Mutations in HOXA1 can lead to diseases such as Bosley–Salih–Alorainy syndrome, involving severe cardiovascular malformations. However, the role of HOXA1 in cardiac morphogenesis remains unclear. hoxa1a is a homologous gene to human HOXA1 in zebrafish. We used CRISPR to make hoxa1a-null zebrafish that exhibited multiple heart malformations. In situ hybridization and sections revealed the morphological changes in mutants: enlarged ventricle with thickened myocardium and increased trabeculae, intensified OFT and inadequate heart looping, with electrocardiography supporting these pathological changes. High-speed photography captured cardiac pumping and revealed a significant decrease in cardiac output. Furthermore, lacking hoxa1a led to posterior body abnormality that affected movement ability, corresponding with the motor development delay in patients. Upregulation of hox paralogues in hoxa1a-null fish implied a compensatory mechanism between hox genes. Accordingly, we successfully constructed a hoxa1a-null model with a cardiac disease pattern which occurred in human HOXA1-associated heart malformation. The study of hoxa1a in zebrafish can further promote the understanding of hox genes and related diseases.

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Background: Bisphenol A (BPA), an environmental endocrine disruptor, is involved in precocious puberty. Chinese Herbs for Nourishing Yin and Purging Fire (NYPF) have been effective in delaying early onset of puberty. However, the underline mechanism is unknown. As epigenetics plays an important role in advanced puberty caused by extraneous factors. We intend to investigate the epigenetic impact of BPA and NYPF herbs on Kiss1 gene, a vital gene driving puberty onset, in hypothalamic neuron cells.Methods: GT1-7 cells derived from GnRH neurons were used in the study. A series of concentration of BPA and serum containing NYPF herbs were prepared in advance. GT1-7 cells were administrated with BPA or vehicle first, followed by NYPF herbs or normal saline (NS) for 24 hours. Afterward, quantification of Kiss1 expression was performed through Realtime-PCR and Weston-blot methods. In addition, epigenetic modifications at Kiss1 promoter were examined through Bisulphite Sequencing (BSP) and Chromatin immunoprecipitation combined real-time PCR（Chip-qPCR）assays.Results: Compared to control group, GT1-7 cells with BPA 10mg/l administration presented lower methylation status (61.25% Vs. 76.56%) concomitantly with enhanced abundance of mixed-lineage leukemia 1(MLL1) (1.05%±0.15 Vs. 0.27%±0.01) and Tri-Methyl-Histone 3 at lysine 4 (H3K4me3) (0.77%±0.51 Vs. 0.02%±0.04) at Kiss1 promoter, leading to elevated expression of Kiss1. On the contrary, TCM for NYPF promoted increased DNA methylation (83.75% Vs. 72.5%), accompanied with less deposition of MLL1/H3K4me3 (MLL1: 0.14%±0.02 Vs. 0.97%±0.09; H3K4me3: 0.01%±0.00 Vs. 0.57%±0.30) at Kiss1 promoter than BPA 10mg/l group, which resulted in markedly inhibition of Kiss1 expression. Conclusions: BPA may induce elevated expression of Kiss1 through influencing DNA methylation and histone modification at Kiss1 promoter. And NYPF herbs reverse the adverse effects of BPA on Kiss1 gene through epigenetic modification as well.