Changes in the frequency of specific transcripts during development of the pancreas.

The sequence complexity and frequency distribution of adult rat pancreas polyadenylated RNAs and the changes in these pancreas transcripts during development have been analyzed by means of complementary DNA-RNA hybridization analysis. The following results were obtained. (a) An abundant set of pancreas polyadenylate# RNAs hybridizes with 90% of pancreas cDNA (the copy of aduit rat pancreas polyadenylated RNA), comprises ahout 2%) of total pancreas RNA, has a sequence complexity of about 2 x 10’ nucleotides, and is largely tissue specific. These and other data imply that these RNAs code primarily for pancreas-specific secretory prateins. (b) A less abundant set of pancreas RNAs hybridizes with HTC cell cl)NA (the copy of HTC cell polyadenylated RNA), comprises about 0.03% of total pancreas RNA, has a sequence complexity of at least 3 x lo” nucleotides, and is more frequent in rapidly proliferating tissues (HTC cells and embryonic pancreas) than in the adult pancreas. (c) In contrast to the pancreas, the relatively undifferentiated HTC cell does not contain a predominant set of polyadenylated RNAs of low complexity. (d) Moderate concentrations of RNAs complementary to pancreas cl)NA are present in pancreatic rudiments at 14 days of gestation when levels of pancreas-specific proteins are low. Between 14 and 20 days of gestation, the concentration of these RNAs increases several hundred-fold in the embryonic pancreas, in parallel with the increased rate of synthesis of pancreas-specific proteins. (e) Pancreas cL)NA hybridizes in situ primarily to the regions of acinar cells actively synthesizing secretory proteins. The very favorable signalto-noise ratio of the technique indicates that it will be useful for detecting low amounts of pancreas-specific RNAs early in development.

The sequence complexity and frequency distribution of adult rat pancreas polyadenylated RNAs and the changes in these pancreas transcripts during development have been analyzed by means of complementary DNA-RNA hybridization analysis. The following results were obtained. (a) An abundant set of pancreas polyadenylate# RNAs hybridizes with 90% of pancreas cDNA (the copy of aduit rat pancreas polyadenylated RNA), comprises ahout 2%) of total pancreas RNA, has a sequence complexity of about 2 x 10' nucleotides, and is largely tissue specific. These and other data imply that these RNAs code primarily for pancreas-specific secretory prateins.
(b) A less abundant set of pancreas RNAs hybridizes with HTC cell cl)NA (the copy of HTC cell polyadenylated RNA), comprises about 0.03% of total pancreas RNA, has a sequence complexity of at least 3 x lo" nucleotides, and is more frequent in rapidly proliferating tissues (HTC cells and embryonic pancreas) than in the adult pancreas. (c) In contrast to the pancreas, the relatively undifferentiated HTC cell does not contain a predominant set of polyadenylated RNAs of low complexity. (d) Moderate concentrations of RNAs complementary to pancreas cl)NA are present in pancreatic rudiments at 14 days of gestation when levels of pancreas-specific proteins are low. Between 14 and 20 days of gestation, the concentration of these RNAs increases several hundred-fold in the embryonic pancreas, in parallel with the increased rate of synthesis of pancreas-specific proteins. (e) Pancreas cL)NA hybridizes in situ primarily to the regions of acinar cells actively synthesizing secretory proteins. The very favorable signalto-noise ratio of the technique indicates that it will be useful for detecting low amounts of pancreas-specific RNAs early in development.  3.2 x lo-' 5.4 x 10" 2 27 1.6 x 10" 1.7 x 10-t 2.9 x 10" 6.9 x 10" 3 24 4.1 x 10' 3.9 x 100 6.6 x 10" n Derived from computer analysis (see "Materials and Methods"). ' (Rot, 2 observed) x (fraction of polyadenylated RNA in oligo(dT)-bound RNA) x (fraction of cDNA hybridized).
' (tR,,t, r corrected)/(R,,t,,, globin)) x (complexity globin) = ((R,,t,,g correctedU(l.1 x lo-" mol s liter-')) x (1860 nucleotides  Although these calculations are rather inexact due to the complex shapes of the curves, the latter value is clearly much less than the percentage of total pancreas RNA complementary to pancreas cDNA (about 2%, see above). If it is assumed that the pancreatic RNA species complementary to HTC cell cDNA are polyadenylated, they maximally comprise about 2% of pancreas polyadenylated RNA.

Cellular Distribution of Abundant Pancreas Polyadenylated RNAs
The distribution of specific transcripts among different pancreatic cell types was examined by means of in situ hybridization.
Pancreas cDNA was incubated with fixed tissue sections of adult pancreas and hybrid structures visualized by autoradiography (Fig. 4). The acinar tissue is very heavily labeled, indicating that the cDNA hybridized extensively in situ. In contrast, the lumen of a blood vessel (b) is unlabeled and the endocrine cells of the islets of Langerhans (i) and connective tissue cells (c) are very weakly labeled. Fig. 5 shows a higher magnification of the autoradiograph. The basal and perinuclear regions of acinar cells are highly labeled. This area contains the majority of the rough endoplasmic reticulum.
In contrast, the nuclei (arrows), cell apices, and the lumens of the acini (1) are poorly labeled. Thus pancreas cDNA hybridizes primarily with RNAs present in the regions of acinar cells actively synthesizing secretory proteins.

Embryonic
Pancreas RNAs Complementary to Pancreas cDNA -In order to determine whether the abundance of RNA species complementary to pancreas cDNA changes during development, RNAs isolated from fetal rat pancreases of increasing gestational age were hybridized with pancreas cDNA. The kinetics of hybridization are shown in Fig. 6. The following results are obtained.
(a) The embryonic hybridization curves shift toward the position of the adult curve on the R,,t scale as development proceeds, indicating that the concentration of RNA sequences complementary to pancreas cDNA increases as a function of gestational age. The complex shapes of the hybridization curves in Fig. 6 preclude a precise calculation of changes in RNA concentration.
However, the 14-and 16-day hybridization curves are approximately 1 log unit apart on the ROt scale as are the 16-and B-day curves, implying about a lofold increase in the concentration of RNAs complementary to pancreas cDNA during each 2 days of development.
The 18and 20-day curves are somewhat less than 1 unit apart on the ROt scale, indicating a smaller increase in RNA concentration. The concentration of RNA species characteristic of adult pancreas therefore increases about 500-fold between 14 days of development and adulthood. (b) The maximum amount of hybridization obtained is not the same for all embryonic RNAs. At the highest R,t values tested, adult, 20-, and 18-day RNAs hybridize with about 90% of pancreas cDNA and 16-and 14-day RNAs hybridize with 60 and 30% of the cDNA, respectively.
(c) The embryonic hybridization curves (Fig. 6) are not all the same shape as the adult hybridization curve (Fig. 2). The 20-and B-day RNAs hybridize over more than 4 orders of FIG 4 (upper).
In situ hybridization of adult pancreas tissue with pancreas cDNA. Tissue was prepared and hybndlzed with 200,000 cpm of pancreas cDNA as described under "Materials and Methods." The acinar tissue is heauzly labeled. The lumen of a blood vessel (b), endocrine cells of the islets of Langerhans (z), and connective tissue cells (c) are relatruely unlabeled.
The autoradiograph was exposed for 2 weeks. Magnification, x 100. Fig. 4 ) cDNA and comprise about 0.03% of total pancreas RNA. Copies of these RNA species may be present in pancreas cDNA but would be difficult to detect unambiguously since they would constitute less than 10% of the cDNA and would hybridize over a wide range of R,t values.

FIG. 5 (lower). A portion of
We estimate that the less abundant set of pancreas RNAs is at least 10 times more complex than the abundant polyadenylated species. The complexity of the abundant RNAs was determined from the kinetics of hybridization of pancreas cDNA with pancreas polyadenylated RNA. The complexity of the less abundant species was derived from the fact that total adult pancreas RNA hybridizes with 40% of HTC cell cDNA which is complementary to a polyadenylated RNA population of high sequence complexity. Complexity values determined from cDNA-RNA hybridization analysis are uncertain for a number of reasons as has been discussed by others (5, 21-23). Of particular importance is the fact that, although hybridization curves containing two or three ideal pseudo-first order kinetic components fit our data closely and thus approximate the frequency distribution of RNA sequences in the population, curves containing greater numbers of components could also have been used. The number of components affects the calculation since the component of greatest complexity largely determines the total. However, HTC cell cDNA hybridizes with its RNA template over at least 3 more log units of R,t values than does pancreas cDNA with its template. Thus a substantial difference in sequence complexity is indicated irrespective of how the data is simplified.
A major difference in the kinetics of hybridization of pancreas cDNA and HTC cell cDNA with their respective tem-plate RNAs is that 90% of pancreas cDNA hybridizes at Rd values at which only 5% of HTC cell cDNA reacts. Thus the abundant pancreatic polyadenylated RNA population has no apparent counterpart in HTC cells. This result probably reflects the specialization of the differentiated pancreas. Not all differentiated tissues (for instance chicken liver (231, mouse liver, and mouse brain (22)) contain such a predominant set of RNA sequences.
The abundant pancreas polyadenylated RNAs probably code primarily for the synthesis of pancreas-specific secretory proteins. This is suggested by the following: (a) mRNAs coding for the in uztro synthesis of pancreatic exocrine proteins bind to oligo(dT)-cellulose, are therefore polyadenylated, and can be copied by RNA-dependent DNA polymerase (3, 4); (b) pancreatic secretory proteins comprise 50 to 90% of total pancreatic protein (24); their mRNAs should be most abundant; (c) about 80% of pancreas cDNA does not hybridize with RNA isolated from other rat tissues or HTC cells at high R,t values and is therefore tissue-specific within the limitation of the analysis. However, since the end point of a hybridization reaction cannot be determined when some cDNA remains unhybridized, our data do not preclude a basal level of synthesis of "pancreas-specific" RNAs in other tissues. Other studies indicate that the pancreatic secretory proteins are probably tissue-specific. For instance, carboxypeptidase A and lipase activities characteristic of pancreas secretion cannot be detected in other tissues (2, 25). Although amylase activity is found in parotid gland and liver as well as pancreas, pancreatic amylase has a different electrophoretic mobility (26-281, and antigenic specificity (27, 29, 30) than both parotid and liver amylases and a different amino acid composition and peptide map pattern than the parotid enzyme (26, 27). (d) In sttu hybridization experiments indicate that the abundant pancreatic RNAs are primarily localized in the cytoplasmic regions of acinar cells which synthesize secretory proteins. (e) About 15 major proteins are isolated from rat zymogen granules. Furthermore, the polyadenylated RNA used as template for the synthesis of pancreas cDNA codes for the in uztro synthesis of a similar number of proteins. One of these has been identified as a precursor of amylase by specific immunoprecipitation and a number of others are inferred to be precursors of other pancreatic secretory proteins3 These results are consistent with the complexity of the abundant pancreas polyadenylated RNAs, estimated here as 2 x lo4 nucleotides, which is sufficient to code for about 15 mRNA sequences of average size. (f, The low sequence complexity of the majority of pancreas polyadenylated RNA is not an artifact of the isolation procedure since HTC cell RNA, isolated and copied into cDNA by the same techniques, clearly contains a polyadenylated RNA population of high sequence complexity. Furthermore, the result is not unique to the rat pancreas. Undegraded RNA can be isolated from dog pancreas by phenol extraction because the dog, unlike the rat, does not synthesize significant amounts of exportable ribonuclease (31). Our unpublished experiments indicate that the kinetics of hybridization of dog pancreas cDNA with phenol-or guanidine HCl/diethylpyrocarbonate-extracted dog oligo(dT)-bound RNA are similar to the kinetics of hybridization of rat pancreas cDNA-RNA shown here.
Changes in Concentration of Pancreas-specific RNAs during Development-Enzymatic activities characteristic of adult pancreatic secretion are present at relatively low, constant levels in the pancreatic rudiment between Days 12 and 14 of gestation during the period of extensive morphogenesis (the protodifferentiated