BMP4 Plays a Key Role in Left–Right Patterning in Chick Embryos by Maintaining Sonic Hedgehog Asymmetry  Anne-Hélène Monsoro-Burq, Nicole M. Le Douarin  Molecular Cell  Volume 7, Issue 4, Pages 789-799 (April 2001) DOI: 10.1016/S1097-2765(01)00223-4

Figure 1 Comparison of Bmp4, Shh, and Fgf8 Expression at HH5 to HH7 Comparison of hybridization for Bmp4 ([A], [E], [I], [M], and [N]) or Shh ([C], [G], and [K]) to double labeling for both genes ([B], [F], and [J]) and expression of Fgf8 ([D], [H], and [L]). Bmp4 expression appears on the right side of the node at HH5− ([A]), is reinforced at HH6/7− ([E]), and is downregulated in the node at HH7+/8 ([I]). This pattern is exactly complementary to that of Shh from HH5 ([B] and [C]) to HH7− ([F] and [G]). At HH6, Fgf8 expression, which was hardly detected before (D), appears strongly in the primitive streak and on the right side of the node ([H]). From HH7+ onward, Shh and Bmp4 asymmetrical expression patterns decrease ([I] and [K]); both genes seem to overlap in the node ([J]). Fgf8 right-sided expression is also less prominent ([L]). Sections realized in a 1ss embryo showed that Bmp4 is strongly expressed in the primitive streak ([M]), where it is symmetrical on L–R axis, whereas in the node ([N]) Bmp4 is found exclusively on the right side. PS, primitive streak; HN, Hensen's node; L, left; and R, right. In (A)–(L), the scale bar is 400 μm, and in (M) and (N) the scale bar is 160 μm Molecular Cell 2001 7, 789-799DOI: (10.1016/S1097-2765(01)00223-4)

Figure 2 Regulation of Bmp4 Gene in Hensen's Node The regulation of Bmp4 gene expression was analyzed by implanting either control ([A], [B], [E], [F], and [I]), Activin A– ([A], [C], and [D]), or SHH- ([E], [G], [H], [J], and [K]) containing beads in an ectopic position on one side of the node. Control beads modified neither Bmp4 nor Shh gene expression patterns ([B], [F], and [I], yellow arrows). Activin A–soaked beads (red arrows) upregulated Bmp4 expression in close vicinity to the node ([C] and [D]) but not in more anterior positions, along the notochord, or in more lateral areas ([D]). The black line indicates the induced signal, and the black arrow points at normal Bmp4 expression on the right side of the node. SHH-containing beads ([G], [H], [J], and [K], red arrows) prevented Bmp4 expression (blue staining) in the node and the most anterior part of the primitive streak ([G] and [J]; dark-field image and close up). In this area, Shh gene (red staining) was expressed without L–R polarization ([H] and [K]; close up). In (A)–(H), the scale bar is 400 μm, and in (I)–(K) the scale bar is 200 μm Molecular Cell 2001 7, 789-799DOI: (10.1016/S1097-2765(01)00223-4)

Figure 3 Shh Expression in the Node Is Controlled by Bmp4 (A–D) The effect of exogenous BMPs on Shh gene expression was tested by implanting either control, BMP2, or BMP4 sources on the left side of the node ([A]). Control cells or beads (yellow arrow) did not modify Shh pattern ([B], the black arrow shows the most caudal level of Shh expression on the left side of the node, while the arrowhead indicates the most posterior level on the right side). BMP2-producing cells ([C], red arrow) induced a mild downregulation of Shh signal on the left ([C], note that the black arrow and the arrowhead are closer, and that labeling intensity is lower on the left side, as compared to [B]). Recombinant BMP4 ([D], red arrows) completely abolished Shh expression in the node (note that the black arrow and the arrowhead are at the same anterior level), showing that BMP4 was sufficient to block Shh gene activation in the node. (E–H) Endogenous BMP activity was blocked by providing the BMP antagonist Noggin on the right side of the node ([E]). When compared to controls ([F]), recombinant Noggin coated on beads (green arrow) resulted in a moderate extension of Shh expression on the right side of the node ([G], note that the arrowhead, indicating the most caudal extension of Shh staining on the left, is closer to the right arrow, which indicates Shh signal on the right). Cells producing the Noggin protein led to a strong Shh expression on the right side of the node, resulting in a symmetrical Shh staining pattern ([H], note that the black arrow and the arrowhead are at the same caudal level). The scale bar is 400 μm Molecular Cell 2001 7, 789-799DOI: (10.1016/S1097-2765(01)00223-4)

Figure 4 Noggin Early Expression in the Axial Mesoderm Noggin expression was first detected at HH4 ([A]) in the anterior part of the node (arrow). Afterwards, Noggin transcripts remained localized in the head process at HH4+ ([B]) and in the prechordal plate and notochord later on ([C]–[E]). The most caudal level of expression was located at the anterior tip of the node (arrows). The scale bar is 500 μm Molecular Cell 2001 7, 789-799DOI: (10.1016/S1097-2765(01)00223-4)

Figure 5 BMPs Can Override BMP Antagonists and Block Shh Expression in the Notochord In order to test the function of the BMP antagonists recorded in the notochord, BMP sources were provided in more anterior locations along the notochord. In control grafts with cells ([A], yellow arrow) or beads ([D]), Shh expression was perturbed neither in the node (indicated by arrows 1 and 2) nor in the notochord. After implanting BMP2-producing cells ([B] and [C], red arrow), Shh expression in Hensen's node was downregulated (between arrows 1 and 2), while the notochord was curved and its Shh staining slightly diminished (between arrows 2 and 3). BMP4-soaked beads (red arrow in [E] and [F]) abolished Shh expression not only in the node (between arrows 1 and 2) but also in the posterior part of the notochord (between arrows 2 and 3). In (A), (C), (D), and (F), the scale bar is 200 μm, and in (B) and (E) the scale bar is 400 μm Molecular Cell 2001 7, 789-799DOI: (10.1016/S1097-2765(01)00223-4)

Figure 6 Fgf8 Expression Is Regulated by BMPs in the Node and Primitive Streak BMP activity on Fgf8 gene expression was analyzed by overexpressing BMPs on the left side of the node ([A] and [D]) or implanting a Noggin source on the right side of the node and primitive streak ([G]). Control grafts did not perturb the right-sided asymmetrical expression of Fgf8 ([B] and [H], yellow arrows). BMP2-secreting cells ([C], red arrow) weakly induced Fgf8 on the left side of the node, resulting in a symmetrical pattern ([C]). BMP4-soaked beads ([E], red arrows) strongly induced Fgf8 on the left side ([E], black arrows) or in the entire node ([F], black arrows). In contrast to control CHO cells ([H], yellow arrows), Noggin-secreting cells ([I], green arrows) abolished Fgf8 expression in the node (black arrow) and strongly downregulated its expression in the primitive streak. The graft of BMP2-producing cells ([J], red arrow) on the left side of the node resulted into a mild induction of Fgf8 on this side ([J], black arrow, blue staining) in a domain where Shh was still expressed ([B], red staining). In (B) and (C), the scale bar is 250 μm, and in (E)–(K) the scale bar is 500 μm Molecular Cell 2001 7, 789-799DOI: (10.1016/S1097-2765(01)00223-4)

Figure 7 Molecular Interactions in Hensen's Node during Early L–R Polarization During the early phase of caudalward regression of Hensen's node, from HH5− to HH7−, we propose that the maintenance of Shh gene asymmetry on the left side of Hensen's node and the induction of the FGF8-dependent cascade on its right result from right-sided BMP4 activity. (A) At HH4, the environment imposes the left–right polarity axis (Pagàn-Westphal and Tabin, 1998). (B) At HH4+/5−, within the node, Bmp4 gene expression is induced on the right by Activin-like signals, while SHH prevents Bmp4 activation on the left side. In turn, BMP4 is required to prevent Shh reactivation on the right. This regulatory loop involving BMP4 and SHH proteins ensures the persistence of the initial asymmetry in Shh and Bmp4 gene expression. BMP antagonists such as Chordin, on the left side of the node, or Noggin, in the axial mesoderm, are likely to block BMP4 molecules diffusing from the right side. (C) At HH6, BMP4 activates the FGF8 signaling cascade in the node, while SHH upregulates Nodal in the paraxial mesoderm (see text for further details) Molecular Cell 2001 7, 789-799DOI: (10.1016/S1097-2765(01)00223-4)