The transcription factor hepatocyte nuclear factor 4 (HNF4) is an orphan member of the nuclear receptor superfamily expressed in mammals in liver, kidney, and the digestive tract. Recently, we isolated the Xenopus homolog of mammalian HNF4 and revealed that it is not only a tissue-specific transcription factor but also a maternal component of the Xenopus egg and distributed within an animal-to-vegetal gradient. We speculate that this gradient cooperates with the vegetally localized embryonic induction factor activin A to activate expression of HNF1␣, a tissue-specific transcription factor with an expression pattern overlapping that of HNF4. We have now identified a second Xenopus HNF4 gene, which is more distantly related to mammalian HNF4 than the previously isolated gene. This new gene was named HNF4␤ to distinguish it from the known HNF4 gene, which is now called HNF4␣. By reverse transcription-PCR, we detected within the 5 untranslated region of HNF4␤ two splice variants (HNF4␤2 and HNF4␤3) with additional exons, which seem to affect RNA stability. HNF4␤ is a functional transcription factor acting sequence specifically on HNF4 binding sites known for HNF4␣, but it seems to have a lower DNA binding activity and is a weaker transactivator than the ␣ isoform. Furthermore, the two factors differ with respect to tissue distribution in adult frogs: whereas HNF4␣ is expressed in liver and kidney, HNF4␤ is expressed in addition in stomach, intestine, lung, ovary, and testis. Both factors are maternal proteins and present at constant levels throughout embryogenesis. However, using reverse transcription-PCR, we found the RNA levels to change substantially: whereas HNF4␣ is expressed early during oogenesis and is absent in the egg, HNF4␤ is first detected in the latest stage of oogenesis, and transcripts are present in the egg and early cleavage stages. Furthermore, zygotic HNF4␣ transcripts appear in early gastrula and accumulate during further embryogenesis, whereas HNF4␤ mRNA transiently appears during gastrulation before it accumulates again at the tail bud stage. All of these distinct characteristics of the newly identified HNF4 protein imply that the ␣ and ␤ isoform have different functions in development and in adult tissues.A central event in early development is the establishment of different cell types from a single cell, the fertilized egg. This cell type is highly specialized, as it contains maternal components that define embryonic induction processes and the initiation of transcriptional cascades. This unique feature of the egg is especially evident in amphibians such as Xenopus laevis. In this species, no gene transcription occurs in early cleavage stages and all early events are regulated by preexisting transcripts and translational products made during oogenesis. Many of these maternal components are transcription factors that can most easily be analyzed in Xenopus (e.g., fos [13], B-myb [3], and Xrel [22]). Recently, we revealed that hepatocyte nuclear factor 4 (HNF4), previously known as a tissue-spe...

The tissue specific transcription factor HNF1 alpha (LFB1) expressed in liver, kidney, stomach and gut gets transcriptionally activated in Xenopus shortly after zygotic transcription starts. By microinjection into fertilized Xenopus eggs, a HNF1 alpha promoter fragment is activated in the middle part of developing larvae, reflecting the activation pattern of the endogenous HNF1 alpha gene. Mutational analysis of the HNF1 alpha promoter shows that HNF1 and HNF4 binding sites are essential for proper embryonic regulation. Since by injecting HNF4 mRNA into fertilized eggs the endogenous HNF1 alpha gene is activated ectopically and HNF4 is present as a maternal protein within an animal to vegetal gradient in the embryo, we assume that HNF4 initiates a transcriptional hierarchy involved in determination of different cell fates.

The transcription factor LFB1 (HNF1) was initially identified as a regulator of liver-specific gene expression in mammals. It interacts with the promoter element HP1, which is functionally conserved between mammals and amphibians, suggesting that a homologous factor, XLFB1, also exists in Xenopus laevis. To study the role of LFB1 in early development, we isolated two groups of cDNAs coding for this factor from a Xenopus liver cDNA library by using a rat LFB1 cDNA probe. A comparison of the primary structures of the Xenopus and mammalian proteins shows that the myosin-like dimerization helix, the POU-A-related domain, the homeodomain-related region, and the serine/threonine-rich activation domain are conserved between X. lwvis and mammals, suggesting that all these features typical for LFB1 are essential for function. Using monoclonal antibodies, we demonstrate that XLFB1 is present not only in the liver but also in the stomach, intestine, colon, and kidney. In an analysis of the expression of XLFB1 in the developing Xenopus embryo, XLFB1 transcripts appear at the gastrula stage. The XLFB1 protein can be identified in regions of the embryo in which the liver diverticulum, stomach, gut, and pronephros are localized. The early appearance of XLFB1 expression during embryogenesis suggests that the tissue-specific transcription factor XLFB1 is involved in the determination and/or differentiation of specific cell types during organogenesis.In recent years, a growing number of transcription factors that coordinate the activity of target genes and are involved in the determination of a particular cellular fate or phenotype have been defined. In some cases, the importance of these regulatory proteins and their corresponding target sequences in the DNA is supported by the observation that the transacting factors and their cis elements have been conserved throughout evolution. Concerning liver-specific gene expression, four different types of regulatory elements and transacting factors have been identified. The prototypes of these factors involved in liver-specific transcription are C/EBP, LFB1 (HNFl), HNF3, and HNF4 (reviewed in references 17 and 27).By analyzing the liver-specific expression of the Xenopus laevis albumin genes, we have identified the hepatocytespecific promoter element HP1, which has been conserved throughout evolution, as it is functional in mammalian cells (41). HP1 not only constitutes one of the most important regulatory elements for liver-specific expression of mammalian albumin genes (15,24,29,39) but also is involved in the expression of a group of genes specifically expressed in hepatocytes, such as the genes encoding a-fetoprotein, a1-antitrypsin, and a-and 3-fibrinogens (9, 14, 24). HP1 is recognized by the liver transcription factor LFB1 (14), also referred to as HNF1 or HNFla (9, 31). The cDNA encoding LFB1 has been cloned from rats (4, 7, 12), mice (26), and humans (2). The deduced amino acid sequences for these three mammalian proteins are highly conserved and revealed that LFB1 is the most ...