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Elucidating the Molecular Regulation of PTHrP+ and Fgfr3+ Chondrocytes in Diverse Endochondral Tissues /

Record Type:	Electronic resources : Monograph/item
Title/Author:	Elucidating the Molecular Regulation of PTHrP+ and Fgfr3+ Chondrocytes in Diverse Endochondral Tissues // Shawn A Hallett.
Author:	Hallett, Shawn A.,
Description:	1 electronic resource (293 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 86-11, Section: B.
Contained By:	Dissertations Abstracts International86-11B.
Subject:	Developmental biology. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=32092681
ISBN:	9798314875674

Elucidating the Molecular Regulation of PTHrP+ and Fgfr3+ Chondrocytes in Diverse Endochondral Tissues /
Hallett, Shawn A.,

Elucidating the Molecular Regulation of PTHrP+ and Fgfr3+ Chondrocytes in Diverse Endochondral Tissues /Shawn A Hallett. - 1 electronic resource (293 pages)

Source: Dissertations Abstracts International, Volume: 86-11, Section: B.

Diseases affecting development and growth of the skeleton pose a massive healthcare burden and require invasive surgical treatment to repair. Of the many cell types involved in endochondral ossification, skeletal stem and progenitor cells (SPPCs) have profound roles in facilitating the development, maintenance and regeneration of skeletal tissues. SSPCs are mainly comprised of precursors of osteoblasts, adipocytes and chondrocytes, which are housed within specific niches throughout the embryonic and postnatal skeletons. Chondrocytes not only form a rudimentary anlage, which will eventually give rise to organized bone, but they also persist throughout adulthood at the articular surface to provide mechanical resistance during locomotion in bipedal mammals. Yet, the molecular regulation of this specialized cell type within the appendicular and craniofacial skeletons remains incompletely understood.The endochondral skeleton contains several unique tissues that are controlled by shared as well as site-specific mechanisms. For example, the long bone growth plate, a cartilaginous structure housed within the epiphysis of the femur and tibia among other endochondral tissues, is comprised of layers of chondrocytes, which are eventually replaced by bone during skeletal maturation in humans. The organization of the growth plate is unidirectional in nature, facilitating growth accordingly. Conversely, housed within the craniofacial skeleton is the cranial base, an endochondral-derived tissue that functions to promote anteroposterior growth of the craniofacial complex. Importantly, the various bones that comprise the cranial base are separated by cartilaginous growth center synchondroses. Like the long bone growth plate, the synchondroses are comprised of distinct layers of chondrocytes. However, synchondrosis chondrocytes are organized bilaterally in a mirror-imaged manner, thus facilitating bidirectional growth of the cranial base. The developmental regulation of the synchondroses has only recently been subject to scientific investigation. The overall goal of this dissertation is to better define the molecular regulation of epiphyseal growth plate and cranial base synchondrosis chondrocytes. Ultimately, these findings aim to provide novel therapeutic targets to regenerate congenitally malformed or damaged endochondral tissues:Aim 1: It has been shown that chondrocytes in the resting zone of the postnatal growth plate are characterized by slow cell cycle progression and encompass a population of parathyroid hormone-related protein (PTHrP) expressing skeletal stem cells that contribute to the formation of columnar chondrocytes. Yet, how these chondrocytes are maintained in the resting zone remains undefined. We undertook a genetic pulse-chase approach to isolate slow cycling, label-retaining chondrocytes (LRCs) using a chondrocyte-specific doxycycline-controllable Tet-Off system regulating expression of histone 2B-linked GFP. Comparative RNA-seq analysis identified significant enrichment of inhibitors and activators for Wnt signaling in LRCs and non-LRCs, respectively. Activation of Wnt/b-catenin signaling in PTHrP+ resting chondrocytes using Pthlh-creER and Apc-floxed alleles impaired their ability to form columnar chondrocytes. Therefore, slow-cycling chondrocytes are maintained in a Wnt-inhibitory environment within the resting zone, unraveling a novel mechanism regulating maintenance and differentiation of PTHrP+ skeletal stem cells of the postnatal growth plate.Aim 2: The cranial base contains a special type of growth plate termed the synchondrosis, which functions as the growth center of the skull. The synchondrosis is composed of bidirectional oppo-site-facing layers of resting, proliferating, and hypertrophic chondrocytes, and lacks a secondary ossification center (SOC). In long bones, the resting zone of the epiphyseal growth plate houses a population of PTHrP expressing chondrocytes that contribute to the formation of columnar chondrocytes. Prior to data presented herein, whether PTHrP+ chondrocytes in the synchondrosis possess similar functions remained unknown. Using Pthrp-mCherry knock-in mice, we found that PTHrP+ chondrocytes predominantly occupied a lateral wedge-shaped area of the synchondrosis, unlike those in the femoral growth plate that reside in the resting zone within the epiphysis. In vivo cell-lineage analyses using a tamoxifen-inducible Pthrp-creER line revealed that PTHrP+ chondrocytes failed to establish columnar chondrocytes in the synchondrosis. Therefore, PTHrP+ chondrocytes in the synchondrosis do not possess column-forming capabilities, unlike those in the resting zone of the long bone growth plate. These findings suggest the possible importance of the SOC within the long bone epiphysis in establishing the stem cell niche for PTHrP+ chondrocytes, the absence of which may be related to the lack of column-forming capabilities of PTHrP+ chondrocytes in the cranial base synchondrosis.Aim 3: In humans, RUNX2 haploinsufficiency causes cleidocranial dysplasia associated with deficient midfacial growth. However, it is not known how RUNX2 regulates chondrocytes in the cranial base synchondrosis. To address this, we inactivated Runx2 in postnatal synchondrosis chondrocytes using a tamoxifen-inducible Fibroblast Growth Factor Receptor 3 (Fgfr3-creER) (Fgfr3-Runx2cKO) mouse model. Fgfr3-Runx2cKO mice displayed skeletal dwarfism and reduced anteroposterior cranial base growth associated with premature synchondrosis ossification due to impaired chondrocyte proliferation, accelerated hypertrophy, apoptosis, and osteoclast-mediated cartilage resorption accompanied by localized angiogenesis. Lineage tracing reveals that Runx2-deficient Fgfr3+ cells fail to differentiate into osteoblasts. Notably, Runx2-deficient synchondrosis chondrocytes show an elevated level of FGFR3 and its downstream signaling components, pERK1/2 and SOX9, suggesting that RUNX2 downregulates FGFR3 in the synchondrosis. This study unveils a new role of Runx2 in cranial base chondrocytes, identifying a possible RUNX2-FGFR3-MAPK-SOX9 signaling axis that may control cranial base growth.

English

ISBN: 9798314875674Subjects--Topical Terms:

592588
Developmental biology.
Subjects--Index Terms:

Craniofacial biology

Elucidating the Molecular Regulation of PTHrP+ and Fgfr3+ Chondrocytes in Diverse Endochondral Tissues /
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520 $a Diseases affecting development and growth of the skeleton pose a massive healthcare burden and require invasive surgical treatment to repair. Of the many cell types involved in endochondral ossification, skeletal stem and progenitor cells (SPPCs) have profound roles in facilitating the development, maintenance and regeneration of skeletal tissues. SSPCs are mainly comprised of precursors of osteoblasts, adipocytes and chondrocytes, which are housed within specific niches throughout the embryonic and postnatal skeletons. Chondrocytes not only form a rudimentary anlage, which will eventually give rise to organized bone, but they also persist throughout adulthood at the articular surface to provide mechanical resistance during locomotion in bipedal mammals. Yet, the molecular regulation of this specialized cell type within the appendicular and craniofacial skeletons remains incompletely understood.The endochondral skeleton contains several unique tissues that are controlled by shared as well as site-specific mechanisms. For example, the long bone growth plate, a cartilaginous structure housed within the epiphysis of the femur and tibia among other endochondral tissues, is comprised of layers of chondrocytes, which are eventually replaced by bone during skeletal maturation in humans. The organization of the growth plate is unidirectional in nature, facilitating growth accordingly. Conversely, housed within the craniofacial skeleton is the cranial base, an endochondral-derived tissue that functions to promote anteroposterior growth of the craniofacial complex. Importantly, the various bones that comprise the cranial base are separated by cartilaginous growth center synchondroses. Like the long bone growth plate, the synchondroses are comprised of distinct layers of chondrocytes. However, synchondrosis chondrocytes are organized bilaterally in a mirror-imaged manner, thus facilitating bidirectional growth of the cranial base. The developmental regulation of the synchondroses has only recently been subject to scientific investigation. The overall goal of this dissertation is to better define the molecular regulation of epiphyseal growth plate and cranial base synchondrosis chondrocytes. Ultimately, these findings aim to provide novel therapeutic targets to regenerate congenitally malformed or damaged endochondral tissues:Aim 1: It has been shown that chondrocytes in the resting zone of the postnatal growth plate are characterized by slow cell cycle progression and encompass a population of parathyroid hormone-related protein (PTHrP) expressing skeletal stem cells that contribute to the formation of columnar chondrocytes. Yet, how these chondrocytes are maintained in the resting zone remains undefined. We undertook a genetic pulse-chase approach to isolate slow cycling, label-retaining chondrocytes (LRCs) using a chondrocyte-specific doxycycline-controllable Tet-Off system regulating expression of histone 2B-linked GFP. Comparative RNA-seq analysis identified significant enrichment of inhibitors and activators for Wnt signaling in LRCs and non-LRCs, respectively. Activation of Wnt/b-catenin signaling in PTHrP+ resting chondrocytes using Pthlh-creER and Apc-floxed alleles impaired their ability to form columnar chondrocytes. Therefore, slow-cycling chondrocytes are maintained in a Wnt-inhibitory environment within the resting zone, unraveling a novel mechanism regulating maintenance and differentiation of PTHrP+ skeletal stem cells of the postnatal growth plate.Aim 2: The cranial base contains a special type of growth plate termed the synchondrosis, which functions as the growth center of the skull. The synchondrosis is composed of bidirectional oppo-site-facing layers of resting, proliferating, and hypertrophic chondrocytes, and lacks a secondary ossification center (SOC). In long bones, the resting zone of the epiphyseal growth plate houses a population of PTHrP expressing chondrocytes that contribute to the formation of columnar chondrocytes. Prior to data presented herein, whether PTHrP+ chondrocytes in the synchondrosis possess similar functions remained unknown. Using Pthrp-mCherry knock-in mice, we found that PTHrP+ chondrocytes predominantly occupied a lateral wedge-shaped area of the synchondrosis, unlike those in the femoral growth plate that reside in the resting zone within the epiphysis. In vivo cell-lineage analyses using a tamoxifen-inducible Pthrp-creER line revealed that PTHrP+ chondrocytes failed to establish columnar chondrocytes in the synchondrosis. Therefore, PTHrP+ chondrocytes in the synchondrosis do not possess column-forming capabilities, unlike those in the resting zone of the long bone growth plate. These findings suggest the possible importance of the SOC within the long bone epiphysis in establishing the stem cell niche for PTHrP+ chondrocytes, the absence of which may be related to the lack of column-forming capabilities of PTHrP+ chondrocytes in the cranial base synchondrosis.Aim 3: In humans, RUNX2 haploinsufficiency causes cleidocranial dysplasia associated with deficient midfacial growth. However, it is not known how RUNX2 regulates chondrocytes in the cranial base synchondrosis. To address this, we inactivated Runx2 in postnatal synchondrosis chondrocytes using a tamoxifen-inducible Fibroblast Growth Factor Receptor 3 (Fgfr3-creER) (Fgfr3-Runx2cKO) mouse model. Fgfr3-Runx2cKO mice displayed skeletal dwarfism and reduced anteroposterior cranial base growth associated with premature synchondrosis ossification due to impaired chondrocyte proliferation, accelerated hypertrophy, apoptosis, and osteoclast-mediated cartilage resorption accompanied by localized angiogenesis. Lineage tracing reveals that Runx2-deficient Fgfr3+ cells fail to differentiate into osteoblasts. Notably, Runx2-deficient synchondrosis chondrocytes show an elevated level of FGFR3 and its downstream signaling components, pERK1/2 and SOX9, suggesting that RUNX2 downregulates FGFR3 in the synchondrosis. This study unveils a new role of Runx2 in cranial base chondrocytes, identifying a possible RUNX2-FGFR3-MAPK-SOX9 signaling axis that may control cranial base growth.
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