Heterozygous mutations in the human POU-homeobox TCF2 (vHNF1, HNF1␤) gene are associated with maturity-onset diabetes of the young, type 5, and abnormal urogenital tract development. Recently, pancreas atrophies have been reported in several maturity-onset diabetes of the young type 5 patients, suggesting that TCF2 is required not only for adult pancreas function but also for its normal development. Tcf2-deficient mice die before gastrulation because of defective visceral endoderm formation. To investigate the role of this factor in pancreas development, we rescued this early lethality by tetraploid aggregation. We show that TCF2 has an essential function in the first steps of pancreas development, correlated with its expression domain that demarcates the entire pancreatic buds from the earliest stages. Lack of TCF2 results in pancreas agenesis by embryonic day 13.5. At earlier stages, only a dorsal bud rudiment forms transiently and expresses the transcription factors Ipf1 and Hlxb9 but lacks the key transcription factor involved in the acquisition of a pancreatic fate, Ptf1a, as well as all endocrine precursor cells. Regional specification of the gut also is perturbed in Tcf2 ؊/؊ embryos as manifested by ectopic expression of Shh and lack of Ihh and Ipf1 in the posterior stomach and duodenum. Our results highlight the requirement of Tcf2 for ensuring both accurate expression of key regulator molecules in the stomach-duodenal epithelium and proper acquisition of the pancreatic fate. This study provides further insights into early molecular events controlling pancreas development and may contribute to the development of cell-replacement strategies for diabetes.diabetes MODY5 ͉ homeodomain transcription factor ͉ pancreas development ͉ gut regionalization ͉ tetraploid aggregation I n mammals, the pancreas emerges as ventral and dorsal evaginations from the foregut-midgut junction that subsequently fused to form a complex organ. The signaling molecule Sonic Hedgehog (SHH) demarcates a molecular boundary between the prepancreatic endoderm and adjacent stomach and duodenal anlagen and exerts an inhibitory action on pancreas development (1-3). Genetic studies in mice have identified a hierarchical regulatory network involved in pancreas morphogenesis, with significant and sequential differences between ventral and dorsal pancreas. In the mouse, the dorsal bud appears at embryonic day 9.5 (E9.5) concomitantly with the first differentiated glucagon-producing cells. The homeobox gene Ipf1(Pdx1) is expressed before and during this budding, and all pancreatic cell types derive from IPF1 ϩ progenitors (4, 5). However, in Ipf1-deficient mice, pancreas development is arrested after budding (6, 7), implying that other factors promote pancreas specification. Recently, the transcription factor Ptf1a (P48) has been shown to be essential for the acquisition of a pancreatic fate by undifferentiated ventral foregut endoderm, being required for the specification of the ventral pancreas and robust outgrowth of the dorsal bud. In its a...

We have constructed a transient expression vector containing 400 bp of rat albumin gene immediate 5′‐flanking sequences inserted 5′ to the bacterial enzyme chloramphenicol acetyl transferase (CAT). We have transfected various clones of rat hepatoma cells representing different states of expression of the liver phenotype with this vector (pALB‐cat) and also with two control vectors containing viral promoters (pSVE‐cat and pRSV‐cat), and measured activity of the bacterial enzyme CAT in cellular extracts 48 h later. The albumin flanking sequences are able to direct highly efficient CAT expression, compared with the control vectors, only in cells which express their own albumin gene: the albumin‐negative hepatoma cells are at least 100 times less efficient in expressing CAT after transfection with the pALB‐cat plasmid than are the albumin‐positive ones. An unexpected result of our study is the total inability of the rat albumin flanking sequences to direct expression in albumin‐producing mouse hepatoma cells.

Numerous studies of cell hybrids have indicated that somatic cells produce negative regulators (extinguishers) that prevent the expression of functions foreign to their own differentiation. Here, we report genetic evidence of such control. In microcell hybrids between well-differentiated rat hepatoma cells and microcells of mouse fibroblast L cells, the extinction of albumin synthesis is directly related to the presence of a single specific chromosome of the mouse fibroblast parent. The expression of several other hepatic functions is not affected. Transfection of these hybrids with a recombinant plasmid, containing a tissue-specific control element of the upstream region of the rat albumin gene linked to coding sequences of the chloramphenicol acetyltransferase gene, reveals that extinction acts on or via this cis-control element.The phenotype of somatic cells at terminal stages of differentiation is characterized by stable and heritable expression of a restricted number of genes coding for a set of differentiated functions. The expression of these genes seems to be controlled at the level of transcription, and cis-acting sequences involved in control of their tissue-specific expression have been identified (1-13).However, little information (14) is available concerning regulatory molecules able to interact with these sequences and/or with components of the transcriptional complex. Somatic cell hybridization has provided evidence for such regulators. For example, the existence of negative regulators (extinguishers) has been deduced from the selective and reciprocal extinction of differentiated functions observed after fusion ofcells of different histotypes (refs. 15 and 16; for review, see refs. 17 and 18). These factors have been thought to prevent the expression of foreign differentiated functions.Extinction is subject to gene-dosage effects (19,20) and is reversible (21,22). Reversibility (i.e., reexpression of the extinguished functions) has been correlated with the loss of chromosomes of the extinguishing parent (21-23). This latter observation indicates that the genetic basis of the extinguishing factors can be investigated. Two types of hypotheses have been proposed to account for extinction. According to the first, trans-acting extinguishers encoded by discrete and specific loci control the expression of each (or of a small group of) differentiated function (24). The independent reexpression of individual functions, in no apparent order, argues for this hypothesis. According to the second hypothesis, in hybrids, every chromosome contributes to extinction in a nonspecific manner, and the ratio between the number of chromosomes of the two parents will impose the phenotype of the hybrid cell (25). The existence of such a generalized gene-dosage effect cannot be rigorously tested in the absence of knowledge concerning putative specific extinguishers.In order to test the hypothesis of specific loci which cause extinction, hybrids were generated between Fao rat hepatoma cells and microcells derived f...

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We have analysed by in situ hybridization the expression of myf-5, the murine homologue of the human myogenic regulatory sequence myf5, during embryogenesis in the mouse. myf-5 sequences were first detected in the earliest somites (from about 8 days p.c.) in the dermomyotome, before formation of the dermatome, myotome and sclerotome. The dermomyotome is classically considered to give rise to the precursor muscle cells of body and limb skeletal muscle. myf-5-positive cells were also detected early in the visceral arches and limb buds. In this case, as in somites, myf-5 expression precedes that of the two related myogenic regulatory sequences, myogenin and MyoD1, and indeed any other skeletal muscle marker examined to date. myf-5 is not detected at any stage in developing cardiac muscle. From 11.5 days p.c., the level of myf-5 transcripts begins to decrease to become undetectable (by in situ hybridization) from 14 days p.c. Both the appearance and disappearance of myf-5 follow the anteroposterior gradient of somite formation and maturation in the embryo. The time and place of myf-5 expression are consistent with a role in the early events of myogenic differentiation, possibly during determination of the myogenic lineage.