We have generated a mouse bearing a null allele of the gene encoding basic helix-loop-helix (bHLH) protein p48, the cell-specific DNA-binding subunit of hetero-oligomeric transcription factor PTF1 that directs the expression of genes in the exocrine pancreas. The null mutation, which establishes a lethal condition shortly after birth, leads to a complete absence of exocrine pancreatic tissue and its specific products, indicating that p48 is required for differentiation and/or proliferation of the exocrine cell lineage. p48 is so far the only developmental regulator known to be required exclusively for committing cells to an exocrine fate. The hormone secreting cells of all four endocrine lineages are present in the mesentery that normally harbors the pancreatic organ until day 16 of gestation. Toward the end of embryonic life, cells expressing endocrine functions are no longer detected at their original location but are now found to colonize the spleen, where they persist in a functional state until postnatal death of the organism occurs. These findings suggest that the presence of the exocrine pancreas is required for the correct spatial assembly of the endocrine pancreas and that, in its absence, endocrine cells are directed by default to the spleen, a site that, in some reptiles, harbors part of this particular cellular compartment.[Key Words: Genetics; gene inactivation; transcription factor; bHLH protein; morphogenesis; pancreas; mouse]Received June 4, 1998; revised version accepted September 30, 1998. The mammalian pancreas develops by fusion of dorsal and ventral primordia that originate from an evagination of the foregut early during development. Both the endocrine and exocrine portions of the gland differentiate from a common pool of endodermal progenitor cells (Pictet and Rutter 1972;Le Douarin 1988). The exocrine acinar cells secrete digestive enzymes into the gastrointestinal tract, while the four types of endocrine islet cells are specialized for the synthesis of different hormones. Islet cell-specific differentiation has been studied extensively in rodents and found to require the action of genes encoding homeotic proteins Pdx-1 and Isl-1. Pdx-1 (previously termed Ipf-1), the ortholog of the XlHbox8 protein of the frog (Wright et al. 1988), is transiently expressed in all pancreatic cells as well as in the epithelial layer of the duodenal mucosa during embryogenesis (Leonard et al. 1993;Ohlsson et al. 1993;Jonsson et al. 1994;Miller et al. 1994). As development proceeds, Pdx-1 becomes progressively confined to endocrine ␤ cells, where it plays a critical role in the transcriptional regulation of the insulin gene (Ohlsson 1993;Guz 1995). Mice carrying a null allele of the gene encoding this protein do not form a pancreatic gland even though the primordia are formed and the dorsal organ undergoes some limited proliferation (Jonsson 1994;Offield 1996). In addition, the duodenum displays a local absence of epithelial lining (Offield 1996). These observations support a model by which Pdx-1 is expressed ...

Sleep is regulated by a homeostatic process that determines its need and by a circadian process that determines its timing. By using sleep deprivation and transcriptome profiling in inbred mouse strains, we show that genetic background affects susceptibility to sleep loss at the transcriptional level in a tissue-dependent manner. In the brain, Homer1a expression best reflects the response to sleep loss. Timecourse gene expression analysis suggests that 2,032 brain transcripts are under circadian control. However, only 391 remain rhythmic when mice are sleep-deprived at four time points around the clock, suggesting that most diurnal changes in gene transcription are, in fact, sleep-wake-dependent. By generating a transgenic mouse line, we show that in Homer1-expressing cells specifically, apart from Homer1a, three other activity-induced genes (Ptgs2, Jph3, and Nptx2) are overexpressed after sleep loss. All four genes play a role in recovery from glutamate-induced neuronal hyperactivity. The consistent activation of Homer1a suggests a role for sleep in intracellular calcium homeostasis for protecting and recovering from the neuronal activation imposed by wakefulness.homeostasis ͉ microarray ͉ mRNA tagging ͉ sleep deprivation ͉ sleep function T wo main processes regulate sleep. A homeostatic process regulates sleep need and intensity according to the time spent awake or asleep. A circadian process regulates the appropriate timing of sleep and wakefulness across the 24-h day. A highly reliable index of the homeostatic process is provided by the amplitude and prevalence of delta (1-to 4-Hz) oscillations in the electroencephalogram (EEG) of nonrapid eye movement (NREM) sleep (hereafter, ''delta power''). Delta power is high at sleep onset and decreases during sleep, in parallel with sleep depth. Sleep deprivations and naps induce a predictable increase or decrease, respectively, in delta power during subsequent sleep. The interaction between homeostatic and circadian processes is mathematically described in the two-process model of sleep regulation, which provides a framework for prediction and interpretation of a large body of experimental data (1).Among hypotheses concerning the physiological function of waking-induced changes in sleep, the most compelling suggests that sleep plays a key role in synaptic plasticity (2, 3). More specifically, EEG delta power during NREM sleep has been shown to play a critical role in learning-induced plasticity (4-6). In general, the prediction is that local neural activation due to specific behavioral (cognitive) demands imposes a burden on the brain which necessitates sleep and which is reflected by the EEG delta power.On the basis of mathematical modeling and experimental data, we have shown that sleep need, as indexed by the EEG delta power, is under genetic control (7), which is of direct relevance for explaining the interindividual vulnerability to sleep loss in human subjects (8, 9). However, deciphering the molecular bases of sleep need is rendered difficult because the contr...

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We report the isolation of cDNA for the p48 DNA‐binding subunit of the heterooligomeric transcription factor PTF1. A sequence analysis of the cDNA demonstrates that p48 is a new member of the family of basic helix‐loop‐helix (bHLH) transcription factors. The p48 bHLH domain shows striking amino acid sequence similarity with the bHLH domain of proteins that act as developmental regulators, including the twist gene product, myogenic factors and proteins involved in hematopoietic differentiation. We show that reduced p48 synthesis correlates with a diminished expression of genes encoding exocrine pancreas‐specific functions. The synthesis of p48 mRNAs, and therefore also the protein, is restricted to cells of the exocrine pancreas in the adult and to the pancreatic primordium in the embryo. Thus the pancreas‐specific DNA‐binding activity of PTF1 originates from the synthesis of at least one cell‐specific component rather than from a cell‐specific assembly of more widely distributed proteins.

Footprint analysis of the 5'-flanking regions of the oa-amylase 2, elastase 2, and trypsina genes, which are expressed in the acinar pancreas, showed multiple sites of protein-DNA interaction for each gene. Competition experiments demonstrated that a region from each 5'-flanking region interacted with the same cell-specific DNA-binding activity. We show by in vitro binding assays that this DNA-binding activity also recognizes a sequence within the 5'-flanking regions of elastase 1, chymotrypsinogen B, carboxypeptidase A, and trypsind genes. Methylation interference and protection studies showed that the DNA-binding activity recognized a bipartite motif, the subelements of which were separated by integral helical turns of DNA. The &-amylase 2 cognate sequence was found to enhance in vivo transcription of its own promoter in a cell-specific manner, which identified the DNA-binding activity as a transcription factor (PTF 1). The observation that PTF 1 bound to DNA sequences that have been defined as transcriptional enhancers by others suggests that this factor is involved in the coordinate expression of genes transcribed in the acinar pancreas.The regulation of transcription in eucaryotes is mediated by protein components that interact with specific DNA sequences and thereby modify the rate at which RNA polymerase initiates at adjacent promoter sites. A considerable number of cis-acting DNA elements and trans-acting protein factors have now been identified in viral and cellular genes (for reviews, see references 10 and 14). The prevailing view is that regulation of transcription from a particular promoter involves the concerted activity of multiple protein components within the transcription complex. The details of how transcription factors interact with each other to form an active transcription complex are still not understood. The apparent complexity of these interactions may be dictated by the need of the cell to modulate the promoter efficiency of many of its genes in a temporal manner during development, cell differentiation, and the cell cycle or in adaptation to different physiological stimuli.Little is known about the regulatory factors involved in the expression of genes encoding the specialized functions of a cell, since elucidation of a general principle is hampered by the fact that cell-specific expression of eucaryotic genes is regulated in a modular way. For instance, expression of the genes encoding liver-specific functions depends on the concerted activity of several factors that bind to different promoter elements (2,8,12). The activities of some of these factors are apparently restricted to hepatocytes (18).We have studied a set of genes that are expressed in acinar pancreatic cells. Expression of the genes for o-amylase 2, elastase 2, and trypsina has been previously shown to be regulated at the transcriptional level by means of strong promoters (6,23). In this paper, we present evidence that the same cell-specific transcription factor binds in vitro to conserved DNA motifs that are located in ...