Functional analyses of Xhairy2 in the formation of cranial sensory organs in Xenopus laevis embryos: Requirements for lens formation

村戸, 康人

Vertebrate head is a complex structure that consists of various kinds of tissues and organs. Among them, cranial sensory organs and ganglions are derived from special cell groups, called
placodes. Based on the facts that they differentiated into multiple cell types and that are only found in vertebrate lineages, placodes are interesting subjects of studies from the perspectives of both ontogeny and phylogeny. Lines of studies have suggested that all placodes are derived from the pre-placodal ectoderm (PPE) that is boundary between future epidermis region and future neural region. A Xenopus gene Xhairy2 is, to our knowledge, the earliest gene that shows clear expression in PPE. The present study aimed at elucidating a function(s) of Xhairy2 in the formation of placodes by examining formation of cranial sensory organs. Morphological analyses of Xhairy2 morphants revealed that ocular lens was severely malformed though retinal structure looked normal. Vertebrate eyes are divided into retina and lens in terms of their origins: retina is formed from a part of diencephalons, while lens is developed from placode. Detailed marker gene analyses of eye in Xhairy2 morphants showed that the expression of all lens marker genes tested was reduced or abolished, while that of retinal marker genes was not affected. These results indicated that Xhairy2
was specifically required for lens development. Ectopic expression of p27xic1 by means of Xhairy2 knockdown was identified as a first trigger of lens malformation. First, apoptosis was induced in
Xhairy2 morphants, which is a known marker of ectopic expression of p27xic1. Second, overexpression of p27xic1 mimicked phenotypes of Xhairy2 morphants. Finally, the phenotypes of Xhairy2 knockdown were partially rescued by simultaneous knockdown of p27xic1. Since p27xic1 is a cell-cycle inhibitor, it was assumed that the number of lens precursor cells was drastically reduced. To test this assumption, cell proliferation within regions of lens induction was examined by means of BrdU incorporation analyses in Xhairy2 morphants. However, no significant changes were observed at the onset of lens induction. Furthermore, lens induction occurred normally in the embryos treated with drugs that directly inhibit cell cycle. Collectively, the results suggested that Xhairy2 repressed
the differentiation inducing activity, another activity of p27xic1. Placodes start differentiation long after the first fate decision. Therefore, it is quite important how they maintain stem-cell-like undifferentiated states. As Xhairy2 did not seem to be a master regulator of cascade of lens transcription factors and was shown to be involved in inhibition of differentiation via repression of p27xic1 expression, it was suggested that the undifferentiated states was actively maintained by early PPE expression of Xhairy2. The cascade of differentiation beginning with a signal input would normally work only when this kind of fundamental cell state, competence, exists.


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