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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

Expression of YAP1 and TAZ proteins during embryonic development. Immunofluorescence using specific antibodies against total TAZ protein, total YAP1 protein and phosphorylated YAP1 (S127) in red. Nuclei are counterstained with DAPI (blue). (A–J) Localization of effectors TAZ (A–E) and YAP1 (F–J) using antibodies recognizing total protein. Note the nuclear localization in periluminal cells of Rathke's pouch (yellow arrowheads in B,C,G,H, white arrowheads in D,E,I,J). No YAP1/TAZ proteins are detected in the rostral tip (arrowheads in B,C,G,H) and there is a reduction in expression in ventral regions (asterisks in B,C,G,H). Note expression in structures resembling capillaries from 15.5dpc (arrows in D,E,J). (K–O) Immunofluorescence to detect the phosphorylated form of YAP1 at S127. Protein detection indicates Hippo kinase cascade activity at all stages, primarily in periluminal RP epithelium (white arrowheads in K,N,O). Note the ventral bias of protein localization at 12.5dpc and 13.5dpc (yellow arrows in L,M) and complete absence of protein from the rostral tip (white arrowheads in L,M). Boxed inserts show higher magnifications of the epithelium. Examples of cytoplasmic localization are noted by white arrows and examples of nuclear localization by yellow arrows. Abbreviations: rp, Rathke's pouch; vd, ventral diencephalon; m, mesenchyme; oe, oral ectoderm; inf, infundibulum; sph, sphenoid; rt, rostral tip; pl, posterior lobe; al, anterior lobe; il, intermediate lobe. Sagittal sections between 10.5dpc and 13.5dpc and frontal between 15.5dpc and 17.5dpc. Axes in (A) applicable to (A–C,F–H,K–M: d, dorsal; v, ventral; r, rostral; c, caudal). Axes in (D) applicable to (D,E,I,J,N,O: d, dorsal; v, ventral; ri, right; le, left). Scale bars 100 μm and 20 μm in boxed inserts.
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Figure 5: Expression of YAP1 and TAZ proteins during embryonic development. Immunofluorescence using specific antibodies against total TAZ protein, total YAP1 protein and phosphorylated YAP1 (S127) in red. Nuclei are counterstained with DAPI (blue). (A–J) Localization of effectors TAZ (A–E) and YAP1 (F–J) using antibodies recognizing total protein. Note the nuclear localization in periluminal cells of Rathke's pouch (yellow arrowheads in B,C,G,H, white arrowheads in D,E,I,J). No YAP1/TAZ proteins are detected in the rostral tip (arrowheads in B,C,G,H) and there is a reduction in expression in ventral regions (asterisks in B,C,G,H). Note expression in structures resembling capillaries from 15.5dpc (arrows in D,E,J). (K–O) Immunofluorescence to detect the phosphorylated form of YAP1 at S127. Protein detection indicates Hippo kinase cascade activity at all stages, primarily in periluminal RP epithelium (white arrowheads in K,N,O). Note the ventral bias of protein localization at 12.5dpc and 13.5dpc (yellow arrows in L,M) and complete absence of protein from the rostral tip (white arrowheads in L,M). Boxed inserts show higher magnifications of the epithelium. Examples of cytoplasmic localization are noted by white arrows and examples of nuclear localization by yellow arrows. Abbreviations: rp, Rathke's pouch; vd, ventral diencephalon; m, mesenchyme; oe, oral ectoderm; inf, infundibulum; sph, sphenoid; rt, rostral tip; pl, posterior lobe; al, anterior lobe; il, intermediate lobe. Sagittal sections between 10.5dpc and 13.5dpc and frontal between 15.5dpc and 17.5dpc. Axes in (A) applicable to (A–C,F–H,K–M: d, dorsal; v, ventral; r, rostral; c, caudal). Axes in (D) applicable to (D,E,I,J,N,O: d, dorsal; v, ventral; ri, right; le, left). Scale bars 100 μm and 20 μm in boxed inserts.

Mentions: In order to infer activity of the Hippo kinase cascade, we investigated the localization of effector proteins TAZ, total YAP1, as well as the inactive phosphorylated form of YAP1 (S127). TAZ and YAP1 had similar localization at 10.5dpc; they appeared nucleo-cytoplasmic with a bias for the apical cytoplasm of RP epithelium (Figures 5A,F, arrowheads). Inactive YAP1, marked by pYAP1 was strongly cytoplasmic and also displayed an apical bias (Figure 5K, arrowheads). All three antibodies marked cells in the mesenchyme and neural tissue. At 12.5dpc and 13.5dpc YAP1 and TAZ both localized mostly in nuclei of cells in RP epithelium, with stronger expression in the dorsal RP epithelium at 12.5dpc, which persisted at 13.5dpc for YAP1 (yellow arrowheads in Figures 5B,G,H). In the ventral portion of the epithelium there was nuclear localization in a thin cell layer surrounding the cleft. Little expression was observed in more ventral regions (asterisk in Figures 5B,C,G,H), and no expression in the rostral tip (arrowheads in Figures 5B,C,G,H). Phosphorylated YAP1 was cytoplasmic in cells both in the dorsal and ventral regions at both stages but completely absent from the rostral tip (arrowheads in Figures 5L,M). Expression was stronger in the ventral epithelium than the dorsal (yellow arrowheads in Figures 5L,M), the reverse of the observed pattern for total YAP1 and TAZ. At 15.5dpc and 17.5dpc YAP1 and TAZ were nucleo-cytoplasmic in the marginal zone epithelium surrounding the cleft on both sides, in the intermediate and anterior lobes (arrowheads in Figures 5D,E,I,J). TAZ protein was detected in a broader domain surrounding the epithelium than YAP1 (Figure 5E). They were both present in cells scattered around the anterior pituitary and in structures resembling blood vessels (arrows in Figures 5D,E,J). Inactive phosho-YAP1 was present in the cytoplasm of cells in the marginal zone epithelium (arrowheads in Figures 5N,O) and in many cells throughout the anterior and intermediate lobes at both stages. The posterior lobe stained with all three antibodies, nucleo-cytoplasmic for TAZ and cytoplasmic for YAP1 and phospho-YAP1.


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)

Expression of YAP1 and TAZ proteins during embryonic development. Immunofluorescence using specific antibodies against total TAZ protein, total YAP1 protein and phosphorylated YAP1 (S127) in red. Nuclei are counterstained with DAPI (blue). (A–J) Localization of effectors TAZ (A–E) and YAP1 (F–J) using antibodies recognizing total protein. Note the nuclear localization in periluminal cells of Rathke's pouch (yellow arrowheads in B,C,G,H, white arrowheads in D,E,I,J). No YAP1/TAZ proteins are detected in the rostral tip (arrowheads in B,C,G,H) and there is a reduction in expression in ventral regions (asterisks in B,C,G,H). Note expression in structures resembling capillaries from 15.5dpc (arrows in D,E,J). (K–O) Immunofluorescence to detect the phosphorylated form of YAP1 at S127. Protein detection indicates Hippo kinase cascade activity at all stages, primarily in periluminal RP epithelium (white arrowheads in K,N,O). Note the ventral bias of protein localization at 12.5dpc and 13.5dpc (yellow arrows in L,M) and complete absence of protein from the rostral tip (white arrowheads in L,M). Boxed inserts show higher magnifications of the epithelium. Examples of cytoplasmic localization are noted by white arrows and examples of nuclear localization by yellow arrows. Abbreviations: rp, Rathke's pouch; vd, ventral diencephalon; m, mesenchyme; oe, oral ectoderm; inf, infundibulum; sph, sphenoid; rt, rostral tip; pl, posterior lobe; al, anterior lobe; il, intermediate lobe. Sagittal sections between 10.5dpc and 13.5dpc and frontal between 15.5dpc and 17.5dpc. Axes in (A) applicable to (A–C,F–H,K–M: d, dorsal; v, ventral; r, rostral; c, caudal). Axes in (D) applicable to (D,E,I,J,N,O: d, dorsal; v, ventral; ri, right; le, left). Scale bars 100 μm and 20 μm in boxed inserts.
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Figure 5: Expression of YAP1 and TAZ proteins during embryonic development. Immunofluorescence using specific antibodies against total TAZ protein, total YAP1 protein and phosphorylated YAP1 (S127) in red. Nuclei are counterstained with DAPI (blue). (A–J) Localization of effectors TAZ (A–E) and YAP1 (F–J) using antibodies recognizing total protein. Note the nuclear localization in periluminal cells of Rathke's pouch (yellow arrowheads in B,C,G,H, white arrowheads in D,E,I,J). No YAP1/TAZ proteins are detected in the rostral tip (arrowheads in B,C,G,H) and there is a reduction in expression in ventral regions (asterisks in B,C,G,H). Note expression in structures resembling capillaries from 15.5dpc (arrows in D,E,J). (K–O) Immunofluorescence to detect the phosphorylated form of YAP1 at S127. Protein detection indicates Hippo kinase cascade activity at all stages, primarily in periluminal RP epithelium (white arrowheads in K,N,O). Note the ventral bias of protein localization at 12.5dpc and 13.5dpc (yellow arrows in L,M) and complete absence of protein from the rostral tip (white arrowheads in L,M). Boxed inserts show higher magnifications of the epithelium. Examples of cytoplasmic localization are noted by white arrows and examples of nuclear localization by yellow arrows. Abbreviations: rp, Rathke's pouch; vd, ventral diencephalon; m, mesenchyme; oe, oral ectoderm; inf, infundibulum; sph, sphenoid; rt, rostral tip; pl, posterior lobe; al, anterior lobe; il, intermediate lobe. Sagittal sections between 10.5dpc and 13.5dpc and frontal between 15.5dpc and 17.5dpc. Axes in (A) applicable to (A–C,F–H,K–M: d, dorsal; v, ventral; r, rostral; c, caudal). Axes in (D) applicable to (D,E,I,J,N,O: d, dorsal; v, ventral; ri, right; le, left). Scale bars 100 μm and 20 μm in boxed inserts.
Mentions: In order to infer activity of the Hippo kinase cascade, we investigated the localization of effector proteins TAZ, total YAP1, as well as the inactive phosphorylated form of YAP1 (S127). TAZ and YAP1 had similar localization at 10.5dpc; they appeared nucleo-cytoplasmic with a bias for the apical cytoplasm of RP epithelium (Figures 5A,F, arrowheads). Inactive YAP1, marked by pYAP1 was strongly cytoplasmic and also displayed an apical bias (Figure 5K, arrowheads). All three antibodies marked cells in the mesenchyme and neural tissue. At 12.5dpc and 13.5dpc YAP1 and TAZ both localized mostly in nuclei of cells in RP epithelium, with stronger expression in the dorsal RP epithelium at 12.5dpc, which persisted at 13.5dpc for YAP1 (yellow arrowheads in Figures 5B,G,H). In the ventral portion of the epithelium there was nuclear localization in a thin cell layer surrounding the cleft. Little expression was observed in more ventral regions (asterisk in Figures 5B,C,G,H), and no expression in the rostral tip (arrowheads in Figures 5B,C,G,H). Phosphorylated YAP1 was cytoplasmic in cells both in the dorsal and ventral regions at both stages but completely absent from the rostral tip (arrowheads in Figures 5L,M). Expression was stronger in the ventral epithelium than the dorsal (yellow arrowheads in Figures 5L,M), the reverse of the observed pattern for total YAP1 and TAZ. At 15.5dpc and 17.5dpc YAP1 and TAZ were nucleo-cytoplasmic in the marginal zone epithelium surrounding the cleft on both sides, in the intermediate and anterior lobes (arrowheads in Figures 5D,E,I,J). TAZ protein was detected in a broader domain surrounding the epithelium than YAP1 (Figure 5E). They were both present in cells scattered around the anterior pituitary and in structures resembling blood vessels (arrows in Figures 5D,E,J). Inactive phosho-YAP1 was present in the cytoplasm of cells in the marginal zone epithelium (arrowheads in Figures 5N,O) and in many cells throughout the anterior and intermediate lobes at both stages. The posterior lobe stained with all three antibodies, nucleo-cytoplasmic for TAZ and cytoplasmic for YAP1 and phospho-YAP1.

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