Sonic hedgehog (Shh) is an important signalling protein expressed extensively in development, throughout tissues of the central nervous system, gut and the posterior of the limb bud. The complicated expression pattern of Shh is regulated by a series of long-range enhancers located flanking and dispersed throughout a 1 Mb genomic desert. Disruption of SHH as a result of mutations within the gene or its enhancers has been implicated in two developmental conditions. These are Holoproencephaly (HPE3) a common developmental defect of the forebrain and frequently the mid-face in humans, and preaxial polydactyly (PPD), a congenital limb abnormality encompassing a varied phenotype affecting the digits on the anterior side of the hands and feet that has been attributed to misexpression of Shh. In order to investigate the Shh regulatory landscape and survey regulatory activity, a transposon-based chromosomal engineering strategy known as the local hopping enhancer detection (LHED) system was employed. Using this method a targeting vector containing a LacZ reporter gene as well as LoxP sites was inserted within the Shh region. The ‘hopping’ nature of the transposable element was then exploited to scatter it throughout the region. Tetraploid complementation embryos derived entirely from ES cells were generated and examined in order to gain an insight into enhancer activity. The region was found to be in an open conformation over its length and is generally susceptible to all Shh enhancers. Genes within the regulatory domain, such as the widely expressed Rnf32 gene, were found to resist Shh enhancer activities by a process of regulatory evasion by the promoter, a mechanism that may be common in large regulatory domains. Finally, at the boundaries of the region Shh activity was found to be lost incrementally at a number of genomic positions. Mouse lines were also generated to look at both enhancer activity and loss of function effects and three deletions of increasing size were generated between Shh and the furthest enhancer, the Zrs. These in turn, delete firstly a gut and pharyngeal epithelial enhancer, secondly the gut, pharyngeal enhancers as well an oral epithelial enhancer and finally all three epithelial enhancers as well as three forebrain enhancers. Reporter gene expression was found to be lost incrementally from those enhancers deleted without disrupting the rest of the region. Previously unidentified notochord enhancer(s) were found to lie within the region 100-530 kb upstream of Shh. Examination of the resultant phenotypes showed that gut and craniofacial defects were found to occur as a result of the loss of enhancers which drive expression within these tissues. Variable phenotypes were found to occur potentially as a result of temporal changes to Shh expression or as a result of threshold levels of HH being required for normal development. Other enhancers within the Shh region and outwith the deletions were not found to be disrupted by these modifications suggesting the enhancers within the region act independently of each other. The largest deletion resulted in bringing the Zrs (which drives Shh limb expression) within 170 kb of the gene, however limb development; was not, found to be affected suggesting distance is not required for Zrs function. Overall, the LHED transposon system has been utilised in order to examine the Shh region in more detail, allowing mapping of enhancer function by reporter gene expression and examination of phenotypes generated by deletion of enhancers.

Correct number and pattern of digits is determined in a time and concentration-dependent manner by a gradient of Sonic Hedgehog (SHH) across the anterior-posterior axis of the embryonic limb bud. Owing to the potent morphogenic/mitogenic capabilities of SHH, transcription of the SHH gene in the limb is tightly regulated by feedback loops with other signalling pathways and by the Zone of Polarising Activity regulatory sequence (ZRS). The ZRS is a long-range, cis-regulatory limb-specific enhancer of SHH, and is essential for correct limb SHH expression. The Silkie, a polydactylous breed of chicken, possesses a C > A mutation in the ZRS, resulting in ectopic SHH expression in the anterior limb and hindlimb-specific polydactyly. We employ the Silkie mutant to investigate how SHH is regulated by the ZRS, and how Hedgehog signalling can modulate SHH expression in an autoregulatory manner. We further characterise the effects that the Silkie mutation has on subsequent limb development; investigating the dependence of increased posterior SHH, increased Hedgehog-dependent growth and necessary genotype in both the posterior and anterior limb bud. Several fundamental questions regarding SHH during limb development have yet to be fully addressed: how much SHH protein is present, and does it form a gradient as hypothesised by Wolpert’s Morphogen Gradient Model? By developing a standard curve-based method to assess absolute quantities of processed SHH protein, N-SHH, we find that the quantity of N-SHH protein increases through limb development, and does indeed form a quantifiable gradient across the posterior limb. By comparing quantity of N-SHH protein in equivalently staged mouse, rat, emu and chicken limbs, we find that there is no significant link between N-SHH protein quantity and digit number between mammalian and avian species, and investigate how digit number is modulated in the late limb. A number of species exhibit reduced numbers of digits, including the wings of the emu, cassowary and kiwi. Unlike in mammalian examples of digit loss (i.e. cow, pig) the emu wing has delayed and significantly reduced SHH expression. Through sequencing and functional in vivo testing of ZRS sequences of ratite bird species, we investigate whether the ZRS has a role in evolutionary digit loss. We also demonstrate the aspects of digit loss and Hh signalling are shared with examples of mammalian digit loss. This thesis presents novel research into multiple aspects of genetic regulation, limb development, and evolutionary developmental biology; elucidating both long held dogmas and upcoming areas of limb development.

In the vertebrate retina, bipolar interneurons consist of at least 13 distinct subtypes, which are classified based on their morphology, behavior and gene expression. The mechanisms underlying the formation of these subtypes is poorly understood. Our previous unpublished work has implicated Sonic Hedgehog (Shh) in the formation of cone and rod bipolar cell subtypes. In this thesis, I characterized the relationship between Hh signaling and bipolar subtype cell development in greater detail. Using an in vivo plasmid-based reporter approach, I show that Hh signaling is active in both retinal progenitor cells (RPCs) and bipolar cells of the postnatal retina. Next, to address function, I used a conditional gene targeting approach to show that activation of Smoothened (Smo), a downstream Hh signaling component, is both necessary and sufficient in postnatal RPCs to promote the formation of cone but not rod bipolar cells. In contrast, activation of Smo in postmitotic bipolar cells that are greater than 24 hours old from cell birth, does not affect bipolar subtype formation. Together, these results suggest that Hh signaling functions in postnatal RPCs (and potentially in early bipolar cell precursors) to promote cone bipolar cell formation. / Graduate / 2018-06-12

Sonic Hedgehog (SHH) is a secreted morphogen that is an essential regulator of patterning and growth. The SHH protein requires cleavage of its full-length precursor (SHHFL) for secretion of biologically active SHH (SHHNp). Mutations in SHH that affect SHH processing are associated with human disease, which highlights the importance of processing for patterning in vivo. We identified Sortilin (SORT1), a member of the VPS10P receptor family, as a novel SHH interacting protein. SORT1 preferentially associates with SHHFL and SORT1 levels correlate inversely with cleavage of SHHFL. Consistent with an antagonistic relationship between SORT1 and SHH processing, loss of SORT1 results in an increase in SHH levels in axons and a partial rescue of Hedgehog-associated patterning defects in a mouse model of deficient SHH processing. Finally, we demonstrate a functional requirement for SORT1-mediated trafficking on SHH-dependent signaling from axons in the developing visual system in vivo. Our findings identify a novel role for SORT1 in the regulation of SHH processing and trafficking.

Medulloblastoma (MB), a cancer of the cerebellum, is the most common solid tumor affecting children. In the cerebellum, Sonic Hedgehog (Shh) drives the proliferative expansion of granule neuron progenitors (GNP). These cells are located in the external granule layer (EGL) and are the cells of origin of Shh-MB. We recently identified Norrie Disease Pseudoglioma (Ndp) as a novel downstream target of Hh signaling in the developing retina. Ndp encodes an X-linked cysteine-rich secreted protein called Norrin, which is best known for its role in angiogenesis and blood brain barrier (BBB) maintenance in the developing retina and cerebellum, respectively. Norrin mediates this effect by binding to its receptor Frizzled4 (Fzd4) and co-receptors LRP5/6 and Tpsan12 to activate the canonical, β-catenin-dependent Wnt signaling pathway in endothelial cells (ECs). We detected the expression of Ndp and all required receptors in mouse GNPs and MB samples. To investigate a potential role for Ndp in Hh-driven MB, we genetically and pharmacologically inactivated Ndp/Fzd4 signaling in Ptch+/- mice (a mouse model for human Gorlin syndrome), which dramatically increased the incidence and reduced the latency of MB. This accelerated rate of tumorigenesis was caused by an increase in the number of preneoplastic lesions (PNLs), the precursor lesions to MB, and a faster conversion of these lesions to MB. We showed that Ndp mediates this increase in tumorigenesis by signaling through endothelial cell receptor Fzd4 to alter the GNP stroma, which is characterised by 5 major alterations: 1) activated angiogenic program, 2) open BBB, 3) aberrant deposition of extracellular matrix, 4) aberrant lymphocyte recruitment and 5) reduction in meningeal lymphatic vasculature. We propose that these stromal alterations are associated with a pro-tumor microenvironment that promotes DNA damage in GNPs and leads to enhanced lesion formation and progression towards MB. This research highlights 1) an unanticipated role for Ndp/Fzd4 signaling in Shh-MB initiation and progression, 2) a role for stromal signaling in the regulation of MB development and 3) a previously undescribed role for Ndp signaling in maintaining meningeal cerebellum lymphatic vessels.

The caudal fin of zebrafish (danio rerio) is often used to study regeneration thanks to its extraordinary regenerative ability, easy access, and relative simplicity in structure. Branching morphogenesis is observed in many organs, including lungs and salivary glands in mammals, as well as the fin rays in zebrafish and is thought to follow unifying principles. An important developmental gene, sonic hedgehog a (shha), has been shown in other studies to play an essential role in the branch formation. Previous studies in our lab have shown that the transient depletion of the shha-expressing cells following laser ablation of the shha-expressing cells in the regenerating caudal fin results in a delay of fin rays branch formation. In order to study the long-term effect of ablating the shha-expressing cells, I generated a new zebrafish transgenic line (Tg)(2.4shha:CFP-NTR-ABC) to perform a conditional cell ablation using the Metronidazole/Nitroreductase (Mtz/NTR) system. Preliminary data suggest that cell ablation using the Mtz/NTR system is successful in the Tg(2.4shha:CFP-NTR-ABC) embryos. In addition, short-term ablation of the shha-expressing cells through Mtz/NTR system delays branch formation during caudal fin regeneration of the Tg(2.4shha:CFP-NTR-ABC) adult fish. Further work will involve the analysis of the effects of the long-term ablation of the shha-expressing cells and the involvement of other signaling pathways in the ray branching formation during zebrafish caudal fin regeneration. This study can provide insights into understanding of the molecular mechanisms underlying branching morphogenesis in various organs. During the course of the above project, I have observed an organ-wide response to local injury in the zebrafish caudal fin. In this study, I have shown, for the first time, an immediate organ-wide response to partial fin amputation characterized by the damage of blood vessels, nerve fibers and the activation of inflammatory response in the non-amputated tissues. I established that the adult zebrafish caudal fin serves as an excellent model for the study of the organ-wide response to local injury, and such study may provide new insights into the field of regenerative medicine in which stimulating regeneration locally may trigger responses in unintended locations.   Résumé La nageoire caudale du poisson zèbre (danio rerio) est souvent utilisée pour étudier les mécanismes de régénération à cause de son extraordinaire capacité de régénération, son accès facile, et sa relative simplicité structurale. La morphogenèse de branches est observée dans plusieurs organes incluant les poumons et les glandes salivaires chez les mammifères ainsi que les rayons des nageoires du poisson zèbre et est supposée suivre des principes communs. Un important gène de développement, sonic hedgehog a (shha), joue un rôle essentiel dans la formation des branches. Des études précédentes effectuées dans notre laboratoire ont montré que l’absence transitoire des cellules exprimant shha dans des expériences d’ablation au rayon laser induit un délai de la formation des branches dans les rayons au cours de la régénération de la nageoire caudale. Afin d’étudier les effets de l’ablation à long terme des cellules exprimant shha, j’ai fait un nouvelle lignée transgénique de poisson zèbre Tg(2.4shha:CFP-NTR-ABC) pour effectuer une ablation cellulaire conditionnelle à l’aide du système Métronidazole / Nitroréductase (Mtz/NTR). Mes données préliminaires suggèrent que l’ablation cellulaire à l’aide du système Mtz/NTR fonctionne sur les embryons Tg(2.4shha:CFP-NTR-ABC). De plus, l’ablation à court terme des cellules exprimant shha à l’aide du système Mtz/NTR induit un délai de la formation des branches au cours de la régénération des rayons la nageoire caudale des poissons adultes Tg(2.4shha:CFP-NTR-ABC). Des études supplémentaires incluront l’analyse des effets de l’ablation à long terme des cellules exprimant shha et le rôle d’autres cascades de signalisation dans la formation des branches des rayons au cours de la régénération de la nageoire caudale du poisson zèbre. Cette étude pourrait fournir des informations concernant la compréhension des mécanismes moléculaires sous-jacents à la formation de branches dans des organes variés. Au cours de l’étude décrite ci-dessus, j’ai fait l’observation d’une réponse globale de toute la nageoire caudale à une blessure locale. Dans cette étude, j’ai montré pour la première fois, une réponse immédiate et globale après amputation partielle de la nageoire. Cette réponse est caractérisée par des lésions des vaisseaux sanguins, des fibres nerveuses et par l’activation d’une réponse inflammatoire dans les tissus non-amputés. J’ai établi que la nageoire caudale du poisson zèbre adulte est un excellent modèle pour l’étude de la réponse globale d’un organe à une lésion locale. Une telle étude pourrait fournir de nouvelles informations pertinentes à la médecine régénérative qui, en visant à stimuler la régénération de façon locale, peut entraîner des réponses dans des domaines non voulus.

Le cancer bronchique non à petites cellules (CBNPC) est particulièrement chimiorésistant. Aucun marqueur robuste de chimiorésistance n'a été validé dans ce type de cancer. Nous avons cherché à décrire dans ce travail des marqueurs innovants de résistance à la chimiothérapie à base de platine dans les CBNPC. Après avoir étudié les caractéristiques cliniques et moléculaires habituelles des patients réfractaires à la chimiothérapie, nous avons étudié le rôle de la voie Sonic Hedgehog (Shh) dans le CBNPC et son impact en termes de chimiorésistance. Nous avons ainsi montré que cette voie de signalisation, connue comme liée aux cellules souches cancéreuses, est corrélée au caractère réfractaire à la chimiothérapie. L'expression de Gli2 est associée à la progression tumorale, à la survie sans progression et à la survie globale. Nous avons également démontré une corrélation entre l'activation de la voie Shh et la transition épithélio-mésenchymateuse, qui est liée à l'agressivité tumorale, le pouvoir métastasiant et la chimiorésistance. Nous avons aussi validé le rôle de la voie Shh dans la prolifération tumorale et la chimiorésistance dans un autre modèle de cancer thoracique, le mésothéliome pleural malin. Enfin, nous nous sommes intéressés à l'expression de hPAF1C (human polymerase II-associated factor 1 complex), facteur suractivé dans les cellules souches cancéreuses et lié à l'activation de la voie Shh. Nous avons montré que l'expression de hPAF1C est associée à un mauvais pronostic et à la prolifération tumorale par interaction avec c-Myc. Ces résultats soulignent le rôle important de la voie Shh dans le CBNPC en termes de chimiorésistance et d'agressivité tumorale. / Non-small cell lung cancer (NSCLC) is known to be chemoresistant. Few robust markers of chemoresistance have been validated so far in this type of cancer. We have described in this work new innovative markers of resistance to cisplatin-based chemotherapy in NSCLC. After the study of usual clinical and molecular caracteristics of patients who were refractory to chemotherapy, we have then explored the role of the Sonic Hedgehog (Shh) pathway in NSCLC and its impact in term of chemoresistance. We have shown that Shh pathway, closely linked with cancer stem cells, was correlated with the refractory property to chemotherapy. Positive Gli2 immunohistochemistry score was associated with tumor progression et progression-free survival. We have also demonstrated a correlation between Shh activation and epithelial-mesenchymal transition, known to be linked with tumor aggressiveness, metastatic ability and chemoresistance. We have then validated the great role of Shh pathway in tumor proliferation and chemoresistance in another thoracic cancer, known to be chemoresistant, the malignant pleural mesothelioma. At last, the impact of ceancer stem cells on tumor aggressiveness and prognosis has been demonstrated through the study of the expression of hPAF1C (human polymerase II-associated factor 1 complex), described as overactivated in cancer stem cells and linked to Shh pathway activation. We have shown that hPAF1C expression was associated with poor prognosis and with tumor proliferation through an interaction with c-Myc. These results underline the major role of Shh pathway and cancer stem cells in SNCLC in term of chemoresistance and tumor aggressiveness.

Indiana University-Purdue University Indianapolis (IUPUI) / Loss of the finite cochlear hair cells of the inner ear results in sensorineural deafness. Human cochlear hair cells do not regenerate, and there is no cure for deafness. Our laboratory has established a three-dimensional culture system for deriving functional sensory hair cells from human pluripotent stem cells. A major limitation of this approach is that derived hair cells exhibit a morphological and gene expression phenotype reflective of native vestibular hair cells. Previous studies have shown that establishment of localized domains of gene expression along the dorso-ventral axis of the developing otic vesicle is necessary for proper morphogenesis of both auditory and vestibular inner ear structures. Sonic hedgehog (SHH) signaling has been shown to play a key role in specification of the ventral otic vesicle and subsequent cochlear development. Here, SHH treatment was pursued as a potential strategy for inducing a patterning phenotype permissive to cochlear induction in vitro. Single-cell RNAsequencing analysis revealed that while treatment with the SHH pathway agonist Purmorphamine reduced expression of markers for the vestibular-yielding dorsal otic vesicle, upregulation of ventral otic marker genes was modest. More strikingly, the number of otic progenitors exhibiting a neuroprogenitor phenotype increased in response to Purmorphamine treatment. These results suggest that SHH pathway modulation in early-stage inner ear organoids may bias their differentiation toward a neural lineage at the expense of an epithelial lineage. The present study is the first to evaluate the patterning phenotype of human stem cell derived otic progenitors, and sheds light on the transcriptomic profile at this critical point of inner ear development. This study may also cultivate future efforts to derive cochlear cell types as well as inner ear neural cell types from human pluripotent stem cells, and contribute to the establishment of a more complete in vitro model of inner ear development. / 2021-08-21

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