Nodal signaling is essential for mesoderm and endoderm formation, as well as neural plate induction and establishment of left-right asymmetry. However, the mechanisms controlling expression of Nodal pathway genes in these contexts are not fully known. Previously, we showed that Cdx1b induces expression of downstream Nodal signaling factors during early endoderm formation. In this study, we show that Cdx1b also regulates epithalamic asymmetry in zebrafish embryos by modulating expression of ndr2 and lft1. We first knocked down cdx1b with translation-blocking and splicing-blocking morpholinos (MOs). Most embryos injected with translation-blocking MOs showed absent ndr2, lft1 and pitx2c expression in the left dorsal diencephalon during segmentation and pharyngula stages accompanied by aberrant parapineal migration and habenular laterality at 72 ​h post fertilization (hpf). These defects were less frequent in embryos injected with splicing-blocking MO. To confirm the morphant phenotype, we next generated both zygotic (Z)cdx1b^−/− and maternal zygotic (MZ)cdx1b^−/− mutants by CRISPR-Cas9 mutagenesis. Expression of ndr2, lft1 and pitx2c was absent in the left dorsal diencephalon of a high proportion of MZcdx1b^−/− mutants; however, aberrant dorsal diencephalic pitx2c expression patterns were observed at low frequency in Zcdx1b^−/− mutant embryos. Correspondingly, dysregulated parapineal migration and habenular laterality were also observed in MZcdx1b^−/− mutant embryos at 72 hpf. On the other hand, Kupffer’s vesicle cilia length and number, expression pattern of spaw in the lateral plate mesoderm and pitx2c in the gut as well as left-right patterning of various visceral organs were not altered in MZcdx1b^−/− mutants compared to wild-type embryos. Chromatin immunoprecipitation revealed that Cdx1b directly regulates ndr2 and lft1 expression. Furthermore, injection of cdx1b-vivo MO1 but not cdx1b-vivo 4 ​mm MO1 in the forebrain ventricle at 18 hpf significantly downregulated lft1 expression in the left dorsal diencephalon at 23–24 ​s stages. Together, our results suggest that Cdx1b regulates transcription of ndr2 and lft1 to maintain proper Nodal activity in the dorsal diencephalon and epithalamic asymmetry in zebrafish embryos.

节点信号对于中胚层和内胚层的形成以及神经板的诱导和左右不对称的建立至关重要。但是，在这些情况下控制Nodal途径基因表达的机制尚不完全清楚。以前，我们表明Cdx1b在早期内胚层形成过程中诱导下游Nodal信号因子的表达。在这项研究中，我们表明Cdx1b还通过调节ndr2和lft1的表达来调节斑马鱼胚胎中的上丘脑不对称性。我们首先用翻译阻断和剪接阻断吗啉代（MOs）敲除cdx1b。注射翻译阻滞的MOs的大多数胚胎显示缺乏ndr2，lft1和pitx2c受精（hpf）后72h分割和咽阶段中左背间脑表达异常，伴有异常的松果体移位和ha骨侧倾。这些缺陷在注射了剪接阻断型MO的胚胎中较少见。为了确认形态表型，我们接下来通过CRISPR-Cas9诱变产生了合子（Z）cdx1b ^-/-和母体合子（MZ）cdx1b ^-/-突变体。Mz cdx1b ^-/-突变体比例较高的左背间脑中没有ndr2，ltt1和pitx2c的表达。但是，背侧双脑皮坑x2c异常在Z cdx1b ^-/-突变体胚胎中低频观察到表达模式。相应地，在72hpf的MZ cdx1b ^-/-突变体胚胎中也观察到了异常调节的松果体移位和哈贝状偏侧。在另一方面，枯否囊泡纤毛长度和数量，的表达模式SPAW在侧板中胚层和pitx2c在肠道以及左右各种内脏器官的图案形成在MZ没有改变cdx1b ^{- / -}突变体相对于野生型胚胎。染色质免疫沉淀表明Cdx1b直接调节ndr2和lft1表达。此外，注入cdx1b-vivo MO1而不是cdx1b-在18 hpf时前脑室中的cdx1b -vivo 4 mm MO1显着下调了23-24岁时左背中脑的lft1表达。在一起，我们的结果表明，Cdx1b调节ndr2和lft1的转录，以在斑马鱼胚胎的后脑和上丘脑不对称中维持适当的Nodal活性。