Bmp2- E7.25 to E10.5
We initially assayed expression of Bmp genes using whole mount in situ hybridization. (For all descriptions of gene expression in embryonic organs and tissues refer to structure annotations found in top panels of each figure, either in schematic or overlaid on photographed embryo, and associated organ or tissue name in figure legends). Our analysis of Bmp gene expression began at E7.25, a time in development when embryonic tissues start to form following gastrulation. We find that at this stage, Bmp2 is primarily expressed in the yolk sac (y) and allantois (a), in the precardiac crescent (cc), just anterior to the anterior intestinal portal, or AIP (aip) (). Expression appears distinctly absent in the medial, open gut region (g) of the embryo, which includes pre-somitic mesoderm, endoderm, and neural tissues. By E8.25, we observe an increase in Bmp2 expression in the linear heart tube (h) and allantois, while high levels of expression are observed in the constricting AIP and sinus venosus (sv), regions immediately ventroposterior to the heart (). In addition, strong expression initiates in the dorsal most tip of the rostroanterior neural folds (nf) and the midline fusion point of the neural folds spanning the length of the embryo. Expression also appears robustly in the lateral plate mesoderm (lpm).
Expression of Bmp ligands and the BMP antagonist noggin during embryogenesis
Slightly later, at E8.75, expression remains strong in the sinus venosus and in the fusing dorsal neural tube (, and ). Strikingly, rather significant expression is also detected in the heart, shortly after the heart takes shape and starts to loop. Expression is particularly strong in the region that joins the left ventricle and the atria, which will later give rise to the atrioventricular canal region, or AVC (avc) (arrow in ), a region previously shown to express both Bmp2
(Christoffels et al., 2004
). Expression is also high in the pericardium, with declining expression in the ‘seam’ of the dorsal brain, which represents the region of the midline fusion of the anterior neural folds.
Expression of Bmp ligands and the BMP antagonist noggin in developing tissues
Sections showing Bmp ligand and noggin expression
Later, at E9.0, Bmp2 remains strongly expressed in the AVC, but has declined from the midline seam of the telencephalon (ms) (, and ). In addition, expression has decreased in the constricting base of the yolk sac (ysc) (or the future umbilical cord), and associated lateral plate mesoderm, as embryonic turning proceeds and the gut tube forms (). Expression is low in the head and branchial arches, as well as the dorsal neural tube at this stage of development. Expression remains strong in the liver diverticulum (l) ( and ). Bmp2 also increases expression in the developing forelimbs (lb) ( and ), the rostral dorsal aortae (ao) of the trunk and the anterior tip of the developing mesenephros (m) (arrowheads in ).
At E9.5, Bmp2
has also begun to be expressed in the apical ectodermal ridge, or AER (aer), of the limb bud, a region long noted for its organizing activity in driving limb development, and the ventral portion of the developing limb bud, as previously noted (Ahn et al., 2001
; Lyons et al., 1995
) (, and ). Notably, expression in the heart AVC peaks at this point. In addition, expression is prominent in subpopulations of cells between the branchial arches (). By E10.5, expression levels have generally declined throughout the head and tail, and are low throughout the gut tube ( and ); however, there is still detectable expression in the constricting yolk sac (). In addition, expression remains relatively high in the heart AVC (inset, ), otic vesicle (ov), and branchial arches. Expression is robust in the AER at this stage and appears in an ectodermal patch on the posterior aspect of the forelimbs. Additionally, expression in the ventromedial somites (s) increases, especially in the anterior trunk ( and ).
Bmp4 – E7.5 to E10.5
Bmp4 is expressed in a pattern initially similar to that of Bmp2; however, it subsequently varies dynamically throughout early embryogenesis. At E7.25, Bmp4 like Bmp2, is strongly expressed in the yolk sac and allantois, and is concentrated in the anterior part of the embryo, including the cardiac crescent and early neural folds (). At E8.25, Bmp4 remains expressed in the allantois, and strong expression initiates in the posterior lateral plate mesoderm, the AIP, and the sinus venosus (). However, yolk sac expression is slightly lower than that of Bmp2.
After turning, around E8.75, expression continues to increase in the posterior lateral plate and remains relatively strong in the sinus venosus, the allantois and the caudal most mesoderm of the tail (t) (). Again similar to Bmp2
is detected in the dorsal most tips of the rostral neural tube, albeit at significantly lower levels ( and ), and expression appears in the seam of the diencephalon (d) (). Thus, Bmp2
are co-expressed in the AIP, the lateral plate and the neural folds of the head during these earliest stages of embryogenesis (E7.25–E8.75). Bmp4
has been reported to be more strongly expressed in the myocardium at this time, however using our whole mount method, we detect only low Bmp4
expression in the heart (Jones et al., 1991
is expressed strongly in the posterior lateral plate mesoderm, allantois, and yolk sac.
At E9.0, additional domains of Bmp4
expression appear in many different regions of the embryo ( and ). These domains include the midline seam of the telencephalon, the pituitary infundibulum (pi), the otic vesicle, the rostral somites, the tip and cleft of the branchial arches (b), the thyroid primordium (th), the heart sinoatrial region, the emerging limb buds, the liver, the dorsal optic cup (oc), and the posterior lateral plate mesoderm (, and ). Additionally, Bmp4
is expressed in the distal portion of the first branchial arch. Most of these domains are maintained in the E9.5 embryo with marked increase in the rostral dorsal somites, seam of the telencephalon, branchial arches, and in populations of cells likely to be neural crest derived neurons near the branchial arches () (and as shown by (Grotewold et al., 2001
). Expression increases in the dorsal portion of the optic cup.
At E10.5, expression of Bmp4 increases in all these domains, particularly in the telencephalon, the first branchial arch, and around the nasal placode (n), as assessed by intensity of staining by in situ hybridization (). Additionally, expression of Bmp4 is strong in the dorsal somites spanning the length of the embryo, the dorsal gut tube mesoderm, the dorsal aspect of the limb, the outflow tract of the heart (ot), the lung buds (lu) and the limb bud AER (, and ).
Bmp7 – E7.5 to E10.5
Similar to Bmp2
expression varies dynamically throughout early embryogenesis. Bmp7
expression can be observed in many of the same developing tissues as Bmp2
, although its levels appear relatively lower during early development. Bmp7
expression is evident at E7.5 broadly expressed in the cardiac crescent, the allantois, anterior embryonic endoderm, yolk sac, and the anterior primitive streak (), as previously described (Solloway and Robertson, 1999
). By E8.25, however, expression remains strong in the yolk sac, but is significantly lower in the AIP, sinus venosus and allantois than either Bmp2
(). Expression is evident in the dorsalmost portion of the rostral neural folds including strong staining in the ectoderm of the future otic vesicle. In contrast to previous reports, we observe only low levels in the developing heart and allantois (Solloway and Robertson, 1999
). By the end of turning, at E8.75, Bmp7
is prominently expressed in the otic vesicle, telencephalon, the dorsal tip of the neural folds, the future pituitary within the head and a portion of the first branchial arch, while it is present at lower levels in the notochord, heart, somites and the lateral plate mesoderm of the trunk (, and ). Overall, these observations are in agreement with previous reports, except that we detect lower expression in foregut endoderm (Solloway and Robertson, 1999
By E9.0, Bmp7
expression continues in these same tissues, however intensifies in the roof of the midbrain, the otic vesicle, telencephalon and branchial arches, with marked expression along the entire length of the developing kidney (mesonephros), while it remains low in the heart (, and ). Expression in the limb buds also initiates at this stage ( and ). By E9.5, expression levels continue to increase in the telencephalon, the dorsalmost neural tube and the heart (). New domains of expression also appear, such as the dorsal somites and the gut endoderm ( and data not shown). Bmp7
continues to intensify in the dorsal forebrain, neuroectoderm, lateral plate mesoderm, mesonephros, forelimb bud ectoderm and the neural tube, as previously shown (Solloway and Robertson, 1999
). By E10.5, Bmp7
is relatively strongly expressed in portions of the branchial arches, mesonephros, telencephalon, both fore- and hindlimbs, otic vesicles, dorsal optic cup, and throughout the dorsal somites ( and ). In addition, it is expressed in the gut tube, around both lung buds and posterior stomach (st), as well as in the myocardium of the heart ( and ).
Noggin – E7.0 to E10.5
To assess regions where BMP activity may be regulated by BMP antagonists, we examined the expression of the high affinity BMP inhibitor noggin. Similar to Bmp ligand expression patterns, noggin transcription occurs in highly restricted embryonic domains. Interestingly, it displays only limited overlap with that of early Bmp2, 4 and 7 expression.
At E7.0 noggin
levels are undetectable in most embryonic tissues, except for strong expression in the node and emerging notochord (). Tissues like the allantois, AIP and yolk sac, which co-express Bmp ligands, show no expression of their antagonist noggin
. At E8.25, noggin
continues to be expressed in node derivatives and is observed throughout the developing notochord (). In fact, we find that at these stages, noggin
is a robust marker of the early notochordal plate and early notochord. In addition, noggin
becomes expressed in the dorsal tips of the neural folds, in a pattern that overlaps that of Bmp2
(compare ). At E8.75, following turning, noggin
expression expands to encompass the entire dorsalmost neural tube, from the tip of the tail to the rostral telencephalon (, and ). In addition, expression begins in the dorsalmost edge of the somites (as previously shown in (Reshef et al., 1998
expression at this stage strongly marks the fusing and fused neural folds, along the entire length of the embryonic axis, and it continues to be maintained in the notochord, although it soon declines in the anterior portion of the embryo.
As development proceeds, at E9.0, noggin
expression appears at low levels in the dorsal optic cup, the dorsal rostral portion of the somites and more generally throughout the dorsalmost region of the fore- and midbrain, while lower levels are detectable in the liver, as well as the myocardium of the heart (, and ). This is in contrast to the high level of noggin
expression previously reported in the early developing heart (Yuasa et al., 2005
). We observe only low levels of noggin
in the heart, starting around E8.75. Strikingly, while expression intensifies in the seam of the head and neural tube, it is always absent from the isthmus, the region between the mid- and hindbrain (arrowheads in and ). At E9.5, noggin
continues to be expressed in the posterior notochord (no), the telencephalon, and the dorsal neural tube, however overall levels in the notochord and neural tube begin to decrease (). Expression within the dorsal tip of the somites, however, peaks at this stage. At E10.5, overall noggin
expression declines slightly from that seen at E9.5. It is expressed in the mesoderm of the lung buds, but is absent from most of the gut tube and heart ( and ). It also declines in the somites, but remains in the dorsal neural tube (nt) ().
Expression of Bmp ligands and noggin in developing organs and tissues
Notable overlaps in expression of Bmp ligands and their antagonist noggin are observed in developing organs and tissues during organogenesis. The first embryonic regions with overlap of multiple Bmp ligands in post-gastrulation tissues are the allantois, yolk sac and cardiac crescent (compare ). Shortly thereafter, a similar direct overlap of Bmp2, 4, 7 and noggin are all observed in the fusing seam of the telencephalon (with Bmp2 being weakest and noggin being strongest) and in the dorsal most tips of the neural folds, both before and after their fusion into the neural tube (compare and ).
Bmp co-expression is also evident in the early developing heart. Expression in the heart is initially distinct: Bmp2 is strongly expressed in the AVC ( inset and ); Bmp4 is localized to both the inflow, or sinus venosus, and the outflow tracts (, and ); Bmp7, in contrast, is found throughout the heart at low levels ( and ). However, later during embryogenesis, Bmp2 continues to be highly localized and robustly expressed within the AVC, while Bmp4 transcripts increase in the outflow tract and Bmp7 become more ubiquitous throughout the myocardium (compare , panels 5 and panels 2). Interestingly, noggin is notably absent from the heart at most of these early stages, with only slight and transient expression observed around E9.0 ( and ).
Another tissue showing evident overlap of the Bmp ligands is the limb bud. Bmp2
, and 7
are all expressed in the limb bud as previously described ( and ) (Ahn et al., 2001
; Francis et al., 1994
initiates around E9.0 in the ventral ectoderm of the limb bud, as previously noted (Lyons et al., 1995
), and can be found shortly thereafter strongly expressed along the AER ( and ). Bmp4
are initiated slightly earlier, as the limb bud emerges, and they are more broadly and more strongly expressed throughout both the epithelium and the underlying mesenchyme of the limb bud ( and ). Notably, noggin
is absent from this domain of Bmp ligand co-expression ( and ).
In somites, all three ligands and their antagonists show dynamic patterns of expression. While they are all expressed at low levels during early somite development, by E9.0, noggin and Bmp4 are expressed robustly in the dorsal portions of the somites, while Bmp2 and 7 are just initiating low levels of expression ( panels 4 and 5; panels 3). By E9.5, Bmp7 and noggin become evident in the ventral- and dorsalmost portions of the somites ( and ). Then at E10.5, all three ligands and their antagonist are strongly expressed in nested and overlapping patterns, as follows ( panels 6): Bmp2, mid-somite region, stronger in anterior trunk of embryo; Bmp4, low ventral somite expression in anterior trunk, robust dorsal somite expression along entire embryonic axis; Bmp7, high expression in both the dorsal and ventral domains of the somites, along entire embryonic axis; noggin, low expression in ventral portion of somites, stronger expression in dorsal edge of somites, especially in anterior trunk ( panels 6, and panels 6).
Bmp ligand expression is also striking in the budding organs of the developing gastrointestinal tract. While Bmp2
is almost undetectable in the foregut ( and ), Bmp4
is expressed in a strikingly unique pattern around the developing midgut ( and ). Strong asymmetric expression is observed on the right dorsal side of the gut tube, along the entire length of the dorsal mesogastrium, along the stomach and pancreas ( and ), which winds around the lateral right edge of the developing anterior gut tube. This asymmetric expression is particularly interesting, because it is associated with the coincident breaking of embryonic symmetry (Hecksher-Sorensen et al., 2004
), when the pancreas (located posterior to stomach) begins to swing left and the gut tube initiates ‘turning’. Expression of Bmp4
in this region is interesting in that BMPs are known to modulate other extrinsic signals, such as Fgfs, which have been reported in this region (Hecksher-Sorensen et al., 2004
) and have been linked to pancreatic development (Bhushan et al., 2001
is notably absent in this midgut region, while Bmp7
is strong but symmetrical in the posterior stomach and pancreatic domain ( and ).
During this time, while Bmp2
is completely absent from the developing lungs, Bmp4
is strongly expressed in the lung bud mesenchyme and Bmp7
initiates in the tips of the lung epithelium ( and ). Noggin
, although expressed at low levels in the distal lung bud mesenchyme, is absent from the epithelium of the lung buds and from the midgut tube ( and ). Thus, Bmp4
, and noggin
are all three expressed in a restricted manner in the lung buds, as previously shown for Bmp4
(Bellusci et al., 1996
), with Bmp4
primarily in the mesoderm (m), and Bmp7
in the epithelium (e) ().
Relative levels of Bmp and noggin expression in different tissues at different stages are compared in . Overall, we can summarize Bmp ligand expression during early organogenesis as dynamic and often highly localized to distinct tissues and organs during development. As expected, we observe that Bmp ligand expression is particularly enriched in areas where bone growth is required, such as the proximal region of the limb buds, where in addition noggin is completely absent. However, we note that noggin overlaps in a few domains where multiple individual Bmp ligands are expressed, including the neural tube and somites during early embryogenesis, but is absent from other tissues high in Bmp ligand expression, such as the limb buds, otic vesicle, or most other mesodermal and endodermal derived tissues.
Summary of Bmp ligand and noggin expression
BmpR1a – E7.5 to E10.5
In contrast to the localized and distinct expression patterns of Bmp2, 4, and 7 during early mouse embryogenesis, the BMP receptor Bmpr1a is expressed more ubiquitously throughout most embryonic tissues. Expression can be detected at E7.25, albeit at low levels (). Around E8.25, slightly increased levels of transcripts can be detected in the neural folds, as well as the lateral plate mesoderm (). However, moderate levels of transcripts can be observed throughout the embryo, when compared to controls ( and data not shown). By the end of embryonic turning (E8.75), BmpR1a is detected in all tissues examined, with the notable exception of the heart atria and ventricle, which have either low or undetectable levels (arrowhead in ). Increased expression can be seen in the telencephalon, the dorsal neural tube, the pharyngeal endoderm and the first two branchial arches ( and ).
Expression of Bmp receptors during embryogenesis
Expression of Bmp receptors in developing tissues
Sections showing Bmp receptor expression
At E9.0, expression is still widespread, but has increased from moderate to high levels, with more intense expression detected in the telencephalon, the first branchial arch and the emerging limb bud (, and as previously shown in (Dewulf et al., 1995
). In addition, higher levels of expression are detected throughout the neural tube, lateral plate mesoderm and limb buds (, and ). Expression continues to appear either low, or absent in the heart myocardium during early heart development (, and ). The increase in limb bud, pharyngeal and neural tube expression intensifies in the following days, from E9.5 to E10.5 (). In particular, the anteriormost portion of the head expresses high levels of BmpR1a
at E10.5. The posterior portion of the tail and most somites also increase expression (). As the hindlimbs (hl) appear, expression of BmpR1a
appears relatively high in this emerging tissue (). At these later stages, BmpR1a
is ubiquitously expressed at lower levels in the stomach, mesogastrium of the stomach, and lung buds (, and ).
BmpR1b – E7.5 to E10.5
In contrast to the relatively ubiquitous expression of BmpR1a
expression is more restricted to specific tissues throughout most of early development. At E7.5 expression is present at low levels throughout the early embryo, but is particularly evident in the forming AIP and the distal yolk sac (). This is in contrast to previous findings that Bmpr1b
expression does not appear until E9.5 (Dewulf et al., 1995
). By E8.5, expression is either low or undetectable in most tissues, however localized expression becomes detectable in the developing neural folds (), including two distinct patches in the hindbrain, in the region of rhombomere 3 (r3) ( inset and ). Yolk sac, neural tube, rostral neural folds, somites and lateral plate mesoderm are notably devoid of detectable expression. At E8.75, expression continues in the hindbrain and developing otic vesicle and begins to increase in the telencephalon and in the lateral plate mesoderm (, and ).
By E9.0, BmpR1b
becomes strongly expressed in the anteriormost telencephalon, the optic cup, the first branchial arch and the hindbrain, while expression has appeared at low levels in the dorsal neural tube, the endoderm, the limb buds, the liver and throughout the somites (, and ). At E9.5, expression increases in the somites, but declines in most of the gastrointestinal tract ( and ). Expression is also observed in the dorsal neural tube, head (hd) and branchial arches as previously shown (Dewulf et al., 1995
). This same pattern is mostly maintained through E10.5, when expression is still evident in the head, branchial arches, somites, dorsal neural tube, and with low levels of expression maintained in the limb buds ( and previously shown by (Dewulf et al., 1995
). Expression in the lungs, gut tube and heart is either low or undetectable (, and ). However, expression continues in both dorsal neural tube and somites ().
BmpRII – E7.25 to E10.5
BmpRII expression resembles BmpR1a expression in its widespread embryonic distribution. From E7.25 to E8.25, expression is almost negligible, notably lower than either BmpR1a or BmpR1b (). However, ubiquitous expression begins after turning, at E8.75 ( and previously shown in Bernard et al., 1997). At this stage, BmpRII can be found at moderate levels in most tissues, with increased expression in the dorsal rostral neural tube, head, tail, and dorsal region of the somites (, and ). However, like BmpR1a and BmpR1b, relatively lower levels are observed in the early heart than those observed for Bmp ligands.
At E9.0, expression continues to increase in all tissues, with a marked increase in the anteriormost telencephalon, branchial arches, limb bud and tail tip mesoderm (, and ). Continued low to negligible levels are observed in the heart ( and ). This expression distribution remains constant through E9.5, with levels increasing in the posterior tail, first branchial arch, developing liver and sinus venosus region (). At E10.5, expression is now evident at low levels in the heart myocardium and gut tube (, and ). Expression in limb buds, both forelimbs and hindlimbs becomes strong by this stage ( and ). In addition, expression in somites intensifies, albeit diffusely, throughout both the dorsal and ventral portions, along the entire embryonic axis ().
Expression of Bmp receptors in developing organs and tissues
Overall, expression of the Bmp receptors is relatively widespread in organs and tissues during early embryonic mouse development. BmpR1a and BmpRII are expressed ubiquitously in most tissues throughout development, whereas expression of BmpR1b displays more restriction to specific tissues. At E8.75, however, all three Bmp receptors are expressed in the fusing neural folds, with each receptor having its own distinct pattern (, and ). For example, BmpR1a is enriched in the cephalic region of the neural folds (absent or low in midbrain region), while BmpR1b is only observed in a set of small patches within rhombomere 3 of the hindbrain region, and BmpRII is expressed throughout the neural folds with increased expression in the dorsalmost neural tube along its length. Similarly, later between E9.5 and E10.5, all three receptors are found in the telencephalon and branchial arches ( panels 5–6 and panels 2). However, BmpR1b and BmpRII are strongly expressed in neuroectoderm of the roof of the mouth anlagen, while BmpR1a is distinctly absent from this region ( panels 2 and data not shown).
Differences in receptor expression can also be observed in the E10.5 gut tube ( panels 4, and panels 4–5). BmpR1a is expressed ubiquitously in all budding organs examined, including the developing lungs and stomach, while BmpR1b is completely absent from the lung buds and only slightly expressed in the stomach. Like BmpR1a, BmpRII expression is widespread in this region, with higher levels near the tips of the lung bud mesoderm and the mesogastrium of the developing gut tube. Interestingly, all three receptors are expressed at notably low to absent levels in the developing heart (–, panels 2 and 5, and panels 2).
Patterns of Bmp receptor gene expression are also distinct, but overlapping in the developing somites. BmpR1a is most strongly expressed throughout the somites, from E9.0 to E10.5 (). While expression of BmpR1b is lower and localized to the medial region of the somites (), that of BmpRII is more widespread in the somites at E9.0, but becomes slightly enriched in the dorsal portion of the anterior trunk somites by E10.5 ().
Nonetheless, Bmp receptors 1a
display overlaps in expression in many tissues and organs. Both receptors have markedly strong expression in the branchial arches at E9.0 ( and ) and are weakly expressed in the heart throughout development from E8.75 to E10.5 (– and ). Interestingly, in the limb buds, while BmpR1a
are strongly expressed in the limb mesenchyme, they are notably absent from the ectoderm of the forelimb, including the AER ( and ). This is surprising given reports that describe inactivation of BmpR1a
in the AER or ventral limb ectoderm, which show that BmpR1a
is required in this tissue (Ahn et al., 2001
; Pajni-Underwood et al., 2007
). However, BmpR1a
possibly plays a greater role in the hindlimbs as these are more severely affected in Msx2-cre;BmpR1aflox/null
mutants (Pajni-Underwood et al., 2007
). Overall, we could generalize that Bmp receptors are expressed in a more widespread manner than their ligands and display more overlap. When we analyze the expression patterns of both ligand and receptors, we find that ligand expression is not confined to regions that completely overlap with domains of receptor expression, suggesting the possibility that other receptors, such as activin receptors, function to transmit BMP signaling in those regions. Relative levels of Bmp receptor co-expression are summarized in .
Summary of Bmp receptor expression