The lymphatic vasculature is a network of unidirectional capillaries and ducts, which serve to return extracellular fluid and macromolecules to the systemic blood circulation. In addition, the lymphatic vessels have important roles in immune surveillance and fat absorption. Dysfunction of lymphatic vessels has profound physiological consequences. Insufficient lymph uptake results in lymphedema, a chronic disabling condition that currently has no cure. Lymphedemcanoccureither due to developmental defects (primary lymphedema) or due to injuries of existing lymphatic vessels (secondary lymphedema). The significance of lymphangiogenesis in tumour metastasis is demonstrated by the finding that increased lymphangiogenesis is associated with a higher rate of metastasis and poorer prognosis in cancer patients. Prox1, a homeoboxgene, regulates the development of the lymphatic vasculature by upregulating the expression of lymphatic endothelial markers and simultaneously repressing the expression of blood endothelial markers. To explore the mechanisms by which Prox1 establishes lymphatic cell fate, we compared Prox1 mediated activation oftwo key lymphatic cell surface receptors: theVascular Endothelial Growth Factor Receptor-3 (VEGFR-3) and theFibroblast Growth Factor Receptor-3 (FGFR-3) genes. Using a combination of luciferase gene reporter assays and immunoblotting, we compared the ability of different Prox1 constructs to activate either VEGFR-3 or FGFR-3 at both the mRNA and protein levels, respectively. Furthermore, we tested whether recombinant PROX1 protein was able to bind to the proximal promoter regions of VEGFR-3 and FGFR-3usingelectrophoretic mobility shift assays (EMSA). DNA binding deficient Prox1 versions did not activate the FGFR-3 promoter. In contrast, these versions of Prox1still efficiently activated transcription of the VEGFR-3 promoter. In agreement with our luciferase reporter gene assays, immunoblotting of HUVECs demonstrated that only infection with wt Prox1adenovirus increased expression of the FGFR-3 protein. Infection of HUVECs with adenoviral vectors encoding eitherwt Prox1orHDPD∆ Prox1was sufficient to induce a significant increase in VEGFR-3 protein levels. Surprisingly, our EMSA results with recombinant PROX1 demonstrated that PROX1 can bind to the promoter region of both VEGFR-3 and FGFR-3 genes via its DNA binding domain. We showed that PROX1 potentially binds to the promoter region of the VEGFR-3 gene via a consensus Prospero binding site (CGCCTCGGC). Our data demonstrates that, in endothelial cells, PROX1 utilizes distinct mechanisms to activate these two key endothelial growth factor receptors.

The lymphatic vasculature is a network of unidirectional capillaries and ducts, which serve to return extracellular fluid and macromolecules to the systemic blood circulation. In addition, the lymphatic vessels have important roles in immune surveillance and fat absorption. Dysfunction of lymphatic vessels has profound physiological consequences. Insufficient lymph uptake results in lymphedema, a chronic disabling condition that currently has no cure. Lymphedemcanoccureither due to developmental defects (primary lymphedema) or due to injuries of existing lymphatic vessels (secondary lymphedema). The significance of lymphangiogenesis in tumour metastasis is demonstrated by the finding that increased lymphangiogenesis is associated with a higher rate of metastasis and poorer prognosis in cancer patients. Prox1, a homeoboxgene, regulates the development of the lymphatic vasculature by upregulating the expression of lymphatic endothelial markers and simultaneously repressing the expression of blood endothelial markers. To explore the mechanisms by which Prox1 establishes lymphatic cell fate, we compared Prox1 mediated activation oftwo key lymphatic cell surface receptors: theVascular Endothelial Growth Factor Receptor-3 (VEGFR-3) and theFibroblast Growth Factor Receptor-3 (FGFR-3) genes. Using a combination of luciferase gene reporter assays and immunoblotting, we compared the ability of different Prox1 constructs to activate either VEGFR-3 or FGFR-3 at both the mRNA and protein levels, respectively. Furthermore, we tested whether recombinant PROX1 protein was able to bind to the proximal promoter regions of VEGFR-3 and FGFR-3usingelectrophoretic mobility shift assays (EMSA). DNA binding deficient Prox1 versions did not activate the FGFR-3 promoter. In contrast, these versions of Prox1still efficiently activated transcription of the VEGFR-3 promoter. In agreement with our luciferase reporter gene assays, immunoblotting of HUVECs demonstrated that only infection with wt Prox1adenovirus increased expression of the FGFR-3 protein. Infection of HUVECs with adenoviral vectors encoding eitherwt Prox1orHDPD∆ Prox1was sufficient to induce a significant increase in VEGFR-3 protein levels. Surprisingly, our EMSA results with recombinant PROX1 demonstrated that PROX1 can bind to the promoter region of both VEGFR-3 and FGFR-3 genes via its DNA binding domain. We showed that PROX1 potentially binds to the promoter region of the VEGFR-3 gene via a consensus Prospero binding site (CGCCTCGGC). Our data demonstrates that, in endothelial cells, PROX1 utilizes distinct mechanisms to activate these two key endothelial growth factor receptors.

Abstract The homeobox gene prox1 is a transcription factor related to the Drosophila gene Prospero. It play an essential role in the development of central nervous system, lens, liver and pancreas. In addition, prox1 is a master gene controlling the early development of the lymphatic vasculature. In tumorigenesis, prox1 has been shown to function as a tumor suppressor gene in hepatocellular carcinoma and breast cancer. Conversely, prox1 is over-expressed in the majority of colorectal cancer (CRC) and it promotes dysplasia, tumor growth and malignant progression. I report the findings here and show that ectopic expression of prox1 in prox1-null DLD-1 colon cancer cells will increase cell invasion but decrease cell adhesion. In addition, prox1 may induce epithelia- mesenchymal transition (EMT) by attenuating E-cadherin expression and up-regulating other EMT markers. On the contrary, knockdown of prox1 increases E-cadherin expression in SW620 cells; reduction of prox1 increases cell adhesion but decreases invasion. In short, the transcription factor prox1 plays an oncogenic role and promotes cancer metastasis in CRC.

PROX1 étant un facteur de susceptibilité au diabète de type 2 (DT2), nousavons réalisé des études génétiques et moléculaires afin de comprendre son rôledans l’étiologie du DT2.Nous avons analysé l’impact de 80 SNPs de PROX1 sur des phénotypescliniques associés au DT2 dans l’étude HELENA (n=1155 adolescents) et montréque trois SNPs (rs340838, rs340837 et rs340836) sont associés à l’insulinémie àjeun. Nous avons évalué la fonctionnalité de 9 SNPs (les 3 SNPs associés et 6 SNPsen déséquilibre de liaison) en utilisant un gène rapporteur Luciférase dans descellules HepG2 et MIN6. Les allèles associés à la diminution de l’insulinémie desSNPs rs340874, rs340873 et rs340835 sont associés à une diminution del’expression du gène rapporteur Luciférase, suggérant que l’expression de PROX1est diminuée chez les individus porteurs des allèles à risque.Nous avons aussi montré que l’inhibition de l’expression de Prox1 par siRNAsdans les cellules INS-1E engendrait une diminution de 1,7 fois de la sécrétiond’insuline en réponse au glucose et qu’une concentration élevée en glucose modulaitpositivement l’expression de la protéine Prox1.Des analyses transcriptomiques réalisées dans les cellules INS-1E ont permisde montrer que certains des gènes cibles de PROX1 dans les cellules bêta sont desgènes impliqués dans des voies de sécrétion d’insuline.Enfin, nous avons également observé que l’agoniste de PPARgamma, latroglitazone, diminuait l’expression de Prox1 dans les cellules INS-1E.Ces résultats suggèrent qu’une altération de l’expression de Prox1 par desvariants cis-régulateurs pourrait conduire à une sécrétion d’insuline en réponse auglucose altérée des cellules bêta, conférant ainsi une susceptibilité au DT2. / As PROX1 is a susceptibility factor for type 2 diabetes (T2D), we conductedgenetic and molecular studies to better understand the role of PROX1 in the etiology of T2D. We assessed the impact of 80 PROX1 SNPs on T2D-related biochemical traits in the HELENA study (n=1155 adolescents) and showed that 3 SNPs (rs340838, rs340837 and rs340836) were significantly associated with fasting plasma insulin levels. We evaluated the functional impact of 9 SNPs (the 3 insulin-associated SNPs plus 6 SNPs in linkage disequilibrium) using a Luciferase reporter gene expression in HepG2 and MIN6 cells. The insulin-lowering alleles of the rs340874, rs340873 and rs340835 SNPs were associated with lower Luciferase gene expression, suggesting that PROX1 expression may be lower in individuals carrying the insulin-lowering alleles. We also showed that the knock-down of Prox1 expression by siRNA in INS-1E cells resulted in a 1.7 fold reduced glucose-stimulated insulin secretion and that high concentrations of glucose positively modulated Prox1 protein expression. Then, microarray analyses performed in INS-1E cells showed that some PROX1 target genes in the _cells are implicated in insulin secretion pathways. Finally, we observed that the PPARgamma agonist, the troglitazone, decreased Prox1 expression in INS-1E cells. Altogether, these results suggest that an altered expression of Prox1 bys cisregulators variants results in an altered -cell glucose-stimulated insulin secretion andthereby confers susceptibility to T2D.

O carcinoma mucoepidermoide (CME) é a neoplasia maligna de glândula salivar mais comum, e com maior frequência de metástase linfonodal. Alterações genéticas estão intimamente associadas à carcinogênese e, também, aos processos de metástase tumoral. Para o CME o tratamento de escolha mais aplicado hoje é a cirurgia seguida de radioterapia, pois a quimioterapia não tem mostrado muita eficiência para o tratamento destas neoplasias. Entre os quimioterápicos mais prescritos para o tratamento de cânceres encontra-se a cisplatina, à base de platina, que atua no DNA da célula, induzindo a apoptose. Pouco se sabe a respeito de seu mecanismo de ação sobre o CME, inclusive sobre os genes homeobox. Estes genes compreendem uma família grande e essencial de reguladores do desenvolvimento que são vitais para o crescimento e diferenciação celular, e a expressão anômala destes genes têm sido implicados na carcinogênese. Assim, este trabalho teve como objetivo avaliar a expressão dos genes homeobox em células derivadas de carcinoma mucoepidermoide tratadas com cisplatina. Os genes avaliados neste trabalho foram: PROX1, MEIS1, HOXB5, HOXB7 e HOXB9 por RT-qPCR. Previamente, as linhagens celulares derivadas de carcinoma mucoepidermoide UM-HMC1 UM-HMC2 e UM-HMC3A foram tratadas com a cisplatina por 24h e posteriormente submetidas aos ensaios de RT-qPCR. Adicionalmente, as amostras tratadas e sem tratamento foram analisadas pelo ensaio de formação de esferas e ensaio de ferida para verificar o efeito da cisplatina sobre propriedades relacionadas às células quimiorresistentes (putativas células tronco tumorais). Como resultados, entre os genes analisados foram expressos PROX1, MEIS1 e HOXB7. A UM-HMC3A apresentou maior expressão destes genes que as demais linhagens. Os genes HOXB5 e HOXB9 não foram expressos nas linhagens analisadas. A cisplatina reduziu a expressão de MEIS1 e aumentou a expressão de HOXB7, em todas as linhagens. O gene PROX1 apresentou expressão variável entre as linhagens, sendo expresso na UM-HMC1 apenas quando são tratadas com cisplatina e reduzido nas UM-HMC2 e UM-HMC3A tratadas. O número de esferas formadas não apresentou diferença significativa para UM-HMC1 e UM-HMC3A, o número de esferas aumentou na linhagem UM-HMC2 tratada com cisplatina. No ensaio de ferida, a cisplatina foi capaz de reduzir a migração celular em todas as linhagens quando comparadas com seus controles. Os resultados sugerem que o PROX1 e HOXB7 podem estar relacionados com carcinomas mucoepidermoides mais invasivos, enquanto que o MEIS1 pode estar relacionado à carcinogênese e autorrenovação tumoral. A cisplatina é capaz de afetar a expressão dos genes homeobox PROX1, MEIS1 e HOXB7, os quais foram encontrados nas linhagens de carcinoma mucoepidermoide analisados. A cisplatina não afeta as células formadoras de esferas, mas reduzir a migração das linhagens de carcinoma mucoepidermoide. / Mucoepidermoid carcinoma (MEC) is the most common malignant neoplasm of salivary gland and presents higher frequency of lymph node metastasis. Genetic alterations are closely associated with carcinogenesis and also with processes of tumor metastasis. For MEC the treatment of choice most applied today is surgery followed by radiotherapy, since chemotherapy has not shown much efficiency for the treatment of these neoplasms. Among the most commonly prescribed chemotherapic drugs for cancer treatment is platinum-based cisplatin, which acts on the cell\'s DNA, inducing apoptosis. There is no information in the literature regarding its action mechanism on MEC including homeobox genes. These genes comprise a large and essential family of developmental regulators that are vital for cell growth and differentiation and the anomalous expression of these genes have been implicated in carcinogenesis. Therefore, this work aimed to evaluate the expression of the homeobox genes in cells derived from mucoepidermoid carcinoma treated with cisplatin. The genes evaluated in this work were: PROX1, MEIS1, HOXB5, HOXB7 and HOXB9 by RT-qPCR. Previously, cell lines derived from mucoepidermoid carcinoma UM-HMC1 UM-HMC2 and UM-HMC3A were treated with cisplatin for 24h and subsequently subjected to the RT-qPCR assays. In addition, treated and untreated samples were analyzed by the sphere-forming assay and wound assay to verify the effect of cisplatin on chemo resistant cell-related (putative tumor stem cell) properties. As results, PROX1, MEIS1 and HOXB7 were expressed among the analyzed genes. UM-HMC3A showed higher expression of these genes than the other lineages. The HOXB5 and HOXB9 genes were not expressed in the analyzed lineages. Cisplatin reduced MEIS1 expression and increased HOXB7 expression in all lineages. The PROX1 gene showed variable expression between the lineages, being expressed in UM-HMC1 only when treated with cisplatin and reduced in treated UM-HMC2 and UM-HMC3A. The number of spheres formed did not show significant difference for UM-HMC1 and UM-HMC3A, the number of spheres increased in the cisplatin-treated UM-HMC2 lineage. In the wound assay, cisplatin was able to reduce cell migration in all lineages when compared to their controls. The results suggested that PROX1 and HOXB7 may be related to more invasive mucoepidermoid carcinoma, while MEIS1 be related to its carcinogenesis and selfrenew tumoral capacity. Cisplatin is capable to affect the expression of the homeobox genes PROX1, MEIS1 and HOXB7, which were found in the mucoepidermoid carcinoma cell lines analyzed. Cisplatin does not affect sphere formation of cells, but seems to reduce the migration of mucoepidermoid carcinoma lineages.

Elevation of VEGFR-3, the primary mediator of lymphangiogenesis (i.e., new lymphatic vessel formation), is frequently associated with inflammation related to chronic disease and cancer. In the latter case, VEGFR-3 dependent lymphangiogenesis induced by inflamed tumors increases the incidence of distant metastasis, leading to decreased patient survival. However, the molecular mechanisms underlying inflammation-induced VEGFR-3 elevation and lymphangiogenesis are currently unknown. Two potential candidate genes that may regulate expression of VEGFR-3 are Prox1, the primary mediator of embryonic lymphangiogenesis, and NF-κB, the key intracellular regulator of inflammation-induced transcription. We hypothesized that the key inflammatory mediator, NF-κB, regulates transcription of key mediators of lymphangiogenesis, VEGFR-3 and Prox1. We further hypothesized that inflammation-induced elevation of VEGFR-3 and Prox1 are essential steps required for robust lymphangiogenesis in response to inflammation. The three primary goals of this study were to (1) delineate the time-course of events leading to inflammation-induced lymphangiogenesis in vivo; (2) clone and characterize the VEGFR-3 promoter and identify factors regulating VEGFR-3 expression in vitro; and (3) characterize the lymphatic phenotype of NF-κB p50 knockout mice. To begin testing these hypotheses, we used a mouse model of peritonitis to characterize induction of lymphangiogenesis and expression kinetics of NF-κB, Prox1 and VEGFR-3. In vivo time-course analysis of inflammation-induced lymphangiogenesis showed activation of NF-κB followed by sequential upregulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Characterization of the VEGFR-3 promoter by luciferase-reporter and ChIP assays showed direct activation by Prox1, NF-κB p50 and p65 transcription factors. This also revealed that Prox1 and NF-κB p50 bind in close proximity and synergistically activate the VEGFR-3 promoter. Characterization of p50 knockout mice revealed significantly decreased lymphatic vessel density in several organs that corresponded to reduced VEGFR-3 and Prox1 expression. Activation of NF-κB by inflammatory stimuli also elevated expression of NF-κB, Prox1 and VEGFR-3 in cultured lymphatic endothelial cells, which enhanced proliferation and migration in response to the VEGFR-3-specific ligand, VEGF-C152S. Collectively, our findings suggest that induction of the NF-κB pathway by inflammatory stimuli activates Prox1, and both NF-κB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of pre-existing lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20568号 / 医博第4253号 / 新制||医||1022(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 原田 浩, 教授 武藤 学, 教授 戸井 雅和 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Os genes homeobox são responsáveis por codificar proteínas nucleares que agem como fatores de transcrição durante o desenvolvimento embrionário, regulando proliferação e diferenciação celular. A expressão alterada do gene homeobox PROX1 já foi identificado em diferentes neoplasias, incluindo mama, esôfago, fígado, sistema biliar, linfomas e cavidade bucal. Este trabalho teve como objetivo avaliar os efeitos da superexpressão do gene PROX1 em linhagem celular derivada de carcinoma epidermóide bucal nos mecanismos de proliferação e diferenciação celular, apoptose e perfil global de expressão gênica. Após a superexpressão deste gene na linhagem celular SCC-9, foi realizada a análise de proliferação por meio dos ensaios de curva de proliferação celular, citometria de fluxo, índice de incorporação de BrdU ao DNA e expressão de Ki67. A diferenciação celular foi verificada por meio de reações imunocitoquímicas para as citoqueratinas 1, 10, 13, 14, 16, 18 e 19 e a apoptose foi avaliada por meio de células positivas para anexina-V e iodeto de propídeo. O ensaio de microarray foi realizado para avaliação do perfil global de expressão gênica na linhagem celular SCC9 com superexpressão do gene PROX1. Observou-se que a superexpressão do gene PROX1 promove redução da proliferação celular, bem como reduz a expressão das citoqueratinas 1, 13, 18 e 19. Não houve alteração na taxa de apoptose entre as células com superexpressão do gene PROX1 e controles. Os resultados do microarray revelaram a expressão diferencial significante de genes envolvidos com os processos de desenvolvimento, adesão e invasão celular. Desta maneira, estes resultados são fortemente sugestivos de que o gene PROX1 inibe a proliferação celular e contribui para a diferenciação do carcinoma epidermóide bucal. / Homeobox genes encode transcription factors with an important role during normal development by controlling cellular proliferation and differentiation. Altered expression of PROX1 homeobox gene is related to many cancers, including those of the breast, esophagus, liver, billiary system and lymphomas. The aim of this study was evaluate the effects of PROX1 overexpression, in an oral squamous cell carcinoma cell line, on cellular proliferation and differentiation, apoptosis as well as gene expression prolfile. After overexpression of PROX1 gene in SCC9 cell line, proliferation was assessed by proliferation curve, flow citometry, BrdU incorporation to DNA and Ki67 expression. Cell differentiation was verified by immunocytochemistry to cytokeratins 1, 10, 13, 14, 16, 18 and 19 and apoptosis was measured by annexin V positive cells. Gene expression profile was analyzed by microarray in PROX1-overexpressing cells and control. PROX1-overexpressing cells showed a statistically significant decrease in proliferation as well cytokeratin 1, 13, 18 and 19 expression. No significant differences from controls and PROX1- overexpressing cells were observed in apoptosis. Microarray analyses showed differential expression of genes related to development, cellular adhesion an invasion. Our results strongly suggest that overexpression of PROX1 inhibit cell proliferation and contributes to differentiation of oral squamous cell carcinoma.

Os genes homeobox são responsáveis por codificar proteínas nucleares que agem como fatores de transcrição durante o desenvolvimento embrionário, regulando proliferação e diferenciação celular. A expressão alterada do gene homeobox PROX1 já foi identificado em diferentes neoplasias, incluindo mama, esôfago, fígado, sistema biliar, linfomas e cavidade bucal. Este trabalho teve como objetivo avaliar os efeitos da superexpressão do gene PROX1 em linhagem celular derivada de carcinoma epidermóide bucal nos mecanismos de proliferação e diferenciação celular, apoptose e perfil global de expressão gênica. Após a superexpressão deste gene na linhagem celular SCC-9, foi realizada a análise de proliferação por meio dos ensaios de curva de proliferação celular, citometria de fluxo, índice de incorporação de BrdU ao DNA e expressão de Ki67. A diferenciação celular foi verificada por meio de reações imunocitoquímicas para as citoqueratinas 1, 10, 13, 14, 16, 18 e 19 e a apoptose foi avaliada por meio de células positivas para anexina-V e iodeto de propídeo. O ensaio de microarray foi realizado para avaliação do perfil global de expressão gênica na linhagem celular SCC9 com superexpressão do gene PROX1. Observou-se que a superexpressão do gene PROX1 promove redução da proliferação celular, bem como reduz a expressão das citoqueratinas 1, 13, 18 e 19. Não houve alteração na taxa de apoptose entre as células com superexpressão do gene PROX1 e controles. Os resultados do microarray revelaram a expressão diferencial significante de genes envolvidos com os processos de desenvolvimento, adesão e invasão celular. Desta maneira, estes resultados são fortemente sugestivos de que o gene PROX1 inibe a proliferação celular e contribui para a diferenciação do carcinoma epidermóide bucal. / Homeobox genes encode transcription factors with an important role during normal development by controlling cellular proliferation and differentiation. Altered expression of PROX1 homeobox gene is related to many cancers, including those of the breast, esophagus, liver, billiary system and lymphomas. The aim of this study was evaluate the effects of PROX1 overexpression, in an oral squamous cell carcinoma cell line, on cellular proliferation and differentiation, apoptosis as well as gene expression prolfile. After overexpression of PROX1 gene in SCC9 cell line, proliferation was assessed by proliferation curve, flow citometry, BrdU incorporation to DNA and Ki67 expression. Cell differentiation was verified by immunocytochemistry to cytokeratins 1, 10, 13, 14, 16, 18 and 19 and apoptosis was measured by annexin V positive cells. Gene expression profile was analyzed by microarray in PROX1-overexpressing cells and control. PROX1-overexpressing cells showed a statistically significant decrease in proliferation as well cytokeratin 1, 13, 18 and 19 expression. No significant differences from controls and PROX1- overexpressing cells were observed in apoptosis. Microarray analyses showed differential expression of genes related to development, cellular adhesion an invasion. Our results strongly suggest that overexpression of PROX1 inhibit cell proliferation and contributes to differentiation of oral squamous cell carcinoma.

<p>Horizontal cells are retinal interneurons that modulate the output from photoreceptors. Two horizontal cell (HC) subtypes are commonly identified in the vertebrate retina: axon-bearing and axon-less HCs. In this work, we have identified Isl1 as a novel HC marker and demonstrated that Lim1 and Isl1 distinguish axon-bearing and axon-less HCs, respectively. In the chick retina, axon-less HCs are furthermore split into two different subtypes based on the expression of GABA and TrkA.</p><p>We have demonstrated that during early chick retinogenesis, HCs expressing either Lim1 or Isl1 are generated consecutively as two equally large sub-groups at different time points. Moreover, these newborn HCs undertake an unexpected bi-directional migration before settling in their final laminar position. Different HC subtypes complete this migration at different times.</p><p>We investigated the role of activin signaling during HC subtype generation. Activin or its inhibitor follistatin was administrated during the main phase of HC generation and analyzed when HCs had completed migration. Activin caused a significant decrease in both HC subtypes and decreased the proliferation of retinal precursor cells. Follistatin increased the number of late born (Isl1+) HCs, which migrated to the HC-layer during a prolonged migration period. Both treatments affected retinal histology, but only activin influenced the generation of retinal populations other than HCs. These effects were most likely mediated by altered proliferation in certain retinal precursor cells.</p><p>The data on HC subtype ratios, birth-dates, migration, apoptosis and extrinsic activin modulation favor a scenario where the mature proportions of HC subtypes are generated sequentially from a specific HC-precursor cell lineage early in development and remain stable thereafter. These proportions are not adjusted by apoptosis, but rather by the combined actions of transcription factors and extrinsic signaling. Our studies on HC subtypes and their development promises to facilitate future studies on HC development, evolution and function.</p>