZ are active in the stem/progenitor cell population throughout development and at postnatal stages, consistent with their role in promoting the stem cell state. Our results demonstrate for the first time the collective expression of major components of the Hippo pathway during normal embryonic and postnatal development of the pituitary gland.

No MeSH data available.


Related in: MedlinePlus

Validation of RNAscope method in the pituitary gland. RNAscope mRNA in situ hybridization using probes against Hesx1 and Sox2 on wild type CD1 embryos at stages between 10.5dpc and 13.5dpc. (A–C) At 10.5dpc Hesx1 is expressed in the oral epithelium and Rathke's pouch (arrowhead), but excluded from the pharyngeal endoderm. The posterior limit of expression is indicated by the arrow in (A). At 12.5dpc and 13.5dpc expression is reduced although still detectable (C′, boxed area in C). (D–F)Sox2 is expressed in Rathke's pouch at 10.5dpc (arrowheads) as well as neural tissue, oral epithelium and pharyngeal endoderm. Expression decreases in the ventral pouch where committed cells arise (asterisk in F) and persists periluminal and in the dorsal RP (arrowheads in E,F). For clarity, the developing anterior pituitary primordium is indicated by the dotted outline in (E,F). Abbreviations: rp, Rathke's pouch; vd, ventral diencephalon; m, mesenchyme; or, oral ectoderm; pe, pharyngeal endoderm; inf, infundibulum; sph, sphenoid. Sagittal sections, axes in (A) applicable to all panels: d, dorsal; v, ventral; r, rostral; c, caudal. Scale bars 200 μm.
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Figure 1: Validation of RNAscope method in the pituitary gland. RNAscope mRNA in situ hybridization using probes against Hesx1 and Sox2 on wild type CD1 embryos at stages between 10.5dpc and 13.5dpc. (A–C) At 10.5dpc Hesx1 is expressed in the oral epithelium and Rathke's pouch (arrowhead), but excluded from the pharyngeal endoderm. The posterior limit of expression is indicated by the arrow in (A). At 12.5dpc and 13.5dpc expression is reduced although still detectable (C′, boxed area in C). (D–F)Sox2 is expressed in Rathke's pouch at 10.5dpc (arrowheads) as well as neural tissue, oral epithelium and pharyngeal endoderm. Expression decreases in the ventral pouch where committed cells arise (asterisk in F) and persists periluminal and in the dorsal RP (arrowheads in E,F). For clarity, the developing anterior pituitary primordium is indicated by the dotted outline in (E,F). Abbreviations: rp, Rathke's pouch; vd, ventral diencephalon; m, mesenchyme; or, oral ectoderm; pe, pharyngeal endoderm; inf, infundibulum; sph, sphenoid. Sagittal sections, axes in (A) applicable to all panels: d, dorsal; v, ventral; r, rostral; c, caudal. Scale bars 200 μm.

Mentions: To determine reliability of the RNAscope in situ hybridization in the pituitary we first validated this method by analysis of Hesx1, which has a known pattern, only expressed during development. It is expressed strongly in oral ectoderm subsequently fated to become Rathke's pouch at 8.5dpc (Cajal et al., 2012), and persists in RP epithelium until 11.5dpc with levels of expression decreasing thereafter (Thomas and Beddington, 1996). Using the RNAscope in situ hybridization method for sensitive mRNA detection, we confirmed strong expression of Hesx1 in RP at 10.5dpc (Figure 1A, arrowhead), which extended rostrally in the oral epithelium. No expression was detected in the pharyngeal endoderm as previously reported (posterior limit of expression noted by arrow). Expression of Hesx1 was barely detectable at 12.5dpc and 13.5dpc (Figures 1B,C) with presence of only sporadic transcripts (Figure 1C′ magnified boxed region in C). Additionally, we investigated expression of Sox2, which marks progenitors/stem cells in the pituitary, using this method. Robust expression of Sox2 transcripts was detected in RP and the developing ventral diencephalon, with complete absence of expression in mesenchyme surrounding the pouch at all stages (Figure 1). Expression of Sox2 is known to be down-regulated in committed lineages of the pituitary gland, which we confirmed at 13.5dpc; there is an absence of transcripts in the ventral anterior pituitary where cells are undergoing commitment (Figure 1F, asterisk). Expression persisted dorsally, specifically in the marginal epithelium surrounding the pouch where the uncommitted cells reside (arrowheads in Figure 1F).


Expression Analysis of the Hippo Cascade Indicates a Role in Pituitary Stem Cell Development.

Lodge EJ, Russell JP, Patist AL, Francis-West P, Andoniadou CL - Front Physiol (2016)

Validation of RNAscope method in the pituitary gland. RNAscope mRNA in situ hybridization using probes against Hesx1 and Sox2 on wild type CD1 embryos at stages between 10.5dpc and 13.5dpc. (A–C) At 10.5dpc Hesx1 is expressed in the oral epithelium and Rathke's pouch (arrowhead), but excluded from the pharyngeal endoderm. The posterior limit of expression is indicated by the arrow in (A). At 12.5dpc and 13.5dpc expression is reduced although still detectable (C′, boxed area in C). (D–F)Sox2 is expressed in Rathke's pouch at 10.5dpc (arrowheads) as well as neural tissue, oral epithelium and pharyngeal endoderm. Expression decreases in the ventral pouch where committed cells arise (asterisk in F) and persists periluminal and in the dorsal RP (arrowheads in E,F). For clarity, the developing anterior pituitary primordium is indicated by the dotted outline in (E,F). Abbreviations: rp, Rathke's pouch; vd, ventral diencephalon; m, mesenchyme; or, oral ectoderm; pe, pharyngeal endoderm; inf, infundibulum; sph, sphenoid. Sagittal sections, axes in (A) applicable to all panels: d, dorsal; v, ventral; r, rostral; c, caudal. Scale bars 200 μm.
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Related In: Results  -  Collection

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Figure 1: Validation of RNAscope method in the pituitary gland. RNAscope mRNA in situ hybridization using probes against Hesx1 and Sox2 on wild type CD1 embryos at stages between 10.5dpc and 13.5dpc. (A–C) At 10.5dpc Hesx1 is expressed in the oral epithelium and Rathke's pouch (arrowhead), but excluded from the pharyngeal endoderm. The posterior limit of expression is indicated by the arrow in (A). At 12.5dpc and 13.5dpc expression is reduced although still detectable (C′, boxed area in C). (D–F)Sox2 is expressed in Rathke's pouch at 10.5dpc (arrowheads) as well as neural tissue, oral epithelium and pharyngeal endoderm. Expression decreases in the ventral pouch where committed cells arise (asterisk in F) and persists periluminal and in the dorsal RP (arrowheads in E,F). For clarity, the developing anterior pituitary primordium is indicated by the dotted outline in (E,F). Abbreviations: rp, Rathke's pouch; vd, ventral diencephalon; m, mesenchyme; or, oral ectoderm; pe, pharyngeal endoderm; inf, infundibulum; sph, sphenoid. Sagittal sections, axes in (A) applicable to all panels: d, dorsal; v, ventral; r, rostral; c, caudal. Scale bars 200 μm.
Mentions: To determine reliability of the RNAscope in situ hybridization in the pituitary we first validated this method by analysis of Hesx1, which has a known pattern, only expressed during development. It is expressed strongly in oral ectoderm subsequently fated to become Rathke's pouch at 8.5dpc (Cajal et al., 2012), and persists in RP epithelium until 11.5dpc with levels of expression decreasing thereafter (Thomas and Beddington, 1996). Using the RNAscope in situ hybridization method for sensitive mRNA detection, we confirmed strong expression of Hesx1 in RP at 10.5dpc (Figure 1A, arrowhead), which extended rostrally in the oral epithelium. No expression was detected in the pharyngeal endoderm as previously reported (posterior limit of expression noted by arrow). Expression of Hesx1 was barely detectable at 12.5dpc and 13.5dpc (Figures 1B,C) with presence of only sporadic transcripts (Figure 1C′ magnified boxed region in C). Additionally, we investigated expression of Sox2, which marks progenitors/stem cells in the pituitary, using this method. Robust expression of Sox2 transcripts was detected in RP and the developing ventral diencephalon, with complete absence of expression in mesenchyme surrounding the pouch at all stages (Figure 1). Expression of Sox2 is known to be down-regulated in committed lineages of the pituitary gland, which we confirmed at 13.5dpc; there is an absence of transcripts in the ventral anterior pituitary where cells are undergoing commitment (Figure 1F, asterisk). Expression persisted dorsally, specifically in the marginal epithelium surrounding the pouch where the uncommitted cells reside (arrowheads in Figure 1F).

Bottom Line: We have also carried out immunolocalisation studies to determine when YAP1 and TAZ, the transcriptional effectors of the Hippo pathway, are active.We find that YAP1/TAZ are active in the stem/progenitor cell population throughout development and at postnatal stages, consistent with their role in promoting the stem cell state.Our results demonstrate for the first time the collective expression of major components of the Hippo pathway during normal embryonic and postnatal development of the pituitary gland.

View Article: PubMed Central - PubMed

Affiliation: Craniofacial Development and Stem Cell Biology, Dental Institute, King's College London London, UK.

ABSTRACT
The pituitary gland is a primary endocrine organ that controls major physiological processes. Abnormal development or homeostatic disruptions can lead to human disorders such as hypopituitarism or tumors. Multiple signaling pathways, including WNT, BMP, FGF, and SHH regulate pituitary development but the role of the Hippo-YAP1/TAZ cascade is currently unknown. In multiple tissues, the Hippo kinase cascade underlies neoplasias; it influences organ size through the regulation of proliferation and apoptosis, and has roles in determining stem cell potential. We have used a sensitive mRNA in situ hybridization method (RNAscope) to determine the expression patterns of the Hippo pathway components during mouse pituitary development. We have also carried out immunolocalisation studies to determine when YAP1 and TAZ, the transcriptional effectors of the Hippo pathway, are active. We find that YAP1/TAZ are active in the stem/progenitor cell population throughout development and at postnatal stages, consistent with their role in promoting the stem cell state. Our results demonstrate for the first time the collective expression of major components of the Hippo pathway during normal embryonic and postnatal development of the pituitary gland.

No MeSH data available.


Related in: MedlinePlus