© 1999 Macmillan Magazines LtdOne possible explanation is that theexpression of this receptor in the develop-ing gizzard reflects a role in transducingBMP signals originating from adjacentregions of the gut, such as the developingsmall intestine where BMP-4 is expressed,thereby mediating the patterning of struc-tures at the border of the gizzard and thesmall intestine.The pyloric sphincter forms at the junc-tion of the gizzard and the small intestine,and acts as a gate to regulate the passage ofingested food from the stomach to the smallintestine. The sphincter differs from the restof the gizzard with regard to the mesoder-mal and endodermal layers5.While searching for a molecular markerfor the pyloric sphincter, we examined theexpression pattern of Nkx2.5, a homeo-domain-containing transcription factorthat is expressed in the midgut6. We foundthat Nkx2.5 is a specific marker for themesoderm of the pyloric sphincter in thechick embryo (Fig. 1e,f). In the early stages,it is expressed adjacent to the area whereBMP-4 is expressed, and overlaps with theexpression pattern of BMPR1B in the pos-terior of its domain (Fig. 1b–d).To investigate the role of BMP-4 in theformation of the pyloric sphincter, we injec-ted a retrovirus containing the mBMP-4complementary DNA7into stage-10 chickembryos8. Ectopic expression of Nkx2.5 wasseen in the gizzard, but not in the smallintestine, of injected embryos, even thoughboth organs were infected with the retro-virus (Fig. 1i,j). Sections through infectedgizzards showed that Nkx2.5, like theendogenous domain, is limited to the meso-dermal layer (Fig. 1g,h). BMP signalling istherefore sufficient to activate Nkx2.5 in thegizzard primordium.We tested whether BMP signalling isnecessary for endogenous Nkx2.5 activationin the developing sphincter by injecting avirus containing the secreted BMP antago-nist Noggin9into chick embryos (n412).Nkx2.5 was downregulated at the border ofthe gizzard and the small intestine in injec-ted embryos (Fig. 1k), indicating that BMPsignalling is required for Nkx2.5 expressionin the gut. Noggin is a specific antagonist ofBMP-2, -4 and -7 (ref. 10), of which onlyBMP-4 is expressed in this region of thegut, indicating that the endogenous signalfor Nkx2.5 induction is BMP-4 from thesmall intestine. Noggin misexpression alsoaltered development in other regions of thegut (data not shown), reflecting differentroles of BMP signalling in gut patterning2.To test the morphological consequenceof altering BMP signalling during the estab-lishment of the pyloric sphincter, weallowed BMP4-infected embryos to developto E9 (Noggin-infected embryos did notsurvive long enough to allow morphologi-cal analysis of guts where BMP signallingwas disrupted). In addition to its role dur-ing sphincter specification, prolonged BMPexpression interferes with muscle develop-ment and affects the rates of mesodermalproliferation and apoptosis2(data notshown). We therefore limited our morpho-logical analysis to the endoderm.Although the gizzard endoderm haslong, thin villi that are completely coveredby a thick layer of a protective keratin-likesubstance7(Fig. 1l), the pyloric sphincterendoderm has short villi, each of which isthin at the base and bulbous at the tip (Fig.1m). Instead of the long, keratin-coveredvilli characteristic of the gizzard, the endo-derm of BMP4-infected gizzards had shortvilli with thin bases and bulbous extensions,748 NATURE|VOL 402|16 DECEMBER 1999|www.nature.comDevelopmental biologyBMP signalling specifiesthe pyloric sphincterSphincters are muscular valves that form atthe boundaries between organs of the gut;for example, the pyloric sphincter forms atthe junction of the small intestine andstomach. We show here that signalling bybone morphogenetic protein (BMP) fromthe avian small intestine induces the cells ofthe adjacent gizzard (the equivalent of thestomach in the chicken) primordium toform a sphincter. This finding and relatedstudies of the role of Hox genes in the speci-fication of the iliocaecal sphincter1provideinsights into the processes by which newcell fates are specified at the bordersbetween distinct embryological domains.To investigate the inductive signallinginvolved in the development of the chickengut, we examined the expression pattern ofseveral members of the BMP family, includ-ing BMP-2, BMP-4, BMP-5 and BMP-7,and compared them with the expressiondomains of the known BMP receptors2.BMP-4 is the only member of the family tobe expressed in the early chicken gut,appearing in the mesoderm of the smallintestine from embryonic day 2.5 (E2.5)(Fig. 1a,b, and data not shown).Several BMP receptors are expressed inthe intestinal mesoderm in a position tomediate BMP-4 signalling in this portion ofthe gut (data not shown). However, the type I receptor, BMPR1B3,4, is specificallyexpressed in the gizzard mesoderm fromE2.5 (Fig. 1c), despite the fact that neitherBMP-4 nor any other related BMP familymember we examined (data not shown) areexpressed in the early gizzard mesoderm.brief communicationsFigure 1 BMP signalling is necessary and sufficient to specify a pyloric sphincter. a, Diagram of the regions of the embryonic gut. b–d, Section in situhybridization (ISH)2of an E4.5 gut with adjacent sectionsstained by using riboprobes for BMP-4 (b), BMPR1B (c) or Nkx2.5 (d). Red arrowheads point to the posterior border of expression of Nkx2.5 and BMPR1B as well as the anterior border of BMP-4 expression. e, Section ISH of an E9.5 chicken gut using an Nkx2.5 riboprobe. f–h, Section ISH of E6 gizzard sections hybridized with an Nkx2.5 riboprobe on a control gut (f, g), or an embryo injected with RCAS–BMP-4(ref. 7) (h) showing ectopic Nkx2.5 expression in the gizzard. Red line in f demarcates the anterior boundary of Nkx2.5 expression in wild-type embryos; in g and f, the blue line shows the plane of sectioning.i–k, Whole-mount ISH11using Nkx2.5 as a riboprobe on an E4.5 control gut (i), an E4.5 embryo injected with RCAS–BMP-4 showing ectopic Nkx2.5 in the gizzard (j), or an E4.5 Noggin9-injected embryo show-ing a decrease in Nkx2.5expression at the pyloric sphincter (k). The red arrowheads indicate the anterior border of Nkx2.5 expression in a control gut. l–n, Sections through an E9 embryo showing morphologyof the endoderm of either the gizzard (l), pyloric sphincter (m) or the gizzard (n) of an embryo injected with BMP-4 with villi resembling that of the pyloric sphincter. Red arrowheads point to villi at the tips