Hhex and Prox1a synergistically dictate the hepatoblast to hepatocyte differentiation in zebrafish.

The specification of endoderm cells to prospective hepatoblasts is the starting point for hepatogenesis. However, how a prospective hepatoblast gains the hepatic fate remains elusive. Previous studies have shown that loss-of-function of either hhex or prox1a alone causes a small liver phenotype but without abolishing the hepatocyte differentiation, suggesting that absence of either Hhex or Prox1a alone is not sufficient to block the hepatoblast differentiation. Here, via genetic studies of the zebrafish two single (hhex-/- and prox1a-/-) and one double (hhex-/-prox1a-/-) mutants, we show that simultaneous loss-of-function of the hhex and prox1a two genes does not block the endoderm cells to gain the hepatoblast potency but abolishes the hepatic differentiation from the prospective hepatoblast. Consequently, the hhex-/-prox1a-/- double mutant displays a liverless phenotype that cannot be rescued by the injection of bmp2a mRNA. Taken together, we provide strong evidences showing that Hhex teams with Prox1a to act as a master control of the differentiation of the prospective hepatoblasts towards hepatocytes.

Cdx1b protects intestinal cell fate by repressing signaling networks for liver specification.

In mammals, the expression of the homeobox family member Cdx2/CDX2 is restricted within the intestine. Conditional ablation of the mouse Cdx2 in the endodermal cells causes a homeotic transformation of the intestine towards the esophagus or gastric fate. In this report, we show that null mutants of zebrafish cdx1b, encoding the counterpart of mammalian CDX2, could survive more than 10 days post fertilization, a stage when the zebrafish digestive system has been well developed. Through RNA sequencing (RNA-seq) and single-cell sequencing (scRNA-seq) of the dissected intestine from the mutant embryos, we demonstrate that the loss-of-function of the zebrafish cdx1b yields hepatocyte-like intestinal cells, a phenotype never observed in the mouse model. Further RNA-seq data analysis, and genetic double mutants and signaling inhibitor studies reveal that Cdx1b functions to guard the intestinal fate by repressing, directly or indirectly, a range of transcriptional factors and signaling pathways for liver specification. Finally, we demonstrate that heat shock-induced overexpression of cdx1b in a transgenic fish abolishes the liver formation. Therefore, we demonstrate that Cdx1b is a key repressor of hepatic fate during the intestine specification in zebrafish.

Unraveling Differential Transcriptomes and Cell Types in Zebrafish Larvae Intestine and Liver.

The zebrafish intestine and liver, as in other vertebrates, are derived from the endoderm. Great effort has been devoted to deciphering the molecular mechanisms controlling the specification and development of the zebrafish intestine and liver; however, genome-wide comparison of the transcriptomes between these two organs at the larval stage remains unexplored. There is a lack of extensive identification of feature genes marking specific cell types in the zebrafish intestine and liver at 5 days post-fertilization, when the larval fish starts food intake. In this report, through RNA sequencing and single-cell RNA sequencing of intestines and livers separately dissected from wild-type zebrafish larvae at 5 days post-fertilization, together with the experimental validation of 47 genes through RNA whole-mount in situ hybridization, we identified not only distinctive transcriptomes for the larval intestine and liver, but also a considerable number of feature genes for marking the intestinal bulb, mid-intestine and hindgut, and for marking hepatocytes and cholangiocytes. Meanwhile, we identified 135 intestine- and 97 liver-enriched transcription factor genes in zebrafish larvae at 5 days post-fertilization. Our findings provide rich molecular and cellular resources for studying cell patterning and specification during the early development of the zebrafish intestine and liver.