Sp7/osterix positively regulates dlx2b and bglap to affect tooth development and bone mineralization in zebrafish larvae

Osterix (or Sp7) is an important transcription factor that promotes osteoblast differentiation by modulating the expression ofa range of target genes. Although many studies have focused on Osterix/Sp7 regulatory mechanisms, the detailed functionshave not been fully elucidated. Toward this end, in this study, we used CRISPR/Cas9 technology to knock out the zebrafishsp7 gene, and then analyzed its phenotype and biological function. Two knockout sp7 mutant lines were successfullyobtained. The bone mineralization level was significantly reduced in the zebrafish sp7-/- homozygote, resulting inabnormal tooth development in the larvae. Quantitative real-time polymerase chain reaction showed that loss of sp7 led todown-regulated expression of the dlx2b and bglap genes related to tooth development and bone mineralization, respectively. Moreover, cell transfection experiments demonstrated that Sp7 directly regulates the expression of dlx2b and bglapthrough Sp7-binding sites on the promoter regions of these two genes. Overall, this study provides new insight into the roleof Sp7 in bone mineralization and tooth development.

Prospecting molecular markers to distinguish Cichla kelberi, C. monoculus and C. piquiti / Prospecção de marcadores moleculares para distinção de Cichla kelberi, Cichla monoculus e Cichla piquiti

Peacock bass, a fish of the genus Cichla, is an exotic species from the upper river Paraná floodplain in which the species Cichla kelberi and C. piquiti have been confirmed, coupled to the specie C. monoculus upstream in the Capivara and Taquaruçu dams. The introduction of this genus has caused negative impacts on the diversity of native species. Current research prospects DNA sequences capable of distinguishing the three species and provide molecular data for the taxonomic characterization of the species in the upper Paraná River basin. Sequencing of nuclear (tmo4c4, dlx2 and bmp4) and mitochondrial (cox1, cytb) loci were done from fish of the three species of the genus Cichla reported in the literature of the upper Paraná River basin. Sequence analysis provided molecular differentiation for the species through the usage of loci cytb, dlx2 and cox1. Since the latter only distinguished C. piquiti from the other Cichla species, the loci bmp4 and tmo4c4 were not adequate to accomplish our aim.

O tucunaré é um peixe pertencente ao gênero Cichla, sendo exótico na planície de inundação do alto rio Paraná, onde foi confirmada a presença das espécies Cichla kelberi e C. piquiti e, logo a montante, C. monoculus, nas represas de Capivara e Taquaruçu. A introdução deste gênero tem ocasionado impacto negativo à diversidade de espécies nativas. Visando providenciar dados moleculares para auxiliar na caracterização taxonômica das espécies deste gênero na bacia do alto rio Paraná, os objetivos deste trabalho foram prospectar sequências de DNA capazes de distinguir as três espécies. Procedeu-se o sequenciamento de loci nucleares (tmo4c4, dlx2 e bmp4) e mitocondriais (cox1, cytb) de peixes das três espécies do gênero Cichla relatadas na literatura da bacia do alto rio Paraná. As análises das sequências possibilitaram a diferenciação molecular para estas espécies pela utilização dos loci cytb, dlx2 e cox1; este último, somente para distinguir C. piquiti das demais espécies de Cichla. A utilização dos loci bmp4 e tmo4c4 não foi adequada para este propósito.