The thymus is the principal site of T cell development and therefore is of central importance within the immune system: congenital athymia results in profound immunodeficiency, while perturbed thymic function can lead to autoimmunity.... more
The thymus is the principal site of T cell development and therefore is of central importance within the immune system: congenital athymia results in profound immunodeficiency, while perturbed thymic function can lead to autoimmunity. Although highly active in early life, the thymus undergoes premature involution, such that de novo T cell development diminishes significantly with age. This has implications for immune function in the aging population, and in clinical procedures such as bone marrow and solid organ transplantation, where thymic function is required for T cell reconstitution and/or tolerance induction. Interest therefore exists in enhancing immune reconstitution through regenerative or cell therapies for boosting thymus activity in vivo, or providing customized in vitro generated T cell repertoires for adoptive transfer. The success of such strategies is likely to depend on a detailed knowledge of the mechanisms regulating thymus development and homeostasis. Here, we review current understanding of cellular and molecular regulation of thymus organogenesis, focusing on the epithelial component of the thymic stroma which provides many of the specialist functions required to mediate T cell differentiation and T cell repertoire selection.
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The thymus is required for generation of a self-tolerant, self-restricted T-cell repertoire. The capacity to manipulate or replace thymus function therapeutically would be beneficial in a variety of clinical settings, including for... more
The thymus is required for generation of a self-tolerant, self-restricted T-cell repertoire. The capacity to manipulate or replace thymus function therapeutically would be beneficial in a variety of clinical settings, including for improving recovery following bone marrow transplantation, restoring immune system function in the elderly and promoting tolerance to transplanted organs or cells. An attractive strategy would be transplantation of thymus organoids generated from cells produced in vitro, for instance from pluripotent stem cells. Here, we review recent progress toward this goal, focusing on advances in directing differentiation of pluripotent stem cells to thymic epithelial cells, a key cell type of the thymic stroma, and related direct reprogramming strategies.
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Abstract 835 The establishment of the thymic microenvironment early in life is crucial for the production functional T cells. Conversely, thymic involution results in a decreased T cell output. Thymic involution has important health... more
Abstract 835 The establishment of the thymic microenvironment early in life is crucial for the production functional T cells. Conversely, thymic involution results in a decreased T cell output. Thymic involution has important health implications especially following bone marrow transplant. Our objective is to determine molecular and cellular mechanisms that will allow for regeneration of involuted thymic tissue, restore production of naïve T cells, and improve immune function while improving our understanding of immunobiology. In this pursuit, we have focused on the Retinoblastoma family of tumor suppressor proteins. The main function of the RB pathway is to restrict passage through the G1/S transition of the cell cycle. RB and its two family members, p107 and p130, mediate the action of a broad range of cellular signals to control the proliferation, survival, and differentiation status of a large number of mammalian cell types. We found that inactivation of the RB pathway in the thymus by early deletion of RB family genes prevents thymic involution, promotes expansion of functional thymic epithelial cells (TECs), and increases thymic T cell output. Moreover, we have identified a direct regulatory relationship between RB and the Foxn1 transcription factor Via E2F transcription factors, where RB/E2F complexes directly repress the Foxn1 promoter, thereby promoting involution. Thus, the RB family is a critical mediator of extra- and intra-cellular signals to regulate thymic epithelial cells and thymus function, and decreasing RB pathway function may promote regeneration of the involuted thymus and restoration of naïve T cell production in patients. Disclosures: No relevant conflicts of interest to declare.
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The thymus and parathyroids originate from bilateral 3rd pharyngeal pouch (pp) endoderm. At E11.5, thymus- and parathyroid-fated domains express Foxn1 and Gcm2 respectively, transcription factors that regulate differentiation but do not... more
The thymus and parathyroids originate from bilateral 3rd pharyngeal pouch (pp) endoderm. At E11.5, thymus- and parathyroid-fated domains express Foxn1 and Gcm2 respectively, transcription factors that regulate differentiation but do not specify organ fate. Tbx1, which encodes a T-box transcription factor required for segmentation of the pharyngeal endoderm is initially widely expressed in the 3rd pp, but by E10.5 is excluded from the ventral, thymus-fated domain. We recently reported that Tbx1 is a negative regulator of thymus development (Reeh et al. Development 141:2950, 2014). Ectopic expression of Tbx1 in the thymus-fated domain of the 3rd pp suppresses Foxn1 and inhibits thymic epithelial cell proliferation and differentiation. MicroRNAs (miRs) are important regulators of gene expression. We find that members of the miR-17-92 cluster are expressed in the ventral domain of the 3rd pp in wildtype embryos. Moreover, miR-17-92 downregulates Tbx1 in cardiac progenitor cells to promote their differentiation (Wang et al. Developmental Cell 19:903, 2010). Therefore, we proposed that miR-17-92 regulates Tbx1 expression in 3rd pp endoderm. In support of this hypothesis, we find that deletion of miR-17-92 enhances TBX1 and reduces FOXN1 in 3rd pp endoderm. The data support a model in which miR-17-92 plays an essential role in thymus development by regulating Tbx1 expression in the 3rd pp.
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SCI-50 The postnatal thymus is the primary source of T cells in vertebrates, and many if not all stages of thymocyte development require interactions with thymic epithelial cells (TECs). There is abundant and growing evidence that the... more
SCI-50 The postnatal thymus is the primary source of T cells in vertebrates, and many if not all stages of thymocyte development require interactions with thymic epithelial cells (TECs). There is abundant and growing evidence that the cellular and compartmental organization of the thymus is crucial for optimal organ function. Changes in the organization and stromal composition of the thymus as a result of genetic changes or during aging-associated involution can negatively impact both T cell production and function. The TEC-specific transcription factor Foxn1 is a critical regulator of these processes. I will present data from our Foxn1 allelic series showing that Foxn1 regulates TEC proliferation and differentiation at both fetal and adult stages, and that Foxn1-dependent TEC differentiation is required to assemble the multiple cell types that make up the complex structure of the functional thymus. Thus, Foxn1 is a central regulator throughout the life cycle of the thymus, and orch...
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Thymic epithelial cells (TEC) are essential for thymocyte differentiation and repertoire selection. Despite their indispensable role in generating functional T cells, the molecular mechanisms that orchestrate TEC development from... more
Thymic epithelial cells (TEC) are essential for thymocyte differentiation and repertoire selection. Despite their indispensable role in generating functional T cells, the molecular mechanisms that orchestrate TEC development from endodermal progenitors in the third pharyngeal pouch (3rd PP) are not fully understood. We recently reported that the T-box transcription factor TBX1 negatively regulates TEC development. Although initially expressed throughout the 3rd PP, Tbx1 becomes downregulated in thymus-fated progenitors and when ectopically expressed impairs TEC progenitor proliferation and differentiation. Here we show that ectopic Tbx1 expression in thymus fated endoderm increases expression of Polycomb repressive complex 2 (PRC2) target genes in TEC. PRC2 is an epigenetic modifier that represses gene expression by catalyzing trimethylation of lysine 27 on histone H3. The increased expression of PRC2 target genes suggests that ectopic Tbx1 interferes with PRC2 activity and implicat...
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Research Interests: Biology, Morphogenesis, Development, Medicine, Gene expression, and 15 moreThyroid, In Situ Hybridization, Biological Sciences, Population, Neural Crest, Thyroid gland, Mice, Animals, Phenotype, Fluorescent Antibody Technique, Base Sequence, Molecular Sequence Data, Thymus Gland, Hypoplasia, and Medical and Health Sciences
Hoxa3(null) mice have severe defects in the development of pharyngeal organs including athymia, aparathyroidism, thyroid hypoplasia, and ultimobranchial body persistence, in addition to defects of the throat cartilages and cranial nerves.... more
Hoxa3(null) mice have severe defects in the development of pharyngeal organs including athymia, aparathyroidism, thyroid hypoplasia, and ultimobranchial body persistence, in addition to defects of the throat cartilages and cranial nerves. Some of the structures altered in the Hoxa3(null) mutant embryos are anterior to the described Hoxa3 gene expression boundary: the thyroid, soft palate, and lesser hyoid horn. All of these structures develop over time and through the interactions of multiple cell types. To investigate the specific cellular targets for HOXA3 function in these structures across developmental time, we performed a comprehensive analysis of the temporal and tissue-specific requirements for Hoxa3, including a lineage analysis using Hoxa3(Cre). The combination of these approaches showed that HOXA3 functions in both a cell autonomous and non-cell autonomous manner during development of the 3rd and 4th arch derivatives, and functions in a neural crest cell (NCC)-specific, non-cell autonomous manner for structures that were Hoxa3-negative by lineage tracing. Our data indicate that HOXA3 is required for tissue organization and organ differentiation in endodermal cells (in the tracheal epithelium, thymus, and parathyroid), and contributes to organ migration and morphogenesis in NCCs. These data provide a detailed picture of where and when HOXA3 acts to promote the development of the diverse structures that are altered in the Hoxa3(null) mutant. Data presented here, combined with our previous studies, indicate that the regionally restricted defects in Hoxa3 mutants do not reflect a role in positional identity (establishment of cell or tissue fate), but instead indicate a wider variety of functions including controlling distinct genetic programs for differentiation and morphogenesis in different cell types during development.
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The transcription factor FOXN1 is essential for fetal thymic epithelial cell (TEC) differentiation and proliferation. In the postnatal thymus, Foxn1 levels vary widely between different TEC subsets, from low or undetectable in putative... more
The transcription factor FOXN1 is essential for fetal thymic epithelial cell (TEC) differentiation and proliferation. In the postnatal thymus, Foxn1 levels vary widely between different TEC subsets, from low or undetectable in putative TEC progenitors to highest in the most differentiated TEC subsets. Correct Foxn1 expression is also required to maintain the postnatal microenvironment, as premature down-regulation of Foxn1 causes a rapid involution-like phenotype, while transgenic over-expression can cause thymic hyperplasia and/or delayed involution. In the current study, we investigated a K5.Foxn1 transgene that drives Foxn1 over-expression in TECs, but does not cause hyperplasia, nor does it delay or prevent aging-related involution. Similarly, this transgene cannot rescue thymus size in Foxn1lacZ/lacZ mice that undergo premature involution due to reduced Foxn1 levels. However, K5.Foxn1 transgenics do maintain TEC differentiation and cortico-medullary organization with aging both...
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Thymic epithelial cells (TECs) are a critical functional component of the thymus’s ability to generate T cells for the adaptive immune system in vertebrates. However, no in vitro system for studying TEC function exists. Overexpression of... more
Thymic epithelial cells (TECs) are a critical functional component of the thymus’s ability to generate T cells for the adaptive immune system in vertebrates. However, no in vitro system for studying TEC function exists. Overexpression of the transcription factor FOXN1 initiates reprogramming of fibroblasts into TEC-like cells (iTECs) that support T cell differentiation in culture or after transplant. In this study, we characterized iTEC reprogramming at the cellular and molecular level to determine how reprogramming proceeds and identify mechanisms that can be targeted for improving this process. These data show that iTEC reprogramming consists of discrete gene expression changes that differ in early and late reprogramming, and that iTECs upregulate markers of both cortical and medullary TEC (cTEC and mTEC) lineages, although mTEC differentiation is blocked at a progenitor stage. We demonstrate that promoting proliferation enhances iTEC generation, and that Notch inhibition allows i...
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Background Localized and total body irradiation are used to treat certain cancers and also used prior to transplantation of stem cells or organs. However, the use of radiation also induces collateral damage to the cells of healthy tissue.... more
Background Localized and total body irradiation are used to treat certain cancers and also used prior to transplantation of stem cells or organs. However, the use of radiation also induces collateral damage to the cells of healthy tissue. Although the acute damage of radiation to oocytes is well known, the long-term effects induced by radiation to stromal cells and their relationship with age are still unclear. Methods A total of 206 two-month-old female mice were whole-body exposed to gamma rays at doses of 0, 0.5, 1, 2, or 4 Gy, respectively. The mice were sacrificed at 3.5, 9, 12, or 18 months of age and pathological changes including cysts and tumors were assessed in the ovary and other organs. Results The overall incidence of visible pathological changes of mice receiving irradiation was 33.7% in the ovary, but much lower in the liver, spleen, lung, thymus, and skin. Among these, the ovarian cyst formation rate was 24.7%, and tumor lesions were 10.2%, respectively, compared to ...
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Current in vitro culture methods for mouse embryos are critically dependent on specially prepared rodent serum. Rodent serum requires careful preparation and stringent assessment of serum quality, while commercially available whole embryo... more
Current in vitro culture methods for mouse embryos are critically dependent on specially prepared rodent serum. Rodent serum requires careful preparation and stringent assessment of serum quality, while commercially available whole embryo culture serum is expensive and shows considerable lot variability. Thus, preparation and testing of suitable serum represents a considerable investment of time and resources, particularly for laboratories with only short-term embryo culture requirements. In addition, serum supplementation of culture medium may introduce unknown serum components that could interfere with interpretation of experimental results, especially where the study is geared towards analysis of a specific growth factor. Here we describe the composition of a standardized serum free culture medium comprised of commercially available stem cell media supplements. With this method, we have successfully cultured midgestation stage mouse embryos and demonstrated, using both morphological and gene expression criteria, that these embryos exhibited proper developmental progression. We believe this method to be a significant advance in whole embryo culture technology that will be of particular use to laboratories needing to utilize whole embryo culture to study midgestation organogenesis.
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The thymus and parathyroids originate from the third pharyngeal pouches, which form as endodermal outpocketings in the pharyngeal region beginning on embryonic day 9 (E9.0) of mouse development. Using organ-specific markers, we have... more
The thymus and parathyroids originate from the third pharyngeal pouches, which form as endodermal outpocketings in the pharyngeal region beginning on embryonic day 9 (E9.0) of mouse development. Using organ-specific markers, we have previously shown that thymus and parathyroid-specific organ domains are established within the primordium prior to formation of the organs proper: Gcm2 expression defines the prospective parathyroid cells in the dorsal pouch from E9.5, while Foxn1 is expressed in the thymus domain from E11.25. Bmp (bone morphogenetic protein) signaling has been implicated in thymic epithelial cell differentiation and thymus organogenesis. In the present study, we report expression patterns of Bmp4 and Noggin, a Bmp4 antagonist, in the third pharyngeal pouch using two lacZ transgenic mouse strains. Results from this gene expression study revealed localization of Bmp4 expression to the ventral region of the third pharyngeal pouch endoderm at E10.5 and E11.5, in those cells that will express Foxn1 and form the thymus. Conversely, the expression of Noggin was confined to the dorsal region of the pouch and primordium at these stages, and thus appeared to be co-expressed with Gcm2 in the parathyroid domain. This represents the first detailed study of Bmp4 and Noggin expression during the early stages of thymus and parathyroid organogenesis.
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The thymus and parathyroids are pharyngeal endoderm-derived organs that develop from common organ primordia, which undergo a series of morphological events resulting in separate organs in distinct locations in the embryo. Previous gene... more
The thymus and parathyroids are pharyngeal endoderm-derived organs that develop from common organ primordia, which undergo a series of morphological events resulting in separate organs in distinct locations in the embryo. Previous gene expression and functional analyses have suggested a role for BMP4 signaling in early thymus organogenesis. We have used conditional deletion of Bmp4 or Alk3 from the pharyngeal endoderm and/or the surrounding mesenchyme using Foxg1-Cre, Wnt1-Cre or Foxn1-Cre. Deleting Bmp4 from both neural crest cells (NCC) and early endoderm-derived epithelial cells in Foxg1-Cre;Bmp4 conditional mutants resulted in defects in thymus-parathyroid morphogenesis. Defects included reduced condensation of mesenchymal cells around the epithelium, partial absence of the thymic capsule, a delay in thymus and parathyroid separation, and failed or dramatically reduced organ migration. Patterning of the primordia and initial organ differentiation were not affected in any of the mutants. Deleting Bmp4 from NCC-derived mesenchyme or differentiating thymic epithelial cells (TECs) had no effects on thymus-parathyroid development, while loss of Alk3 from either neural crest cells or TECs resulted in only a mild thymic hypoplasia. these results show that the processes of cell specification and morphogenesis during thymus-parathyroid development are independently controlled, and suggest a specific temporal and spatial role for BMP4-mediated epithelial-mesenchymal interactions during early thymus and parathyroid morphogenesis.
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Pbx1 is a TALE-class homeodomain protein that functions in part as a cofactor for Hox class homeodomain proteins. Previous analysis of the in vivo functions of Pbx1 by targeted mutagenesis in mice has revealed roles for this gene in... more
Pbx1 is a TALE-class homeodomain protein that functions in part as a cofactor for Hox class homeodomain proteins. Previous analysis of the in vivo functions of Pbx1 by targeted mutagenesis in mice has revealed roles for this gene in skeletal patterning and development and in the organogenesis of multiple systems. Both RNA expression and protein localization studies have suggested a possible role for Pbx1 in pharyngeal region development. As several Hox mutants have distinct phenotypes in this region, we investigated the potential requirement for Pbx1 in the development of the pharyngeal arches and pouches and their organ derivatives. Pbx1 homozygous mutants exhibited delayed or absent formation of the caudal pharyngeal pouches, and disorganized patterning of the third pharyngeal pouch. Formation of the third pouch-derived thymus/parathyroid primordia was also affected, with absent or hypoplastic primordia, delayed expression of organ-specific differentiation markers, and reduced proliferation of thymic epithelium. The fourth pouch and the fourth pouch-derived ultimobranchial bodies were usually absent. These phenotypes are similar to those previously reported in Hoxa3(-/-) single mutants and Hoxa1(-/-);Hoxb1(-/-) or Hoxa3(+/-);Hoxb3(-/-);Hoxd3(-/-) compound mutants, suggesting that Pbx1 acts together with multiple Hox proteins in the development of the caudal pharyngeal region. However, some aspects of the Pbx1 mutant phenotype included specific defects that were less severe than those found in known Hox mutant mice, suggesting that some functions of Hox proteins in this region are Pbx1-independent.
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Previous studies have implicated Sonic hedgehog (Shh) as an important regulator of pharyngeal region development. Here we show that Shh is differentially expressed within the pharyngeal endoderm along the anterior-posterior axis. In... more
Previous studies have implicated Sonic hedgehog (Shh) as an important regulator of pharyngeal region development. Here we show that Shh is differentially expressed within the pharyngeal endoderm along the anterior-posterior axis. In Shh-/- mutants, the pharyngeal pouches and arches formed by E9.5 and marker expression showed that initial patterning was normal. However, by E10.5-E11.0, the first arch had atrophied and the first pouch was missing. Although small, the second, third, and fourth arches and pouches were present. The expression patterns of Fgf8, Pax1, and Bmp4 suggested that pouch identity was abnormal at E10.5 and that Shh is a negative regulator of these genes in the pouches. Despite the loss of pouch identity and an increase in mesenchymal cell death, arch identity markers were expressed normally. Our data show that a Shh-dependent patterning mechanism is required to maintain pouch patterning, independent or downstream of arch identity. Changes in the distribution of Bmp4 and Gcm2 in the third pouch endoderm and subsequent organ phenotypes in Shh-/- mutants suggested that exclusion of Shh from the third pouch is required for dorsal-ventral patterning and for parathyroid specification and organogenesis. Furthermore, this function for Shh may be opposed by Bmp4. Our data suggest that, as in the posterior gut endoderm, exclusion of Shh expression from developing primordia is required for the proper development of pharyngeal-derived organs.
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Tracheal agenesis/atresia (TA) is a rare but fatal congenital disease in which the breathing tube fails to grow. The etiology of this serious condition remains largely unknown. We found that Bmp signaling is prominently present in the... more
Tracheal agenesis/atresia (TA) is a rare but fatal congenital disease in which the breathing tube fails to grow. The etiology of this serious condition remains largely unknown. We found that Bmp signaling is prominently present in the anterior foregut where the tracheal primordium originates and targeted ablation of Bmp4 (Bmp4(cko)) resulted in a loss-of-trachea phenotype that closely resembles the Floyd type II pathology, the most common form of TA in humans. In Bmp4(cko) embryos, tracheal specification was not affected; however, its outgrowth was severely impaired due to reduced epithelial and mesenchymal proliferation. In agreement, we also observed significant reduction in the expression of Cyclin D1, a key cell cycle regulator associated with cellular proliferation. However, the proliferative effect of Bmp signaling appears to be independent of Wnt signaling. Interestingly, we found significantly reduced expression of activated extracellular signal-regulated kinase (Erk) in the Bmp4(cko) ventral foregut, suggesting that Bmp signaling promotes Erk phosphorylation which has been associated with cellular proliferation. This study provides the first evidence linking Bmp signaling to tracheal formation by regulating the proliferative response of the anterior ventral foregut. Our finding sheds light on human tracheal malformations by providing a novel mouse model implicating Bmp signaling, non-canonical Erk activation and cellular proliferation.
Research Interests: Developmental Biology, Immunohistochemistry, In Situ Hybridization, Bone Morphogenetic Proteins, Signal Transduction, and 13 moreBiological Sciences, Mice, Wnt Signaling, Animals, Beta-Catenin, Mouse Model, Digestive System, Body Patterning, Trachea, Gene Targeting, Cell Cycle regulation, Cell Proliferation, and Embryos
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The thymus and parathyroid glands in mice develop from a thymus/parathyroid primordium that forms from the endoderm of the third pharyngeal pouch. We investigated the molecular mechanisms that promote this unique process in which two... more
The thymus and parathyroid glands in mice develop from a thymus/parathyroid primordium that forms from the endoderm of the third pharyngeal pouch. We investigated the molecular mechanisms that promote this unique process in which two distinct organs form from a single primordium, using mice mutant for Hoxa3 and Pax1. Thymic ectopia in Hoxa3(+/-)Pax1(-/-) compound mutants is due to delayed separation of the thymus/parathyroid primordium from the pharynx. The primordium is hypoplastic at its formation, and has increased levels of apoptosis. The developing third pouch in Hoxa3(+/-)Pax1(-/-) compound mutants initiates normal expression of the parathyroid-specific Gcm2 and thymus-specific Foxn1 genes. However, Gcm2 expression is reduced at E11.5 in Pax1(-/-) single mutants, and further reduced or absent in Hoxa3(+/-)Pax1(-/-) compound mutants. Subsequent to organ-specific differentiation from the shared primordium, both the parathyroids and thymus developed defects. Parathyroids in compound mutants were smaller at their formation, and absent at later stages. Parathyroids were also reduced in Pax1(-/-) mutants, revealing a new function for Pax1 in parathyroid organogenesis. Thymic hypoplasia at later fetal stages in compound mutants was associated with increased death and decreased proliferation of thymic epithelial cells. Our results suggest that a Hoxa3-Pax1 genetic pathway is required for both epithelial cell growth and differentiation throughout thymus and parathyroid organogenesis.
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Embryos that are homozygous for Splotch, a null allele of Pax3, have a severe neural crest cell (NCC) deficiency that generates a complex phenotype including spina bifida, exencephaly and cardiac outflow tract abnormalities. Contrary to... more
Embryos that are homozygous for Splotch, a null allele of Pax3, have a severe neural crest cell (NCC) deficiency that generates a complex phenotype including spina bifida, exencephaly and cardiac outflow tract abnormalities. Contrary to the widely held perception that thymus aplasia or hypoplasia is a characteristic feature of Pax3(Sp/Sp) embryos, we find that thymic rudiments are larger and parathyroid rudiments are smaller in E11.5-12.5 Pax3(Sp/Sp) compared to Pax3(+/+) embryos. The thymus originates from bilateral third pharyngeal pouch primordia containing endodermal progenitors of both thymus and parathyroid glands. Analyses of Foxn1 and Gcm2 expression revealed a dorsal shift in the border between parathyroid- and thymus-fated domains at E11.5, with no change in the overall cellularity or volume of each shared primordium. The border shift increases the allocation of third pouch progenitors to the thymus domain and correspondingly decreases allocation to the parathyroid domain. Initial patterning in the E10.5 pouch was normal suggesting that the observed change in the location of the organ domain interface arises during border refinement between E10.5 and E11.5. Given the well-characterized NCC defects in Splotch mutants, these findings implicate NCCs in regulating patterning of third pouch endoderm into thymus- versus parathyroid-specified domains, and suggest that organ size is determined in part by the number of progenitor cells specified to a given fate.
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The origins of the non-hematopoietic cell types that comprise the thymic stroma remain a topic of considerable controversy. Three recent studies, using lineage analysis and other methods to determine the developmental potential of... more
The origins of the non-hematopoietic cell types that comprise the thymic stroma remain a topic of considerable controversy. Three recent studies, using lineage analysis and other methods to determine the developmental potential of specific cell types within the thymus, have provided strong evidence of a single endodermal origin for all thymic epithelial cells. Together with other investigations that merge immunological and developmental biology approaches, these studies have suggested a new model of thymus organogenesis, and have begun to uncover the molecular pathways that control this process.
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Exogenous retinoic acid is teratogenic in animals and man, causing a spectrum of abnormalities termed retinoic acid embryopathy. Using a mouse model of retinoic acid embryopathy, our results show that exposure to all-trans retinoic acid... more
Exogenous retinoic acid is teratogenic in animals and man, causing a spectrum of abnormalities termed retinoic acid embryopathy. Using a mouse model of retinoic acid embryopathy, our results show that exposure to all-trans retinoic acid (RA) on gestational day (gd) 9 results in thymic ectopia, hypoplasia, and thymocyte maturational defects. Immunohistochemical and flow cytometric analyses showed aberrant expression of stromal and thymocyte markers, and abnormalities in thymocyte development. RNA in situ hybridization for the transcription factors Hoxa3 and Pax1 was used to investigate the basis of this defect. Hoxa3 and Pax1 have been shown to be required for normal thymus development, and are normally expressed in the cells of the third pharyngeal pouch and third and fourth pharyngeal arches, involved in thymus organogenesis RA-exposed embryos showed an increased level of Hoxa3 expression in the neural tube and caudal pharyngeal arches as soon as 6 hr after exposure. The Pax1 expression pattern, in conjunction with analysis of the external pharyngeal morphology, showed that the development and structure of the third pharyngeal pouch and cleft were disrupted, resulting in a reduced third pharyngeal arch and/or fusion of the third and fourth arches. Changes in the expression of cellular retinoic acid binding protein (CRABP) and in the morphology of the cranial ganglia were consistent with altered neural crest cell migration from the caudal hindbrain after RA exposure. Together, our findings suggest that the teratogenic effects of RA on thymus development include changes in both the cranial neural crest and pharyngeal endoderm that contribute to thymus development. Further, the observed defects in thymus development may be mediated by RA-induced alterations in the expression of Hoxa3.