Cells that Express MyoD mRNA in the Epiblast are Incorporated into the Developing Eye
MyoD+ epiblast cells were identified in the stage 2 epiblast based on their expression of the G8 antigen and MyoD mRNA () (Gerhart et al., 2006
). Living stage 2 embryos were fluorescently labeled with the G8 MAb in order to track the sites of incorporation of MyoD+ cells during eye development. In a previous study we determined that cells labeled with the G8 MAb applied to the stage 2 epiblast were integrated into the developing somites, heart and central nervous system (Gerhart et al., 2006
; Gerhart et al., 2007
; Gerhart et al., 2008
). Epiblast cells that expressed the G8 antigen within three hours after the embryo was incubated with the G8 MAb were not tagged by residual unbound antibodies or the transfer of antibodies from other cells (Gerhart et al., 2006
Identifying and tracking MyoD+/G8+ cells from the stage 2 epiblast into tissues of stages 5 and 7 embryos
All MyoD+ epiblast cells were present in the posterior/marginal region of the stage 2 epiblast (). Approximately 24 hours after applying the G8 MAb and fluorescent secondary antibodies (stage 5-5+), fluorescent cells were observed along the posterior margin of the epiblast, within the primitive streak (not shown), lateral to the streak and in the head process, a mesodermal structure that forms the notochord (). By stage 7, fluorescent cells were observed in the unsegmented paraxial mesoderm (not shown) and prechordal mesoderm (), the tissues of origin of the extraocular muscles (Couly et al., 1992
; Gage et al., 2005
; Jacob et al., 1984
; Johnston et al., 1979
; Noden, 1983
). G8 labeled cells also were found in the region of the ectoderm containing the primordia of the sensory placodes (Litsiou et al., 2005
), including the lens placode (), and in the anterior/lateral neural plate from which the optic vesicle evaginates (Chow and Lang, 2001
) (). The movements of G8 labeled cells from the posterior epiblast suggest that some MyoD+ epiblast cells enter the mesoderm while others remain in the ectoderm.
By stage 17, MyoD+ cells tagged with the G8 MAb were found as single cells or in foci of approximately two to eight cells within the eyes (). G8 labeled cells (G8+) constituted a minor subpopulation in the eye, and therefore, not all sections contained these cells in the same locations ( and ). In the lens vesicle, G8+ cells were present in the equatorial zone where lens epithelial cells initiate differentiation, as well as in the anterior epithelium and posterior primary lens fiber region (). The optic cup contained foci of G8+ cells in its anterior margin () and within the posterior region of the developing neural retina (). The G8 expressing cells in the chick retina may be related to the rare population of retina cells that weakly express GFP under the regulation of enhancer and promoter elements of the MyoD gene (Kirillova et al., 2007
). Cells labeled with the G8 MAb also were present within the periocular mesenchyme surrounding the lateral and posterior regions of the optic cup (). The results of these experiments demonstrate that MyoD+/G8+ cells are incorporated into skeletal muscle (periocular mesenchyme) and non-skeletal muscle forming tissues (lens and optic cup) during eye development.
Distribution of G8+ epiblast cells in the eyes of stages 17 and 18 embryos
Co-expression of G8, MyoD mRNA and Noggin in the developing eye
G8+ Cells Express MyoD mRNA and Noggin in the Developing Eye
Cells labeled with the G8 MAb were examined for the expression of MyoD mRNA and Noggin mRNA and protein in representative sections through the eyes of three stages 17–18 and three stages 23–24 embryos (). All G8+ cells expressed MyoD mRNA. At stage 23–24, Noggin mRNA was found in all G8+ cells in the eyes and only 7% of the cells expressing Noggin lacked detectable levels of G8. Greater than 80% of the cells that expressed MyoD mRNA were stained with an antibody to Noggin protein. Two thirds of the cells with Noggin mRNA or protein contained G8 or MyoD mRNA.
Expression of the G8 antigen, MyoD mRNA and Noggin in the developing eye
The distribution of double and single labeled cells is shown in . G8+/MyoD mRNA+ cells (), G8+/Noggin mRNA+ cells (), MyoD mRNA+/Noggin protein+ cells (), and Noggin protein+/MyoD mRNA-cells () were found in the lens, anterior margin and posterior derivatives of the optic cup, and periocular mesenchyme of stages 17–18 and 23–24 embryos. A few Noggin mRNA+/G8− cells () and Noggin protein+/MyoD mRNA− cells were present in the lens and optic cup derivatives (). These double labeling experiments demonstrate that G8+/MyoD+ cells are the major source of Noggin in ocular tissues.
Ablation of G8+/MyoD+ Cells in the Epiblast
MyoD+ cells were ablated in the epiblast by lysing G8 labeled cells with complement (Gerhart et al., 2006
; Gerhart et al., 2008
). Consistent with our previous experiments (Gerhart et al., 2006
), the stage 2 epiblast contained 67 ± 3 G8+ cells (n = 6 embryos). All G8+ cells were located in the posterior/medial region of the epiblast. Cell lysis was demonstrated by incubation in trypan blue (). Embryos treated with G8 and complement contained 66 ± 2 lysed cells (n = 4), whereas only one lysed cell was observed in two embryos treated with complement alone.
Lysis of MyoD+ cells in the epiblast and its effects on eye morphogenesis
To determine whether more cells emerged in the epiblast after ablation, a secondary antibody conjugated with fluorescein was applied to the embryo directly after washing out the complement to label cells that had bound the G8 MAb. Three hours later, the embryos were re-stained with another application of the G8 MAb and a secondary antibody conjugated with rhodamine. Cells tagged with fluorescein only or both fluorochromes corresponded to the original population of G8 labeled cells lysed with complement in the stage 2 epiblast, whereas cells stained with rhodamine only had expressed G8 after the initial labeling period (). There were 11 ± 3 cells (n = 5 embryos) that were stained with rhodamine only, and therefore, had emerged in the epiblast after the ablation procedure (). In four out of five embryos, 64–100% of these newly labeled cells were present on the left side of the embryo in a field adjacent to the lysed cells. This experiment demonstrates that additional cells express the G8 antigen after lysis of the original population, and in the majority of these embryos, the newly emerging G8 expressing cells were present on the left side of the posterior epiblast. These results suggest that subtle differences in the timing of ablation (early to late stage 2) could lead to phenotypes of varying severity.
Ablation of G8+/MyoD+ Cells in the Epiblast Adversely Affects Eye Development
On the third day after ablating MyoD+ cells in the epiblast, the eyes appeared normal externally. Examination of the complete set of serial tissue sections from two of these embryos revealed that the left eyes had a normal morphology while the lens vesicles of both of the right eyes were more densely stained than the control lenses and lacked a lumen (). By the fifth day after ablation, all of the right eyes and 27% of the left eyes had externally visible eye defects, including clinical anopthalmia, micropthalmia, an abnormal shape and/or altered pigmentation ( and ; ). The right eyes were more severely affected than the left eyes ( and ). The asymmetry in the severity of the defects may reflect the emergence of additional G8+ cells on the left side of epiblast after the ablation procedure.
Histological appearance of the eye defects that arise after ablating MyoD+ cells in the epiblast
Incidence of eye defects in ablated embryos in the presence or absence of exogenous Noggin
Effects of ablating MyoD+ epiblast cells on ocular morphology
None of the eyes in control embryos treated with buffer, the G8 MAb or complement alone, or the E12 MAb and complement had visible eye defects ( and ). The E12 MAb binds specifically to a subpopulation of epiblast cells that expresses the NeuroM transcription factor, but not MyoD or G8 (Gerhart et al., 2006
; Strony et al., 2005
). Ablation of the E12+ and G8+ subpopulations of epiblast cells results in different malformations, thereby demonstrating the specificity of the ablation procedure (Gerhart et al., 2006
Histological analyses of the ablated embryos revealed eye defects of varying severity ( and , ). All of the right eyes and 64% of the left eyes were abnormal. Half of the left eyes that appeared normal externally had internally visible defects. A frequently observed malformation was folding of the neural retina within the vitreous body (, ). In some embryos, retinal folding occurred in right and left orbits of normal size, suggesting that the retinal tissue had undergone abnormal expansion. Retinal folds were present in eyes with either a normal lens (), or more commonly, eyes with lens defects (). The lens malformations included alterations in size (enlarged or reduced) or an absence of identifiable lens tissue ().
One of the ablated embryos contained a normal lens accompanied by retinal folds in the left eye (embryo #8, ). The lens and optic cup in the right eye of this embryo were small and malformed and appeared to be arrested in development. Pigment was present throughout the optic cup (, embryo #8). Another embryo with retinal folds and no lens in the left eye lacked internally visible ocular tissue on the right (, embryo #10). The RPE had either a normal morphology or contained folds that were less pronounced than those of the neural retina (). The embryo with the most severe phenotype had only a small, dense piece of pigmented tissue on both sides of the head (, embryo #11). The conclusion from these experiments is that ablation of MyoD+ cells in the stage 2 epiblast disrupts the morphogenesis of the lens and/or optic cup derivatives. While both eyes are affected by the loss of MyoD+ epiblast cells, the defects in the right eye are more severe than those in the left eye.
Noggin Producing MyoD+ Cells are Reduced in the Eyes of Ablated Embryos
Determination of whether the severity of eye defects correlated with the numbers of residual MyoD+ cells was carried out by comparing the number of G8+ and/or MyoD+ cells in every section through the eyes of stages 23–24 control (unablated) and ablated embryos (). All three control embryos contained fewer MyoD+ cells in the right lens than the left lens. Although the right lenses were smaller than the left lenses in two embryos (#1 and #3: number of sections containing lens tissue: right 30 and 28, left 55 and 36), both lenses were similar in size in the third embryo (#2: right 26, left 27). Two of the control embryos (#2 and #3) contained fewer MyoD+ cells in the right than the left neural retina and periocular mesenchyme. Differences in the numbers of MyoD+ cells in the right and left eyes did not correlate with the differences in the overall size of the eyes in control embryos, as one of the right eyes was larger than the left eye (embryo #2), another was smaller on the right (embryo #1), and the third pair of eyes was similar in size (embryo #2). Therefore, differences in the number of MyoD+ cells in the two eyes does not consistently correlate with the size of the tissue in which they were located.
Distribution of MyoD+/G8+ epiblast cells in control and ablated eyes
MyoD+ and G8+ cells were reduced or eliminated in ocular tissues of ablated embryos compared to control embryos ( and ). One ablated embryo contained residual MyoD+/G8+ cells in the lens and retina (embryo #2, ). Both of these tissues appeared to have a normal morphology (). MyoD+/G8+ cells were not found in the periocular mesenchyme of this eye, suggesting that sufficient numbers of these cells were present in the lens and retina to support their morphogenesis independently of the mesenchyme.
Distribution of Noggin+ cells in the eyes of ablated embryos
Although the second embryo had the same number of residual G8+/MyoD+ cells in the retina of the left eye as the first ablated embryo, its retina had a folded appearance (embryo #7, and ). Only two G8+/MyoD+ cells remained in the lens of this eye. The left eye of the third embryo also contained retinal folds even though the folds contained more MyoD+/G8+ cells than the retina of the eye that lacked folds (embryos #9 and #2). The lens of this eye had only two MyoD+/G8+ cells but appeared to have a normal morphology. It is therefore possible that a critical number of residual MyoD+/G8+ cells in either the retina or the lens may compensate to some degree for their loss in the adjacent tissue.
The right eyes of ablated embryos contained fewer residual MyoD+/G8+ cells than the left eyes (). Whereas MyoD+/G8+ cells were present in the left eyes of all three embryos, these cells were not detected in the lens or retina of two of the right eyes of ablated (embryos #2 and #7, ). One of these eyes lacked detectable lens tissue and the other had a misshapen lens (). A few MyoD+/G8+ cells were found in the right retina and lens of the third embryo (#9). This eye also had a misshapen lens. All of the right eyes exhibited retinal folding. These results demonstrate that the asymmetry in the severity of the eye defects correlates with a greater reduction in the number of residual MyoD+/G8+ cells in the right eye than the left eye.
The results displayed in demonstrate that MyoD+/G8+ cells are the primary source of Noggin in stages 23–24 eyes. The eyes of ablated embryos were examined for Noggin expression to determine whether other cells compensated for the loss of Noggin producing, MyoD+/G8+ cells (). Six Noggin+ cells were observed in the left eye of embryo #7 and half of these cells expressed MyoD. Only one Noggin+ cell was present in the right eye of this embryo. This cell lacked detectable levels of MyoD mRNA. The second embryo contained 17 Noggin+ cells. Most of these cells were present in the retina and all but one Noggin+ cell was stained for G8. Five Noggin+/G8+ cells were detected in the right eye of this embryo. This analysis revealed that ablation of MyoD+ cells in the epiblast results in a reduction in Noggin producing cells in the eye. Comparison of the phenotype of the ablated eyes with the number of residual Noggin producing MyoD+ cells indicates that the severity of ocular malformations increases with decreasing numbers of a source for Noggin.
Exogenous Noggin Compensates for the Ablated MyoD+ Cells
Determination of whether exogenous Noggin could compensate for the missing MyoD+ cells was carried out by implanting beads soaked in Noggin into stages 10–13 ablated embryos after the lens placode and optic vesicle were specified within the ecotoderm and neuroectoderm, respectively. The most cranial implantation site was adjacent to the first somite on the right side of the embryo (). This implantation site was expected to limit the amount of Noggin that diffused to the eye, thereby reducing the likelihood that eye defects would result from excessive blockage of BMP signaling, as occurs when Noggin is over-expressed in the eye (Huillard et al., 2005
; Zhao et al., 2002
). The beads eventually dislodged from the tissue but were secured in the vicinity of the implantation site by the embryonic membranes. As the embryo developed, the first bead was present in the cervical flexure adjacent to the eye ().
Whereas the eyes of ablated embryos implanted with beads soaked in buffer continued to exhibit the same defects as ablated embryos lacking beads (), the external morphology of both eyes from five stages 23–24 ablated embryos appeared normal after supplementation with exogenous Noggin (, ). Histological analyses revealed that the right and left eyes of three out of four ablated embryos that received Noggin soaked beads appeared normal (, ). The ability of exogenous Noggin to restore normal eye development after ablation in the majority of embryos, combined with the results of the previous experiment in which it was found that Noggin producing, MyoD+/G8+ cells were reduced or absent in ocular tissues of ablated embryos, suggests that MyoD expressing cells are a critical source of Noggin during eye development.