neural crest origin of the endocrine polypeptide …differentiation of high protein turnover cells...

Gut, 1971, 12, 783-788

Neural crest origin of the endocrine polypeptide(APUD) cells of the gastrointestinal tract andpancreas

A. G. E. PEARSE AND JULIA M. POLAK

From the Department ofHistochemistry, Royal Postgraduate Medical School, Hammersmith Hospital, London

SUMMARY A method of labelling known to be appropriate for the demonstration of endocrinepolypeptide (APUD) cells was found to label the cells of the neural crest in the chick embryo afteras little as 72 hours' development. The method depends on the production, from an exogenousprecursor, of an amine which is stored in specific granules and which is convertible by treatmentwith hot formaldehyde vapour into a fluorescent derivative. The whole technique is described asAPUD-FIF.The application of APUD-FIF to mouse embryos shows that at the 7-8 somite stage (eight days)

labelled neural crest cells migrate in large masses in a ventrad direction. At around the ninth daythey colonize the developing foregut and its derivatives, including pharynx, stomach, duodenum,ultimobranchial body, and pancreas. In subsequent stages of development (up to 12 days) the cellsare seen in comparatively large numbers in the gastrointestinal tract and in the pancreas.

Complete proof that these early APUD cells, which demonstrably arise from the neural crest,are the precursors of all the endocrine polypeptide cells of the adult pancreas, stomach, duodenum,and small and large intestine, is not at present available. Notwithstanding a great deal of earlierevidence to the contrary, the premise seems likely to be true.

Three types of method have been used for tracingthe cells of the neural crest from their origin tointermediate and final destinations. The first of thesedepends on the employment of 'hot' homografts(radioactive labelling, autoradiography), the secondon the technique of heterospecific grafting (allo-grafts), and the third on induced amine formationand storage. In cells possessing the cytochemicalcharacteristics described as APUD (Amine Pre-cursor Uptake and Decarboxylation) includedamines can be demonstrated by formaldehyde-induced fluorescence (FIF, Eranko, 1967), and thewhole process can therefore be described as APUD-FIF.The first approach was used by Johnston (1966),

who introduced a radioactive marker (tritiatedthymidine) into the nuclei of an early chick embryobefore transplanting the region containing thecranial neural crest into another embryo. Thelabelled cells moved rapidly in a ventrad directionand Johnson concluded that they gave rise, inter alia,to the chondrocytes of the visceral arch cartilages.Received for publication 27 July 1971.

The second approach, employed by Le Douarinand Le Lievre (1970), involved transplantation ofthe neural tube region from 7-10-somite Japanesequail embryos into coeval embryos of the domesticfowl. Quail cells can be distinguished from those ofthe fowl by their possession of a large centralchromatin mass. Following transplantation andfurther incubation it was possible to demonstratethe participation of grafted quail cells in the forma-tion of the ultimobranchial body in the chick. Theauthors concluded that the C cells of this organ wereof neuroectodermal origin.The third approach (APUD-FIF) was used by

Pearse and Carvalheira (1967) to demonstrate thatthe calcitonin-secreting C cells of the mammalianthyroid gland entered that organ with the ventral(ultimobranchial) component of the last pharyngealpouch. Since their studies, carried out on mouseembryos, extended from the 10th (now recognizedto have been the 12th) day of gestation onwards,they noted that possibly the true origin of the C cellswas 'outside the pharyngeal pouches'. It had beensuggested (Pearse, 1966a and b) that C cells, and

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A. G. E. Pearse and Julia M. Polak

other endocrine polypeptide-secreting cells subse-quently included in the APUD series (Pearse, 1968),arose from the neural crest. This hypothesis was notoriginal. A neural crest origin for the C cells wasimplied by the work of Sunder-Plassmann (1939)and Danisch (1924) had made a similar postulate forintestinal enterochromaffin cells. Moreover theendocrine cells of the adrenal medulla, and themelanocytes, which had been shown to belong tothe APUD series, are universally accepted as neuralcrest derivatives.A pilot study, using chick embryos, was carried

out by Polak, Rost, and Pearse (1971) and thisindicated that the cells of the neural crest possessAPUD characteristics as early as 72 hours. A similarstudy of early stages of development in the mouseembryo (Pearse and Polak, 1971) had shown thatthe developing ultimobranchial pouch was colonized,at the 10-somite stage (nine-10 days) by cells fromthe neural crest, confirming the findings of LeDouarin and Le Lievre (1970). It was thereforedecided to extend the work to cover the develop-ment of the gastrointestinal tract and pancreas.

Material and Methods

Seventy-eight embryos from 10 pregnant albinomice were studied and the age of each litter wascalculated either from a witnessed mating or bycomparison with previous litters. Where possiblethe number of somites was counted. The period ofgestation extended from the seventh to the 12th day.The pregnant mice received a single intraperitoneal

injection of L-DOPA (100 mg/kg) and all were killedone hour later. Control embryos from uninjectedmothers were provided from a series previouslystudied.

Immediately after removal the embryos werequenched by immersion in melting Arcton (Freon)22 and freeze-dried in a thermoelectric tissue dryerat -40° for 16 hours (overnight). Subsequently thedried embryos were exposed to formaldehydevapour at 600 or 800 for four hours. All were thenembedded in paraffin wax, careful attention beingpaid to their orientation so as to provide serial(5 ,tm) transverse sections. After cutting, the latterwere placed on prewarmed slides, directly from theknife, and mounted in Styrolite.

Fluorescence microscopy was carried out using aZeiss (Oberkochen) standard universal microscopewith an HBO 200 lamp, BG12 and BG38 excitationfilters, and a K530 barrier filter (50% transmissionat about 530 nm).

Every third section was stained with haematoxylinand eosin in order to facilitate identification ofstructures seen in the fluorescence microscope.

Photomicrographs were taken on Ilford FP4(fluorescence) or Pan F films.

Results

At the earliest stages of investigation (seven to eightdays) the developing pharynx was readily identified(Fig. 1) together with various pouches, the last ofwhich becomes the ultimobranchial body. Groupsof labelled (APUD) cells, travelling down as thecentral stream of the neural crest, traverse themesenchymal mass between the neural tube andpharynx (Fig. 2) though they are not detectable inconventionally stained preparations.At a slightly later stage (10 somites) we observed

colonization of the IVth pouch and the wall of thepharyngeal tube by cells from the neural crest, aspreviously reported by Pearse and Polak (1971). Thisprocess is shown in Fig. 3, where the degree ofcolonization is observed to be particularly intensein both anterior and posterior walls of the pharynx.

Close examination of serial sections of the deve-loping foregut of 8-9-day embryos revealed anepithelium-lined pouch springing from the dorsalwall of the duodenum. This is clearly illustrated anddescribed by Wessells and Rutter (1969) as the dorsalpancreas. Our APUD-FIF sections showed coloni-zation of the gut wall (future stomach and duo-denum) by fluorescent neural crest cells and invasionalso of the presumptive dorsal pancreas. Theseprocesses are illustrated in Fig. 4 where the fluores-cent cells can be seen in the mesenchyme which iscondensing around the duodenal pouch, as well asin the wall of the foregut. Because the developingacinar cells can be recognized clearly at the 10th dayon account of their (transient) uptake of L-DOPA,and because the dorsal pouch of the duodenum inour serial sections showed no such uptake. thepossibility exists that it represents the anlage of thecommon duct and gallbladder rather than of thepancreas. On balance the latter is still the morelikely of the two interpretations.At a later stage of development (10-11 days) the

wall of the definitive stomach as well as that of theduodenum (Fig. 5) contained numerous fluorescentAPUD cells. Most of these still occupied the basallayers of the epithelium but many already possessedlong processes extending towards the lumen. Thedeveloping pancreas at this stage (10-11 days) isshown in Figure 6. Dorsal and ventral pancreas nowconsist of isolated groups of developing acini, ducts,and islets. Brightly fluorescent APUD cells arevisible, mainly in areas where the structure of theepithelial cells is duct-like. Some fluorescence isvisible in the presumptive acinar tissue. As observedby Alm, Ehinger, and Flack (1969), acinar cells can

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Fig. 1 Mouse embryo, 7-8 days. Freeze-dried, Fig. 2 As Figure 1. Shows a long stream offluorescentformaldehyde vapour-fixed. Shows the developing APUD-FIF labelled cells. These are definitive neuralpharynx (P) at the level of the I Vth pharyngeal pouch. crest cells (nc) travelling ventradfrom their origin at theThe relationships ofthe neural tube (NT), notochord (NC), epithelio-neural junction. Part of the neural tube (NT)andpharynx are shown, as is the mesenchymal mass is visible at upper right. x 140.lying on either side of the mid-line. Haematoxylin andeosin. x140.

Fig. 3 Mouse embryo, 9-10 days. Freeze-dried, formaldehyde vapour-fixed, examinedby fluorescence microscopy. APUD-FIF-labelled cellsfrom the neural crest haveinvaded the pharynx (P), particularly on itsanterior andposterior aspects. The region ofthe IVth pouch is labelled. x 290.



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A. G. E. Pearse and Julia M. Polak

Fig. 4 Mouse embryo,8-9 days, processedforAPUD-FIF. Shows cellsfrom the central stream ofthe neural crest (nc), in themesenchyme condensingaround the (presumptive)dorsalpancreatic rudiment.Fluorescent APUD cellsare visible also in the latterand in the foregut, whichis here destined to becomeKmainly duodenum. Theprotuberance from the gutwall (extreme left) is

... possibly the ventralpancreas. x 340.

Fig. 6

Fig. 5 Mouse embryo, 11-12 days, processedfor APUD-FIF. The definitive duodenum contains manylabelled cells and these are not confined to the basal layers. x 400.Fig. 6 Mouse embryo, 11-12 days, processedfor APUD-FIF. The developing pancreas now consists ofcords ofpresumptive acinar cells, here showing moderate fluorescence, from which duct-like structures are arising.The labelled endocrine cells are mainly, if not exclusively, associated with 'ducts'. x 400.

Fig. 5

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Neural crest origin of the endocrine polypeptide (APUD) cells of the gastrointestinal tract andpancreas 787

take up and decarboxylate DOPA. They do not,however, possess the amine-storage mechanismcharacteristic of endocrine polypeptide cells fromwhich they can thus be distinguished by rapid lossof the label. Proliferating cells of many differenttypes are observed to take up the amino-acid pre-cursors of fluorogenic amines. These are rapidlymetabolized, possibly without decarboxylation.Storage is minimal and prolonging the postadminis-tration period of the precursor provides cleardifferentiation of high protein turnover cells fromAPUD cells.

Discussion

The APUD characteristics of the neural crest cellswhich enable them to be traced effectively over anembryologically long period of time are, in effect,precisely those emphasized by Willmer (1951) whenhe noted that 'the cells of the neural crest, and itsimmediate neighbours, seem to have made a cornerin a special form of tyrosine metabolism which mayculminate either in the formation of melanin oradrenaline'. It is fortunate for us that they haveretained this ancestral habit through 300 millionyears or so of development. Our findings indicateclearly that invading cells from the central, andperhaps from the peripheral, stream of the neuralcrest colonize the foregut and its derivatives, themost important of which is certainly the pancreas.Concomitant studies have shown that the tracheaand primary bronchi are also invaded. The con-clusion of Le Douarin and Le Li6vre (1970) that'les APUD cells de Pearse ont une origine embryo-logique ectodermique' is thus almost certainlycorrect, though marginally premature. What can-not yet be determined is whether all the endocrinepolypeptide cells of the adult gastrointestinal tractand pancreas are descendants of neural crest APUDcells or whether some are derived by differentiationfrom the epithelial cells of the gut and pancreaticducts, as is usually presumed. A list of the endocrinepolypeptide cel!s of the stomach and intestine,modified from the original Wiesbaden list (Creutz-feldt, Gregory, Grossman, and Pearse, 1970), isgiven in the Table.

It is comparatively easy to imagine environmentaldifferences modifying an originally single cell typeso that, for instance, the thyroid or ultimobranchialC cell produces calcitonin while the pyloric G cellproduces gastrin. It is less easy to see how cells of asingle type sharing the same environment aremodified to produce a number of different hormoneseven if, as is often the case, their primary structurescontain identical amino-acid sequences. Differencesin innervation offer a possible solution.

Wiesbaden Equivalent Cell Product orNomenclature in Pancreas Possible Product

rG Not known GastrinST D D (Enterogastrone)l

M ECL Not presentAC A A EnteroglucagonH

L EC EC-

N L A EnteroglucagonTE S Not present SecretinST x D (CCK-PZ)IN EC EC-E LTable Endocrine polypeptide (APUD) cells of thegastrointestinal tract'Possible product is enclosed in brackets.

A number of discrepancies remain to be explained,particularly those relating to the development of theislets. The current view, held almost since theiroriginal discovery, is that they arise by buddingfrom the pancreatic ducts. It will clearly be necessaryto trace back the definitive endocrine cell types ofthe adult gastrointestinal tract and pancreas totheir earliest forms, using immunofluorescence,electron microscopy, and immunoelectron cyto-chemistry. While these methods probably offer noquick solution to the problem, the concept of anancestral relationship between all the polypeptidehormone-producing cells of the foregut and itsderivatives may provide reasons for relationshipsbetween individual cell types which are otherwisedifficult to understand. The APUD cells appear tobe brothers, under the skin, and it is perhaps notsurprising that they appear to recognize, and torespond to each others' products.

This work was carried out with the help of a grantfrom the Wellcome Trust. We are grateful toCaroline Maunder for assistance with the dissectionof the embryos.

ReferencesAlm, P., Ehinger, B., and Falck, B. (1969). Histochemical studies on

the metabolism ofL-DOPA and some related substances in theexocrine pancreas. Acta physiol. scand., 76, 106-120.

Eranko, 0. (1967). The practical histochemical demonstration ofcatecholamines by formaldehyde-induced fluorescence. J. roy.microsc. Soc., 87, 259-276.

Danisch, F. (1924). Ztir histogenese der sogenannten Appendix-karzinoide. Beitr. path. Anat., 72, 687-709.

Johnston, M. C. (1966). A radioautographic study of the migrationand fate of cranial neural crest cells in the chick embryo.Anat. Rec., 156, 143-155.

Le Douarin, N., and Le Li6vre, C. (1970). Demonstration de l'origineneural des cellules a calcitonine du corps ultimobranchial chezl'embryon du poulet. C.R. Acad. Sci. (Paris), D., 270, 2857-2860.



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Pearse, A. G. E. (1966a). 5-Hydroxytryptophan uptake by dog thyroidC cells and its possible significance in polypeptide hormoneproduction. Nature (Lond.), 211, 598-600.

Pearse, A. G. E. (1966b). Common cytochemical properties of cellsproducing polypeptide hormones, with particular reference tocalcitonin and the thyroid C cells. Vet. Rec., 79, 587-590.

Pearse, A. G. E. (1968). Common cytochemical and ultrastructuralcharacteristics of cells producing polypeptide hormones (theAPUD series) and their relevance to thyroid and ultimo-branchial C cells and calcitonin. Proc. roy. Soc. B., 170, 71-80.

Pearse, A. G. E., and Carvalheira, A. F. (1967). Cytochemical evi-dence for an ultimobrandrial origin of rodent thyroid C cells.Nature (Lond.), 214, 929-930.

Pearse, A. G. E., and Polak, Julia M. (1971). Cytochemical evidencefor the neural crest origin of mammalian C cells. Histochemie,27, 96-102.

Polak, J. M., Rost, F. W. D., and Pearse, A. G. E. (1971). Fluoro-genic amine tracing of neural crest derivatives forming theadrenal medulla. Gen. comp. Endocr., 16, 132-136.

Sunder-Plassmann, P. (1939). tYber neuro-hormonale Zellen desVagussydtems in der Schilddri:se. Dtsch. Z. Chir.,252, 210-223.

Wessells, N. K., and Rutter, W. J. (1969). Phases in cell differentiation.Sci. Amer., 220, 36-44.

Willmer, E. N. (1951). Some aspects of evolutionary cytology. InCytology and Cell Physiology, 2nd ed., edited by G. Bourne,pp. 444-493. Clarendon Press, Oxford.



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