BIOLOGICAL OVERVIEW

A Drosophila gene, Hnf4, was selected by cross-hybridization with a probe to rat HNF4 (hepatocyte nuclear factor 4), a member of the steroid hormone receptor superfamily that plays an important role in liver-specific gene expression. Drosophila Hnf4 is transcribed maternally, and disappears soon after cellularization. It reappears zygotically, first in the posterior midgut and subsequently in the anterior midgut, salivary glands and at the growing tip of the Malpighian tubules. Although early gut development occurs normally, Drosophila mutants in Hnf4 show defects in midgut, Malpighian tubule and salivary gland development. HNF4 mRNA is distributed in a different pattern from that of Forkhead, the Drosophila homolog of the vertebrate Forkhead domain protein HNF3. Whereas forkhead is involved in early gut invagination and is expressed most strongly in the ectodermal hindgut and foregut, Drosophila Hnf4 transcription is involved in gut endoderm determination and is confined to the endodermal midgut (Zhong, 1993).

With such limited information, many questions may be asked, but few answers are currently available. Most of the unanswered questions arise from work on the vertebrate homolog of Drosophila HNF4. Recent experiments show strong parallels between Drosophila and vertebrate HNF4 biology; it is these parallels that raise the yet to be answered questions.

Xenopus HNF4 is present as a maternal protein and accumulates in growing oocytes. Xenopus HNF4 protein distributes in an animal to vegetal gradient in the embryo. This distribution remains through the 64 cell stage at which time a proportion of the protein becomes transported to the nucleus. Transcripts are absent in early embryos but become detectable in the early gastrula, when zygotic transcription has started, and accumulate during further development. Xenopus HNF4 is involved in the activation of an endodermal specific transcription factor HNF1alpha, a homeoprotein (Holewa, 1996).

Activation of HNF1alpha early in Xenopus development is via an HNF4-binding site. This HNF4 binding site is identified as an activin A responsive element in the Xenopus HNF1alpha promoter. This means that activin A signaling, apparently through Tgfß type 1 and type 2 receptors, is required for HNF4 to activate the HNF1alpha promoter. One possibility for activation of Xenopus HNF4 might be its post-transcriptional modification (Weber, 1996). The activation of HNF4 by phosphorylation makes sense as tyrosine phosphorylation of HNF4 is required for DNA binding and consequently for activation of HNF4 in cell-free systems as well as in cultured mammalian cells (Ktistaki, 1995).

The existence of maternal Xenopus HNF4 and Drosophila Hnf4 raises the question of the functions these transcription factors carry out early in development. The Xenopus protein may play a role in initiating a transcriptional hierarchy involved in the determination of endoderm. What is the role of maternally coded Drosophila Hnf4 early in development, and is there any similarity to the presumed role of the Xenopus protein? Might activation of Drosophila Hnf4 be one of the early events signaling activation of the egg following fertilization? If so, does its function depend of phosphorylation, is it a target of Decapentaplegic (a TGFß family member) signaling, and what are its targets?

Murine HNF4 is expressed in the visceral endoderm as early as 4.5 days after fertilization, during implantation and well before gastrulation (Duncan, 1994). Disruption of that gene leads to cell death in the embryonic ectoderm at 6.5 days of development, at a time when these cells normally initiate gastrulation. The result is impaired gastrulation of mouse embryos. As assessed by expression of Brachyury and HNF3ß, primitive streak formation and initial differentiation of mesoderm do occur, but are delayed by about 24 hours. This work shows that expression of murine HNF4 in the visceral endoderm is essential for embryonic ectoderm survival and normal gastrulation (Chen, 1994).

What place does zygotic Drosophila Hnf4 transcription have in the hierarchy of gene activation in the endoderm? Is it directly activated by Huckebein, or is it downstream of Serpent? Serpent is responsible for the repression of forkhead in the midgut (Reuter, 1994). Does Serpent induce Hnf4, or is Hnf4 transcription independent of Serpent? Similar questions can be asked for the murine HNF4. Where does it lie in the early developmental hierarchy of the endoderm? Resolution of these questions would go a long way to resolving the origin of vertebrate endoderm. Also, what are the interactions in mouse development that result in ectodermal cell death in murine HNF4 deficient embryos? There must be a signaling pathway between endoderm and ectoderm, but the existence of such a pathway is currently undocumented.

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 666

Structural Domains

The Drosophila gene matches the mouse gene in 60 out of 66 amino acids in the zinc finger DNA binding domain, and in 140 out of 206 amino acids in the domain that specifies dimerization and ligand binding. The 12 amino acids immediately C-terminal to the zinc finger are also completely conserved. The Drosophila cDNA codes for a protein that is more than 200 amino acids longer than the rat and mouse proteins. The genomic coding structure is the same for mammalian and fly HNF4s. One exon encodes five of the eight cysteines found in the two zinc fingers; the next exon encodes the remaining three cysteines of the second zinc finger. In most steroid family members, each zinc finger (four cysteines) is encoded in a separate exon (Zhong, 1993).

date revised: 13 FEB 97 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.