Vessel formation in branchial arches
The development of vessels in the branchial region was extensively described in chicken embryos (Hiruma and Hirakow, 1995
), and is very similar in the quail embryo, although the timing of their development is somewhat different. Generally, in quail embryos the aortic arches remodel faster; they form approximately one stage later and regress few stages earlier than in chick embryos. At stage 15, the first aortic arch is present as a vessel connecting the ventral and dorsal aorta, and the anterior cardinal vein is already developed (). Immediately after the first aortic arch has formed, the second arch starts to develop. The third aortic arch develops around stage 18 () and the fourth one develops at stage 20. At stage 21, the fifth and sixth aortic arches start to develop and fully formed at stage 23 (). The first and second aortic arches start to regress at stage 18. The fifth aortic arch, a minor bypass of the sixth aortic arch, starts to regress at stage 24. While the first, second and fifth aortic arches are transient structures; the third, fourth and sixth arches persist.
(A–D) Normal development of head and neck vessels in quail embryos. Vessels stained with QH1 Ab. (E–F) Expression of Shh, visualized with 5E1 Ab.
At stage 15, only the aortic arches supply the branchial arches (), while at stage 18, the dorsal aorta, the aortic arches as well as the anterior cardinal vein give off smaller branches and capillaries (). At stages 21 and 23 the development of small vessels and capillaries continue in the whole region, resulting in a dense capillary network in branchial arch mesenchyme and in the regions surrounding the brain and eyes ().
Besides luminized vessels and capillaries, we observed also an increasing number of isolated angiogenic cells at stages 18–23 (). These cells are mainly present around the anterior cardinal vein and in branchial arches, while in the area in the vicinity of the dorsal aorta only few of these cells are detected. These isolated angiogenic cells are evenly distributed in the mesenchyme.
Inhibition of Shh signaling with anti Shh Antibodies produced by 5E1 hybridoma cells
In general, we detected Shh in a pattern and timing consistent with that observed in the chick (Roelink et al., 1995
). The notochord is a prominent site of early expression, and starting at stage 15, Shh is expressed in endoderm lining the branchial arches and the foregut (). To conditionally attenuate Shh signaling, we injected 5E1 (anti Shh) hybridoma cells under the vitelline membrane of stage 10–12 embryos, which were analyzed at stages 18–23. 5E1 hybridoma cell-derived anti-Shh antibodies distribute widely in injected embryos. Visualizing the 5E1 antibodies by simply using an anti IgG secondary antibody on sectioned embryos, staining is detected at the sites of Shh
expression, such as the notochord, floor plate and endoderm of the branchial arches and foregut. The secondary antibodies also react with 5E1 hybridoma cells, which always remain at the embryonic surface (), just as the control 12CA5 hybridoma cells ().
Inhibition of the Shh response by 5E1 (anti-Shh) Ab produced by hybridoma cells
To verify if the Shh response is efficiently blocked after 5E1 injection, we analyzed the expression of the gene coding for its receptor Ptch1
by mRNA in situ hybridization. Ptch1
expression is invariably upregulated in response to Shh signaling (Marigo and Tabin, 1996
). In control embryos, Ptch1
is expressed in areas adjacent to Shh sources, such as branchial arch mesenchyme, around the foregut, in the ventral part of the neural tube, and around domains of Shh production in the brain (). In embryos injected with 5E1 cells, Ptch1
expression in the neural tube and endoderm is decreased and no Ptch1
expression is found in the abutting mesenchyme (), demonstrating a significant attenuation of the Shh response. Residual expression of Ptch1
could be the result of incomplete inhibition, but also be caused by other Hh ligands, which are not recognized by 5E1 (Goodrich et al., 1997
; Carpenter et al., 1998
Embryos injected with 5E1 and with control 12CA5 hybridoma cells develop slightly slower than uninjected embryos, and staging was performed based on anatomical landmarks. The 5E1 antibody injected embryos largely exhibit a normal gross morphology, although at least half of them have a smaller head compared to control (). Similar cephalic phenotypes have been reported (Ahlgren and Bronner-Fraser, 1999
). Embryos injected with control 12CA5 hybridoma cells have a macroscopic and microscopic anatomy identical to untreated embryos.
Ink injection into blood vessels at stage 18
Vessel malformations in anti-Shh antibody treated embryos
Even at the anatomical level, the effect of inhibiting Shh on vessel development is remarkable. Generally, in embryos injected with 5E1 hybridoma cells development of the aortic arches is delayed. While in stage 18 control embryos the first and the second aortic arches start to regress into capillary plexi, these aortic arches are still present in 5E1 injected embryos, indicating a delay in remodeling ().
A consequence of Shh inhibition is the failure of the anterior cardinal vein and its branches to form functional vessel walls. Hemorrhages were observed frequently (), and these vessels were permeable to ink, unlike the control hybridoma injected animals, which were able to contain the ink within the vessels (). In addition to the ink-permeability, the lumen of either anterior cardinal vein in 5E1 injected embryos is sinusoidal with endothelium-lined protrusions (). This demonstrates that Shh plays an important role in the establishment of a functional wall in the anterior cardinal veins and its tributaries.
QH1 staining of vessel endothelium showing lumen malformations in anti-Shh hybridoma cell injected embryos
Several arteries show abnormal development as a consequence of 5E1 injection as well. The internal carotid arteries are characterized by the presence of a transverse septum over a length of up to 40μm (in 2 of 6 embryos). This septum consists of two layers of endothelium, with mesenchyme in between, dividing the internal carotid artery into two separate vessels, which merge again further rostrally (). Similarly, an abnormal septum is present in the dorsal aorta (in 4 out of 6 embryos). This aortic septum is usually about 60μm long, and is covered with endothelium on both sides (). Although we assume that this septation is a result of delayed fusion, it remains possible that it has formed after the initial fusion of the left and right dorsal aortae. The same domain of the dorsal aorta in control embryos is already fused and has a single lumen. Vessel abnormalities are also found in outflow tracts of the heart. Besides vessels with irregular lumina and unusual invaginations (), we have also observed curving strands of endothelial cells in successive sections, possibly malformed vessels with an incomplete vessel wall () (in 4 of 6 embryos). Embryos injected with the control hybridoma cells had no obvious vessel malformations.
To further assess malformations in vessel endothelium, we determined the expression VEGFR2, which is expressed in endothelial cells (Jaffredo et al., 1998
) albeit not ubiquitously. At stage 18 VEGFR2 is expressed in all endothelial cells of small developing vessels a capillaries, such as brain capillaries. While the only about half the endothelial cells endothelial cells of bigger arteries express VEGFR2. Similarly, the dorsal aorta is lined by VEGFR2 positive cells, but only in its ventrolateral aspect, where the aortic arches are connected. Besides endothelial cells VEGFR2 is highly expressed in the outflow tract myocardium, while outflow tract endothelium contains only few VEGFR2 positive cells. These myocardial cells are probably derived from endothelium (Wilting et al., 1997
). VEGFR2 was also expressed in the notochord, as it was previously reported (Nimmagadda et al., 2004
). We did not detect any difference in VEGFR2 expression in 5E1 injected embryos compared to control, indicating that VEGF is not a critical mediator of the effects of Shh (not shown).
Following the formation of an endothelial layer, the smooth muscle cells start to surround the forming vessels. At stage 18, smooth muscle cells completely cover the endothelial lining of the dorsal aorta and the internal carotid arteries (). The aortic arches have smooth muscle cells only on their lateral side, while an incomplete layer of smooth muscle cells is associated with the outflow tract. Smooth muscle actin is also present in the myotome (). At stage 23, a continuous smooth muscle layer surrounds aortic arches and outflow tracts (). Also all branches of the internal carotid arteries have a continuous layer of smooth muscle cells, while the anterior cardinal veins and their tributaries are devoid of smooth muscle cells. The formation of the smooth muscle cell lining of the vessels is unaffected by injection of 5E1 hybridoma cells (), despite the presence of obvious vessel malformations. Smooth muscle cells also line the abnormal aortic septum (). This is consistent with our observation that the formation of the smooth muscle lining of the dorsal aortae precedes the subsequent fusion of these vessels. Altogether, this indicates that the loss of Shh signaling has little effect on the process in which smooth muscle cells form around new vasculature.
Distribution of vascular smooth muscle cells visualized with smooth muscle actin Ab
Angiogenic and macrophage-like cells in anti-Shh antibody treated embryos
The main effect of conditional Shh inhibition on blood vessel development in embryos injected at later stages (injected in stage 13–15 and harvested at stage 21–23) is the presence of an increased number of free, round endothelial cells, positive for QH1. These cells are not integrated into functional vessel lumina, but aggregate into multicellular clusters. Such aggregates usually are found around the anterior cardinal veins, around the dorsal aorta and in the branchial arches around the aortic arches. Such cell aggregates are not present in control 12CA5 hybridoma injected embryos, where angiogenic cells are fewer and isolated. The increased number of aggregates is most significant in the sixth branchial arch (), around the anterior cardinal vein and just ventral to the dorsal aorta (), while the anterior branchial arches are the least affected. In the first and second branchial arch, we have not observed any significant difference compared to control in the number of endothelial cell aggregates (data not shown). This might either indicate that at the moment of injection the Shh requirement for initiatial vessel formation has passed, or that the absence of de-novo vascularization associated with the regression of the first and second aortic arches results in a absence of these aggregates of QH1 positive cells.
Figure 6 QH1-positive cell abnormalities in branchial arches at stage 21 (A–D, F) and stage 23 (E, G–U); Characterization of QH1-positive cells: macrophage-like cells stained for acid phosphatase (M–O); apoptotic cells positive for cleaved (more ...)
Figure 7 Abnormalities in the number of QH1-positive cells around main vessel trunks at stage 23 – the anterior cardinal vein and the dorsal aorta (A–L); Characterization of QH1-positive cells: macrophage-like cells stained for acid phosphatase (more ...)
In the third and fourth branchial arches Shh inhibition results in an increased number of larger endothelial aggregates compared to 12CA5 injected control embryos. These aggregates are mainly localized in the mesenchyme, sometimes in close proximity to branchial arch ectoderm and endoderm. Such aggregates are not present in control embryos, where we observed only solitary QH1 positive cells, in comparable numbers to the solitary cells in anti-Shh treated embryos ().
The sixth branchial arch is the most affected as measured by the number of QH1 positive cells not part of an obvious vessel wall. These cells are present along the whole extent of the arches and are concentrated in mesenchyme as aggregates and solitary cells. In control embryos only solitary QH1 positive cells are observed. The total number of nonintegrated QH1 positive cells is significantly higher in 5E1 hybridoma injected embryos than in controls ().
The biggest increase of nonintegrated endothelial cells as a consequence of 5E1 injection is detected ventral to the dorsal aorta (). It is possible that this abundance of angiogenic cells is related to the area of hemangiogenesis within the wall of the ventral aorta (Jaffredo et al., 1998
). Increased numbers of endothelial cell aggregates were also found in the vicinity of the anterior cardinal veins. The QH1 positive cells in this area () are the only cells that are predominantly identified as macrophage-like cells, rather than angiogenic cells (). Macrophage-like cells are the phagocytic cells of the early embryo, are derived from hemangioblasts (Cuadros et al., 1992
) and are characterized by their expression of acid phosphatase (). Direct co-localization of acid phosphatase and QH1 staining is not possible due to incompatible fixation and processing requirements.
Apoptosis and proliferation in anti-Shh Ab treated embryos
In many instances during development, loss of Shh leads to decreased proliferation and increased apoptosis, possibly explaining some of the vessel malformations we observed as a consequence of 5E1 injection.
A significant increase in the number of apoptotic, caspase-3 positive cells () is observed around the anterior cardinal veins in 5E1 injected embryos, suggesting a role for Shh in cell survival in this region and, consequently, an increased number of macrophage-like cells is detected. In contrast, in the branchial arches and around the dorsal aorta the frequency of cleaved caspase-3 positive apoptotic cells do not differ from control embryos (). Since the domains of apoptosis coincide with the areas where vessel integrity is compromised, it remains a possibility that the hemorrhages increase apoptosis in the surrounding tissues (, ).
Coincident with the increased apoptosis we observed decreased proliferation near the anterior cardinal vein (, ), but not in the other regions studied.