- Email: [email protected]
Cellular reorganization during embryonic midfacial narrowing
Abstracts
PS5.23 Effects of vascular formation during alveolar bone process morphogenesis in mice Seo-Young Ana, Ye-Ji Leea, Sanjiv Neupanea, Jong-Hwa Junb, Ji-Youn Kimc, Youngkyun Leea, Karp-Shik Choia, Chang-Hyeon Ana, Jo-Young Suha, Hong-in Shina, Wern-Joo Sohna, Jae-Young Kima a
Kyungpook National University, Daegu, South Korea Keimyung University, Daegu, South Korea c Gachon University, Incheon, South Korea b
The alveolar bone process is the thickened ridge of bone that bears the teeth and known to be dynamic structures in function with surrounding tissues. However, detailed morphological changes of alveolar bone process and its underlying molecular mechanisms in morphogenesis are not elucidated properly yet. In this study, we examined the detailed morphological changes of alveolar bone process using HE and MTC staining. In addition, we evaluated the precise localization patterns of various signaling molecules with blood vessel formation factors including CD31, α-SMA, VEGF, AM143, PERIOSTIN and TGF-β. These morphological and immunohistochemical results suggested that there are intimate relationships between alveolar bone process and blood vessel formation. To examine the developmental function of a blood vessel forming factor, VEGF, in alveolar bone process formation, we designed and employed the microinjection model system using anti-VEGF, bevacizumab, at PN5 for 5 days. When compared with the control, anti-VEGF treated specimens showed the disturbance in integrations of bony tissues to form the alveolar bone process structures, which should contain the periodontal ligaments. Based on these results, we conclude that the specific morphogenesis of alveolar bone process would be formed by interactions with the blood vessels formation.
doi:10.1016/j.mod.2017.04.426
PS5.24 Analysis of CRISPR genome-edited founder mice shows relationship between somatic mosaicism of Pax6 mutation and variable developmental eye abnormalities Akihiro Yasuea, Hitomi Konob, Munenori Habutab, Tetsuya Bandob, Keita Satob, Junji Inoueb, Seiichi Oyadomaric, Sumihare Nojid, Eiji Tanakaa, Hideyo Ohuchib a
Institute of Biomedical Sciences, Tokushima University Graduate School, Japan b Okayama University Graduate School of Medicine, Okayama, Japan c Institute for Advanced Enzyme Research, Tokushima University, Japan d Tokushima University, Japan The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a rapid genetargeting technology that does not require embryonic stem cells. To demonstrate dosage effects of the Pax6 gene on eye formation, we generated Pax6-deficient mice with the CRISPR/Cas system. Eyes of founder embryos at embryonic day (E) 16.5 were examined and categorized according to macroscopic phenotype as class 1 (small eye with distinct pigmentation), class 2 (pigmentation without eye globes), or class 3 (no pigmentation and no eyes). Histologically, class 1 eyes were abnormally small in size with lens still attached to the cornea at E16.5. Class 2 eyes had no lens and distorted convoluted retinas. Class 3 eyes had only rudimentary optic vesiclelike tissues or histological anophthalmia. Genotyping of neck tissue
S151
cells from the founder embryos revealed somatic mosaicism and allelic complexity for Pax6. Relationships between eye phenotype and genotype were developed. The present results demonstrated that development of the lens from the surface ectoderm requires a higher gene dose of Pax6 than development of the retina from the optic vesicle. We further anticipate that mice with somatic mosaicism in a targeted gene generated by CRISPR/Cas-mediated genome editing will give some insights for understanding the complexity in human congenital diseases that occur in mosaic form.
doi:10.1016/j.mod.2017.04.427
PS5.25 Developmental shift of Rx expression from non-photoreceptor to photoreceptor lineage as a mechanism for photoreceptor cell specification in Ciona intestinalis Kouhei Oonuma, Takehiro G. Kusakabe Konan University, Kobe, Japan The brain of the ascidian Ciona intestinalis larva contains two distinct photoreceptor organs, a conventional pigmented ocellus and a nonpigmented ocellus. The ciliary photoreceptor cells of these ocelli resemble visual cells of the vertebrate retina. In both ascidians and vertebrates, a retinal homeobox Rx gene is required for development of the photoreceptor cells. Precise identification of the lineage of the photoreceptor cells in the ascidian embryo is key to understanding developmental mechanisms of these cells as well as evolutionary relationships between photoreceptor organs of ascidians and vertebrates. Previous studies inferred that the right a9.33 and a9.37 cells, descendants of a blastomere of the anterior animal hemisphere (a-lineage), give rise to the photoreceptor cells of the pigmented ocellus. The cell lineage is, however, not conclusive because it was the results of speculation based on the observation of different individuals fixed at successive stages without the use of cell differentiation markers. Here, we traced the developmental fates of neural plate cells from the late gastrula through larval stages by labeling particular cells of intact (non-dechorionated) embryos at single-cell resolution using the photo-convertible fluorescent protein Kaede along with photoreceptor-specific markers. Our results conclusively indicate that the photoreceptor cells of both pigmented and nonpigmented ocelli develop from the right anterior vegetal hemisphere (A-lineage), but not from a-lineage. Because Rx has been thought to be expressed in a-lineage cells, which turn out to be "non-photoreceptor" lineage, we reexamined Rx expression during photoreceptor development. Our results clearly indicate that the expression of Rx shifts from a-lineage to A-lineage cells during development of the pigmented ocellus. We also report a transcriptional regulatory mechanism underlying the developmental shift of Rx during photoreceptor development.
doi:10.1016/j.mod.2017.04.428
PS5.26 Cellular reorganization during embryonic midfacial narrowing Joy Richman, Johnathan Woo, Adrian Danescu Life Sciences Institute, University of British Columbia, Vancouver, Canada The most rapid and significant morphological changes affecting embryonic faces are the relocation of the nasal pits from the side of
S152
Abstracts
the head to the midline, fusion of the primary and secondary palate (mammals). Narrowing of the midline is a universal feature of amniotes but the mechanisms are unknown. Our objectives were 1) to use the chicken embryo to tease out the extrinsic versus intrinsic factors and 2) to investigate the role of planar cell polarity (PCP) in mesenchymal cell behaviors. In-vivo results: Heads were scanned with an Optical Projection Scanner, the frontonasal mass was segmented out, landmarks applied and 3D geometric morphometrics carried out using MorphoJ. We found that the frontonasal mass (equivalent to the medial nasal prominences) narrows medio-laterally (ML) while increasing dorsoventrally (DV) and craniocaudally (CrCa). The most rapid changes occurred during a 12 hour period between stage 28 and 29. In vitro results: Faces were dissected and placed into organ culture with or without the eyes and forebrain. Another set was treated with a ROCK inhibitor. Significant narrowing occurred over 48h, independent of the eyes/brain. narrowing was blocked by the ROCKi. Time-lapse tracking of mesenchymal nuclei over 6h showed intercalation of cells in the CrCa axis. The ROCKi prevented cells from intercalating which correlates with the flattening observed in organ cultures. Conclusions. The data demonstrate that intrinsic mechanisms lead to convergent extension (CE) of the frontonasal mass (narrowing in one axis with growth occurring in the perpendicular axes). CE is linked to intercalation of cells in the CrCa axis. The molecular pathway is likely to involve planar cell polarity signaling via small GTPases. Live cell imaging techniques permit the measurement of cell behaviours in growing facial mesenchyme. Supported by CIHR doctoral award to AD (RN 0000183232) and CIHR operating grant to JMR (MOP-123536). doi:10.1016/j.mod.2017.04.429
PS5.27 Wnt signal modulates proliferation of spinal cord ependymal cells by promoting morphological change of the source cells Takuma Shinozukaa,b, Ritsuko Takadab, Shinji Takadaa,b a
The Graduate University for Advanced Studies, Okazaki, Japan Okazaki Institute for Integrative Bioscience, Okazaki, Japan
shape change and direction of roof plate rearrangement. Thus, Wnt signal plays a role in proper elongation of the roof plate by regulating change of the morphology of these cells and specific localization of Wnt proteins appears to be correlated with this change.
doi:10.1016/j.mod.2017.04.430
PS5.28 The role of transcription factor Foxi3 in early hair follicle morphogenesis Vera Shirokova, Ibrahim Sultan, Vinod Kumar, Marja Mikkola University of Helsinki, Institute of Biotechnology, Finland Initiation of hair follicle development is marked by the formation of an epithelial placode and condensation of underlying mesenchymal cells. In the next stage, placode cells invaginate to form a hair bud. Previous studies have shown that placode cells are largely nonproliferative, whereas the bud stage follicle is characterized by a small population of Wnt-high, Sonic hedgehog-expressing nonproliferating cells located at the base of the follicle. These cells are essential for the specification of future hair follicle stem cells (HFSCs) residing in the upper portion of the hair bud. Transcription factor Foxi3 is necessary for hair follicle formation since heterozygous mutation of Foxi3 causes hairless phenotype in dogs. We have previously shown that Foxi3 is expressed in hair placodes and at the base of the hair bud, and that Foxi3 deficiency in mice abrogates hair follicle morphogenesis and compromises HFSC specification. However, the exact function of Foxi3 in hair placode specification and morphogenesis is unknown. Here, we use confocal 3D microscopy to address these questions. We show that hair follicle development is impaired already at the placode stage in Foxi3-deficient mice The study will further focus on the role of Foxi3 in segregation of the Wnt-high/Shh+ and stem cell populations in the hair placode.
doi:10.1016/j.mod.2017.04.431
b
The roof plate, which secretes BMPs and Wnts, acts as a signaling center to specify fates of dorsal interneurons. On the other hand, it remains unclear whether these signals play a role after this event. Here, we examined role of Wnt signal in the morphological change and function of roof plate cells in late embryonic stages. After E12.5, the lumen of the spinal cord reduces along the dorso-ventral axis. According to this reduction, each roof plate cell elongated along the dorso-ventral axis, probably leading to the formation of the median septum. Immunohistological analysis with normal and EGFP-Wnt3a knock-in embryos revealed that Wnt proteins are still expressed in elongated roof plate cells. Analysis of roof plate specific cKO embryos for Wls, which is required for Wnt secretion, revealed that Wnt signal is required for proper elongation of roof plate and septum formation. To gain more insight into the molecular and cellular mechanisms of roof plate elongation by Wnt signal, we examined earlier processes of the roof plate elongation. During the elongation, each roof plate cell rearranged along the dorso-ventral axis in normal embryos, but it was disturbed in Wls cKO embryos. Prior to this rearrangement, the morphology of roof plate cells change to wedge-shaped form converging at the position from which roof plate cells are elongated in normal embryos, but not in Wls cKO embryos. Interestingly, Wnt proteins are specifically accumulated at the converting tip of roof plate cells coincident with the wedge-shaped change, suggesting that this accumulation of Wnt proteins is correlated with the cell
PS5.29 An integrated cell, tissue and whole organ profile of kidney morphogenesis Alexander Combesb, James Lefevrec, Kieran Shorta, Adler Juc, Kylie Georgasc, Timothy Lambertonc, Oliver Cairncrossc, Bree Rumballec, Andrew McMahond, Melissa Littleb, Nicholas Hamiltonc, Ian Smytha a
Monash University, Biomedicine Discovery Institute, Melbourne, Australia Murdoch Childrens Research Institute, Melbourne, Australia c Institute for Molecular Bioscience, University of QLD, Brisbane, Australia d Broad-CIRM Center for Regenerative Medicine and Stem Cell Research, Los Angeles, United States b
While cell, tissue and even organism level analyses of morphogenesis are feasible in invertebrates, the size, opacity and complexity of mammalian organs has impeded systematic analyses of developmental processes critical to organ function. Here, we integrate optical projection tomography, single-cell resolution confocal microscopy and quantitative image analysis to comprehensively document mouse kidney organogenesis across time. This reveals a previously unappreciated structurally stereotypic organ architecture undergoing a temporally non-uniform process of development with respect to rates of cellular proliferation, dominant morphogenetic processes and spatial relationships between key cellular compartments. The
PS5.23 Effects of vascular formation during alveolar bone process morphogenesis in mice Seo-Young Ana, Ye-Ji Leea, Sanjiv Neupanea, Jong-Hwa Junb, Ji-Youn Kimc, Youngkyun Leea, Karp-Shik Choia, Chang-Hyeon Ana, Jo-Young Suha, Hong-in Shina, Wern-Joo Sohna, Jae-Young Kima a
Kyungpook National University, Daegu, South Korea Keimyung University, Daegu, South Korea c Gachon University, Incheon, South Korea b
The alveolar bone process is the thickened ridge of bone that bears the teeth and known to be dynamic structures in function with surrounding tissues. However, detailed morphological changes of alveolar bone process and its underlying molecular mechanisms in morphogenesis are not elucidated properly yet. In this study, we examined the detailed morphological changes of alveolar bone process using HE and MTC staining. In addition, we evaluated the precise localization patterns of various signaling molecules with blood vessel formation factors including CD31, α-SMA, VEGF, AM143, PERIOSTIN and TGF-β. These morphological and immunohistochemical results suggested that there are intimate relationships between alveolar bone process and blood vessel formation. To examine the developmental function of a blood vessel forming factor, VEGF, in alveolar bone process formation, we designed and employed the microinjection model system using anti-VEGF, bevacizumab, at PN5 for 5 days. When compared with the control, anti-VEGF treated specimens showed the disturbance in integrations of bony tissues to form the alveolar bone process structures, which should contain the periodontal ligaments. Based on these results, we conclude that the specific morphogenesis of alveolar bone process would be formed by interactions with the blood vessels formation.
doi:10.1016/j.mod.2017.04.426
PS5.24 Analysis of CRISPR genome-edited founder mice shows relationship between somatic mosaicism of Pax6 mutation and variable developmental eye abnormalities Akihiro Yasuea, Hitomi Konob, Munenori Habutab, Tetsuya Bandob, Keita Satob, Junji Inoueb, Seiichi Oyadomaric, Sumihare Nojid, Eiji Tanakaa, Hideyo Ohuchib a
Institute of Biomedical Sciences, Tokushima University Graduate School, Japan b Okayama University Graduate School of Medicine, Okayama, Japan c Institute for Advanced Enzyme Research, Tokushima University, Japan d Tokushima University, Japan The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a rapid genetargeting technology that does not require embryonic stem cells. To demonstrate dosage effects of the Pax6 gene on eye formation, we generated Pax6-deficient mice with the CRISPR/Cas system. Eyes of founder embryos at embryonic day (E) 16.5 were examined and categorized according to macroscopic phenotype as class 1 (small eye with distinct pigmentation), class 2 (pigmentation without eye globes), or class 3 (no pigmentation and no eyes). Histologically, class 1 eyes were abnormally small in size with lens still attached to the cornea at E16.5. Class 2 eyes had no lens and distorted convoluted retinas. Class 3 eyes had only rudimentary optic vesiclelike tissues or histological anophthalmia. Genotyping of neck tissue
S151
cells from the founder embryos revealed somatic mosaicism and allelic complexity for Pax6. Relationships between eye phenotype and genotype were developed. The present results demonstrated that development of the lens from the surface ectoderm requires a higher gene dose of Pax6 than development of the retina from the optic vesicle. We further anticipate that mice with somatic mosaicism in a targeted gene generated by CRISPR/Cas-mediated genome editing will give some insights for understanding the complexity in human congenital diseases that occur in mosaic form.
doi:10.1016/j.mod.2017.04.427
PS5.25 Developmental shift of Rx expression from non-photoreceptor to photoreceptor lineage as a mechanism for photoreceptor cell specification in Ciona intestinalis Kouhei Oonuma, Takehiro G. Kusakabe Konan University, Kobe, Japan The brain of the ascidian Ciona intestinalis larva contains two distinct photoreceptor organs, a conventional pigmented ocellus and a nonpigmented ocellus. The ciliary photoreceptor cells of these ocelli resemble visual cells of the vertebrate retina. In both ascidians and vertebrates, a retinal homeobox Rx gene is required for development of the photoreceptor cells. Precise identification of the lineage of the photoreceptor cells in the ascidian embryo is key to understanding developmental mechanisms of these cells as well as evolutionary relationships between photoreceptor organs of ascidians and vertebrates. Previous studies inferred that the right a9.33 and a9.37 cells, descendants of a blastomere of the anterior animal hemisphere (a-lineage), give rise to the photoreceptor cells of the pigmented ocellus. The cell lineage is, however, not conclusive because it was the results of speculation based on the observation of different individuals fixed at successive stages without the use of cell differentiation markers. Here, we traced the developmental fates of neural plate cells from the late gastrula through larval stages by labeling particular cells of intact (non-dechorionated) embryos at single-cell resolution using the photo-convertible fluorescent protein Kaede along with photoreceptor-specific markers. Our results conclusively indicate that the photoreceptor cells of both pigmented and nonpigmented ocelli develop from the right anterior vegetal hemisphere (A-lineage), but not from a-lineage. Because Rx has been thought to be expressed in a-lineage cells, which turn out to be "non-photoreceptor" lineage, we reexamined Rx expression during photoreceptor development. Our results clearly indicate that the expression of Rx shifts from a-lineage to A-lineage cells during development of the pigmented ocellus. We also report a transcriptional regulatory mechanism underlying the developmental shift of Rx during photoreceptor development.
doi:10.1016/j.mod.2017.04.428
PS5.26 Cellular reorganization during embryonic midfacial narrowing Joy Richman, Johnathan Woo, Adrian Danescu Life Sciences Institute, University of British Columbia, Vancouver, Canada The most rapid and significant morphological changes affecting embryonic faces are the relocation of the nasal pits from the side of
S152
Abstracts
the head to the midline, fusion of the primary and secondary palate (mammals). Narrowing of the midline is a universal feature of amniotes but the mechanisms are unknown. Our objectives were 1) to use the chicken embryo to tease out the extrinsic versus intrinsic factors and 2) to investigate the role of planar cell polarity (PCP) in mesenchymal cell behaviors. In-vivo results: Heads were scanned with an Optical Projection Scanner, the frontonasal mass was segmented out, landmarks applied and 3D geometric morphometrics carried out using MorphoJ. We found that the frontonasal mass (equivalent to the medial nasal prominences) narrows medio-laterally (ML) while increasing dorsoventrally (DV) and craniocaudally (CrCa). The most rapid changes occurred during a 12 hour period between stage 28 and 29. In vitro results: Faces were dissected and placed into organ culture with or without the eyes and forebrain. Another set was treated with a ROCK inhibitor. Significant narrowing occurred over 48h, independent of the eyes/brain. narrowing was blocked by the ROCKi. Time-lapse tracking of mesenchymal nuclei over 6h showed intercalation of cells in the CrCa axis. The ROCKi prevented cells from intercalating which correlates with the flattening observed in organ cultures. Conclusions. The data demonstrate that intrinsic mechanisms lead to convergent extension (CE) of the frontonasal mass (narrowing in one axis with growth occurring in the perpendicular axes). CE is linked to intercalation of cells in the CrCa axis. The molecular pathway is likely to involve planar cell polarity signaling via small GTPases. Live cell imaging techniques permit the measurement of cell behaviours in growing facial mesenchyme. Supported by CIHR doctoral award to AD (RN 0000183232) and CIHR operating grant to JMR (MOP-123536). doi:10.1016/j.mod.2017.04.429
PS5.27 Wnt signal modulates proliferation of spinal cord ependymal cells by promoting morphological change of the source cells Takuma Shinozukaa,b, Ritsuko Takadab, Shinji Takadaa,b a
The Graduate University for Advanced Studies, Okazaki, Japan Okazaki Institute for Integrative Bioscience, Okazaki, Japan
shape change and direction of roof plate rearrangement. Thus, Wnt signal plays a role in proper elongation of the roof plate by regulating change of the morphology of these cells and specific localization of Wnt proteins appears to be correlated with this change.
doi:10.1016/j.mod.2017.04.430
PS5.28 The role of transcription factor Foxi3 in early hair follicle morphogenesis Vera Shirokova, Ibrahim Sultan, Vinod Kumar, Marja Mikkola University of Helsinki, Institute of Biotechnology, Finland Initiation of hair follicle development is marked by the formation of an epithelial placode and condensation of underlying mesenchymal cells. In the next stage, placode cells invaginate to form a hair bud. Previous studies have shown that placode cells are largely nonproliferative, whereas the bud stage follicle is characterized by a small population of Wnt-high, Sonic hedgehog-expressing nonproliferating cells located at the base of the follicle. These cells are essential for the specification of future hair follicle stem cells (HFSCs) residing in the upper portion of the hair bud. Transcription factor Foxi3 is necessary for hair follicle formation since heterozygous mutation of Foxi3 causes hairless phenotype in dogs. We have previously shown that Foxi3 is expressed in hair placodes and at the base of the hair bud, and that Foxi3 deficiency in mice abrogates hair follicle morphogenesis and compromises HFSC specification. However, the exact function of Foxi3 in hair placode specification and morphogenesis is unknown. Here, we use confocal 3D microscopy to address these questions. We show that hair follicle development is impaired already at the placode stage in Foxi3-deficient mice The study will further focus on the role of Foxi3 in segregation of the Wnt-high/Shh+ and stem cell populations in the hair placode.
doi:10.1016/j.mod.2017.04.431
b
The roof plate, which secretes BMPs and Wnts, acts as a signaling center to specify fates of dorsal interneurons. On the other hand, it remains unclear whether these signals play a role after this event. Here, we examined role of Wnt signal in the morphological change and function of roof plate cells in late embryonic stages. After E12.5, the lumen of the spinal cord reduces along the dorso-ventral axis. According to this reduction, each roof plate cell elongated along the dorso-ventral axis, probably leading to the formation of the median septum. Immunohistological analysis with normal and EGFP-Wnt3a knock-in embryos revealed that Wnt proteins are still expressed in elongated roof plate cells. Analysis of roof plate specific cKO embryos for Wls, which is required for Wnt secretion, revealed that Wnt signal is required for proper elongation of roof plate and septum formation. To gain more insight into the molecular and cellular mechanisms of roof plate elongation by Wnt signal, we examined earlier processes of the roof plate elongation. During the elongation, each roof plate cell rearranged along the dorso-ventral axis in normal embryos, but it was disturbed in Wls cKO embryos. Prior to this rearrangement, the morphology of roof plate cells change to wedge-shaped form converging at the position from which roof plate cells are elongated in normal embryos, but not in Wls cKO embryos. Interestingly, Wnt proteins are specifically accumulated at the converting tip of roof plate cells coincident with the wedge-shaped change, suggesting that this accumulation of Wnt proteins is correlated with the cell
PS5.29 An integrated cell, tissue and whole organ profile of kidney morphogenesis Alexander Combesb, James Lefevrec, Kieran Shorta, Adler Juc, Kylie Georgasc, Timothy Lambertonc, Oliver Cairncrossc, Bree Rumballec, Andrew McMahond, Melissa Littleb, Nicholas Hamiltonc, Ian Smytha a
Monash University, Biomedicine Discovery Institute, Melbourne, Australia Murdoch Childrens Research Institute, Melbourne, Australia c Institute for Molecular Bioscience, University of QLD, Brisbane, Australia d Broad-CIRM Center for Regenerative Medicine and Stem Cell Research, Los Angeles, United States b
While cell, tissue and even organism level analyses of morphogenesis are feasible in invertebrates, the size, opacity and complexity of mammalian organs has impeded systematic analyses of developmental processes critical to organ function. Here, we integrate optical projection tomography, single-cell resolution confocal microscopy and quantitative image analysis to comprehensively document mouse kidney organogenesis across time. This reveals a previously unappreciated structurally stereotypic organ architecture undergoing a temporally non-uniform process of development with respect to rates of cellular proliferation, dominant morphogenetic processes and spatial relationships between key cellular compartments. The