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Thesis (Ph. D.) -- California Institute of Technology, 2010. / Title from home page (viewed 03/25/2010). Advisor and committee chair names found in the thesis' metadata record in the digital repository. Includes bibliographical references.

Thesis (M.S.)--Brigham Young University. Dept. of Physiology and Developmental Biology, 2006. / Includes bibliographical references (p. 49-52).

Thesis (Ph. D.)--Brigham Young University. Dept. of Physiology and Developmental Biology, 2006. / Includes bibliographical references (p. 109-113).

Thesis (Ph. D.)--Brigham Young University. Dept. of Physiology and Developmental Biology, 2006. / Includes bibliographical references (p. 108-115).

Le poisson Astyanax mexicanus présente, au sein de la même espèce, plusieurs populations de poissons de rivières (SF) et de poissons de grottes aveugles (CF). Chez les poisons cavernicoles aveugles, les yeux se développent presque normalement pendant l’embryogenèse. Mais 24 heures après la fécondation (hpf), quand l’embryon éclot, le cristallin entre en apoptose, ce qui déclenche la dégénérescence progressive de l’œil entier. Mon projet de thèse visait à comprendre le mécanisme conduisant à l’apoptose du cristallin, jusqu’alors totalement incompris, en partant du postulat selon lequel le défaut devait avoir lieu pendant les stades précoces du développement du cristallin. Le cristallin se développe à partir d’une placode, un épaississement de l’ectoderme au stade neurula. Toutes les placodes, qui donnent naissance à des organes des sens de la tête, sont issues du champ panplacodal, situé à la bordure de la plaque neurale antérieure à 10 hpf. Nous avons comparé la régionalisation de ce champ chez les deux morphes, par hybridations in situ de gènes marqueurs des différentes placodes. Chez le CF, le territoire présomptif du cristallin est réduit à 10 hpf, et le cristallin est plus petit à tous les stades étudiés. D’autre part, la placode olfactive est étendue, et donne naissance à un épithélium olfactif plus large chez le CF. Les modifications de taille de ces deux placodes pourraient être le résultat évolutif d’un « trade-off » entre ces deux composantes sensorielles. La régionalisation modifiée du champ panplacodal chez le CF est due au moins partiellement à des différences spatiales et temporelles d’expression des molécules de signalisation Shh, Fgf, et peut-être Bmp4.Nous avons pensé que la petite taille du cristallin pouvait être la cause directe de son entrée en apoptose, par un défaut d’effet de communauté. Nous avons réalisé une ablation laser partielle des cellules précurseurs du cristallin à 12-14 hpf chez l’embryon SF, mimant ainsi la taille du cristallin CF. L’apoptose dans le petit cristallin des larves SF à 60 hpf n’a pas été augmentée, ce qui montre que la petite taille n’est pas suffisante pour induire l’apoptose.L’apoptose du cristallin pourrait aussi provenir de défauts de morphogenèse ou d’un problème de lignage cellulaire. Nous utilisons donc l’imagerie biphoton in vivo sur des embryons SF et CF, de 10 à 24 hpf, préalablement injectés au stade une cellule avec des ARNm de H2B-mCherry et Ras-GFP pour marquer les noyaux et les membranes. Les premiers résultats sur les poissons de surface montrent que nous pouvons suivre à rebours les cellules du cristallin de la fin du film jusqu’au champ panplacodal, et étudier la morphogenèse et les divisions.La différenciation du cristallin est également affectée chez le CF : au moins 5 cristallines, qui sont des protéines structurales du cristallin, ne sont pas exprimées correctement chez le CF, d’après des hybridations in situ et des qPCR. Cependant, le rôle fonctionnel de deux de ces modifications d’expression a été testé, et individuellement, elles n’expliquent pas le phénotype apoptotique. Nous émettons l’hypothèse qu’une combinaison de défauts d’expression de plusieurs cristallines serait à l’origine de l’apoptose du cristallin CF. Enfin, et plus largement, les forces évolutives qui ont conduit à la perte de l’œil chez Astyanax mexicanus ne sont pas encore comprises. Par une étude d’évolution moléculaire à l’échelle du transcriptome nous avons identifié des mutations fixées entre SF et CF, et avons pu mettre en évidence une accumulation de mutations dans des « gènes d’yeux » chez les CF. Cela suggère un relâchement de la pression de sélection sur ces gènes, peut-être devenus inutiles dans l’obscurité. De même, les séquences des cristallines de CF paraissent accumuler des mutations fixées à un taux élevé vu leur bas niveau de polymorphisme. / The fish Astyanax mexicanus presents, within the same species, several populations of river-dwelling surface fish (SF) and blind cave-living fish (CF). In blind cavefish, the eyes first develop almost normally during embryogenesis. But 24 hours after fertilization (hpf), when the embryo hatches, the lens enters apoptosis, which triggers the progressive degeneration of the entire eye. My thesis project aimed at understanding the mechanism leading to lens apoptosis, which was so far unknown. We reasoned that the defect(s) should take place during the early stages of lens development. The lens develops from a placode, a thickening of the ectoderm at the neurula stage. All placodes, giving rise to sense organs of the head, originate from the “panplacodal” field, located at the border of the anterior neural plate at 10 hpf. We compared the patterning of the panplacodal field in the 2 morphs, using in situ hybridizations for placodal marker genes. In CF, the lens placode territory is reduced at 10 hpf, and the lens is smaller at all stages examined. Conversely, the olfactory placode is enlarged, and gives rise to a bigger olfactory epithelium in CF. The modifications in size of these two placodes could result evolutionarily from a trade-off between these two sensory components. Developmentally, the modified patterning of the panplacodal field in CF is at least partly due to the spatial and temporal differences in the expression of Shh and Fgf (and perhaps Bmp4) signaling molecules.We hypothesized that the small size of the lens could be the direct cause of its apoptosis, through a lack of community effect. We performed partial laser ablation of lens precursor cells at 12-14hpf in surface fish (thereby mimicking the CF lens size). Apoptosis in the resulting small lens of SF larvae at 60hpf was not enhanced, showing that small size is not sufficient to induce apoptosis. Lens apoptosis could also result from morphogenesis defects or from a problem in cell lineage. We are performing two-photon live imaging, from 10 to 24 hpf, of SF and CF embryos previously injected at the one cell stage with H2B-mCherry and Ras-GFP mRNAs to label nuclei and membranes. First results on surface fish show that we can back-track lens cells to the panplacodal field, and follow morphogenesis and divisions. Lens differentiation is also affected in cavefish: at least 5 crystallins, which are lens structural components, are not expressed correctly in CF, based on in situ hybridization and qPCR data. However the functional role of two of these expression modifications / losses was tested and, individually, they don’t seem to explain the apoptosis phenotype. We propose that a combination of several crystallins expression defects would explain CF lens apoptosis.Finally, and more globally, evolutionary forces that led to eye loss in Astyanax mexicanus are not yet understood. Through a transcriptome-wide molecular evolution approach, we identified fixed mutations in transcripts between SF and CF, and we could show an accumulation of mutations in “eye genes” in CF. This suggests that the selection is relaxed on these genes, that have maybe become useless in the dark. Similarly, CF crystallin sequences seem to accumulate fixed mutations at a high rate, considering their low polymorphism level.

The vertebrate nervous system is extremely complex and contains a wide diversity of cell types. The formation of a functional nervous system requires the differential specification of progenitor cells at the right time and place. The generation of many different types of neurons along the rostro-caudal axis of the CNS begins with the initial specification of a few progenitor domains. This initial coarse pattern is refined by so-called secondary organizers arising at boundaries between these domains. The Isthmic Organizer (IsO) is a secondary organizer located at the boundary between the midbrain and the hindbrain. Although the function and maintenance of the IsO are well understood, the processes underlying its initial specification have remained elusive. In the present work we provide evidence that convergent Wnt and FGF signals initiate the specification of the IsO during late gastrulation as part of the neural caudalization process. The initial step in the generation of the nervous system is the division of the embryonic ectoderm into three cell populations: neural cells giving rise to the CNS, neural plate border cells giving rise to the peripheral nervous system, and epidermal cells giving rise to the outer layer of the skin. While the choice between neural and epidermal fate has been well studied, the mechanism by which neural plate border cells are generated is less well understood. At rostral levels of the neuraxis, the neural plate border gives rise to the olfactory and lens placodes, thickenings of the surface ectoderm from which sensory organs are derived. More caudally, the neural plate border generates neural crest cells, a transient population that migrates extensively and contributes to neurons and glia of the peripheral nervous system. How the early patterning of the central and peripheral nervous systems are coordinated has remained poorly understood. Here we show that the generation of neural plate border cells is initiated at the late blastula stage and involves two phases. During the first phase, neural plate border cells are exposed to Wnt signals in the absence of BMP signals. Simultaneous exposure to Wnt and BMP signals at this early stage leads to epidermal induction. Wnt signals induce expression of Bmp4, thereby regulating the sequential exposure of cells to Wnt and BMP signals. During the second phase, at the late gastrula stage, BMP signals play an instructive role to specify neural plate border cells of either placodal or neural crest character depending on the status of Wnt signaling. At this stage, Wnt signals promote caudal character simultaneously in the neural plate border and in the neural ectoderm. Thus, the choice between epidermal and neural plate border specification is mediated by an interplay of Wnt and BMP signals that represents a novel mechanism involving temporal control of BMP activity by Wnt signals. Moreover, the early development of the central and peripheral nervous systems are coordinated by simultaneous caudalization by Wnt signals.

Transcription factors of the helix-loop-helix (HLH) gene family are widespread in the animal kingdom. Among them, members of HLH subfamilies such as ASCL, Neurogenin, NeuroD, COE, Atonal, Oligo, NSCL, Hairy/E(spl) and Hey (here referred to as neural HLH genes) have been shown to be fundamental for the development of the nervous system. They are expressed at different time periods of neuronal differentiation, from the specification of ectoderm towards a neural lineage, to the ultimate differentiation of neurons. Few HLH genes have been identified in the lamprey; however, considering the wide diversity of HLH gene subfamilies in metazoans, including vertebrates, it is very likely that lampreys possess a large repertoire of HLH genes in their genome. In the present study, the identification of several HLH genes in the lamprey genome, as well as the isolation and expression of different lamprey neural HLH genes is reported. As expected, a wide repertoire of HLH genes was identified in the sea lamprey (Petromyzon marinus) genome. On the other hand, the identification and expression analysis of different neural HLH genes of the ASCL, Neurogenin, COE and Hairy/E(spl) in the brook lamprey Lampetra planeri showed an overall conservation with other vertebrates, both at the sequence and expression pattern levels. In addition, novel features of the lamprey nervous system are revealed, such as the identification of possible new sensory cranial placodes in pharyngeal arches. Furthermore, these genes can serve as molecular markers for different cranial placodes and dorsal root ganglia (DRG), and their expression also highlights the presence of a ventricular zone in the brain and spinal cord, along with a complementary marginal zone. Finally, with the use of a Notch pathway inhibitor in developing L. planeri embryos, the regulation of expression of the isolated genes by the Notch signaling pathway was shown to be generally conserved between lampreys and gnathostomes in the spinal cord. This functional study also revealed that the lamprey spinal cord likely presents an independent developmental programme from the brain. All together, the present study shows that the analysis of neural HLH genes represents an excellent tool to understand the lamprey nervous system.