Holoprosencephaly with a Special Form of Anophthalmia Result from Experimental Induction of bmp4, Oversaturating BMP Antagonists in Zebrafish

Abstract

Vision is likely our most prominent sense and a correct development of the eye is at its basis. Early eye development is tightly connected to the development of the forebrain. A single eye field and the prospective telencephalon are situated within the anterior neural plate (ANP). During normal development, both domains are split and consecutively, two optic vesicles and two telencephalic lobes emerge. If this process is hampered, the domains remain condensed at the midline. The resulting developmental disorder is termed holoprosencephaly (HPE). The typical ocular finding associated with intense forms of HPE is cyclopia. However, also anophthalmia and coloboma can be associated with HPE. Here, we report that a correct balance of Bone morphogenetic proteins (BMPs) and their antagonists are important for forebrain and eye field cleavage. Experimental induction of a BMP ligand results in a severe form of HPE showing anophthalmia. We identified a dysmorphic forebrain containing retinal progenitors, which we termed crypt-oculoid. Optic vesicle evagination is impaired due to a loss of rx3 and, consecutively, of cxcr4a. Our data further suggest that the subduction of prospective hypothalamic cells during neurulation and neural keel formation is affected by the induction of a BMP ligand.

Keywords: Bone Morphogenetic Protein (BMP); antagonists to Bone Morphogenetic Protein (BMP antagonists); crypt-oculoid; eye field; forebrain; holoprosencephaly; rx2; rx3.

Figure 1

(A–Q): expression of bmp-antagonists nog2, chrd, fsta and grem2b in the ANP at 11 and 12 hpf. (A–D): ISH for nog2. (A,B): 11 hpf; (C,D): 12 hpf. (E–H): ISH for chrd. (E,F): 11 hpf; (G,H): 12 hpf. (I–M): ISH for fsta. (I,K): 11 hpf; (L,M): 12 hpf. (N–Q): ISH for grem2b. (N,O): 11 hpf; (P,Q): 12 hpf. (A,C,E,G,I,L,N,P): dorsal view. (B,D,F,H,K,M,O,Q): lateral view. Scalebars indicate 200 µm. (R): summary of experimental procedure. (S–Bb): Embryos were heat shocked at 8.5 hpf and observed at 24 hpf or 48 hpf, resp. (S,T): bright-field image of wildtype at 48 hpf. (U,V): bmp4-induced larva at 48 hpf. (S,U): dorsal view. (T,V): lateral view. Scalebars indicate 250 µm. (W,X): transversal confocal sections of larvae with rx2:GFP at 24 hpf. injection of LY-tdTomato mRNA in zygote. (W): wildtype. (X): bmp4-induced, scalebars indicate 50 µm. (Y–Bb): ISH for rx2 at 24 hpf. (Y,Z): wildtype. (Aa,Bb): bmp4-induced. (Y,Aa): dorsal view; (Z,Bb): lateral view. Scalebars indicate 250 µm.

Figure 2

ISH at 11 hpf after bmp4-induction at 8.5 hpf. left columns wildtypes, right columns bmp4-induced. first and third column dorsal view, second and fourth column lateral view. (A): summary of experimental procedure. Embryos were heat-shocked at 8.5 hpf and analyzed at 11 hpf. (B–E): Expression of emx3 is condensed at the midline after bmp4-induction (arrows). (F–I): the foxg1a domain in the forebrain (arrow) is lost after bmp4-induction. (K–N): expression of eye-field marker six3b is also condensed at the midline after bmp4-induction. (O–R): expression of eye-field marker rx3 (arrow) is lost after bmp4-induction. Scalebars indicate 200 µm.

Figure 3

(A): summary of experimental procedure: embryos were injected at 1-cell-stage and analyzed at 24 hpf or 48 hpf. (B–E): Brightfield-images at 48 hpf. We observed anophthalmia in F0 rx3 Crispants. (F–I): ISH for rx2 at 24 hpf. The expression of rx2 is absent in F0 rx3 Crispants at 24 hpf, correlating with the severity of the phenotype. (B,C,F,G): wildtype. (D,E,H,I): rx3-crispant. (B,D,F,H) dorsal view. (C,E,G,I) lateral view. scalebars indicate 250 µm.

Figure 4

ISH at 11 hpf after bmp4-induction at 8.5 hpf. (A): summary of experimental procedure: embryos were heat-shocked at 8.5 hpf and analyzed at 11 hpf; left columns wildtypes, right columns bmp4-induced;first and third column dorsal view, second and fourth column lateral view. (B–E): Expression of shha is reduced in the area of the prechordal plate (arrows) after bmp4-induction yet still present. (F–I): shhb expression is also still present after bmp4-induction but reduced in intensity (arrows). (K–N): Expression of zic2a is absent in the ANP domain (arrows) after bmp4-induction. (O–R): expression of cxcr4a in the eye field is strongly reduced after bmp4-induction. scalebars indicate 200 µm.

Figure 5

Analysis of marker genes of telencephalic, retinal and hypothalamic precursors after bmp4-induction. (A): summary of experimental procedure: embryos were heat-shocked at 8.5 hpf and analyzed at 24 hpf. Left columns wildtypes, right columns bmp4-induced embryos. First and third column dorsal view, second and fourth column lateral view. (B–E): ISH for pax6a at 24 hpf. Pax6a (dotted lines) is expressed in the crypt-oculoid and in the diencephalic domain after bmp4-induction. (F–I): ISH for pax2a (dotted lines) at 24 hpf. The expression of pax2a in the MHB is conserved after bmp4-induction but the optic stalk domain is lost. (K–N): ISH for emx3 at 24 hpf. Emx3 is present after bmp4-induction but the expression pattern is changed (arrows). (O–R): ISH for her13 at 24 hpf. The expression of her13 is lost in anterior regions after bmp4-induction. (S–V): ISH for fgf8a at 24 hpf. Two expression domains posterior to the crypt-oculoid are visible after bmp4-induction (arrows). The ventral expression domain (left arrow in T) is ceased after bmp4-induction. Scalebars indicate 200 µm.

Figure 6

Scheme, summary of major findings. (A): Experimental induction of bmp4 results in a suppression of rx3 and shha/b with a consecutive suppression of cxcr4a and zic2a. The ANP is not splitting in a proper manner, resulting in a crypt-oculoid. Six3b and emx3 are found condensed at the midline. (B): scheme showing the pathological development of the ANP resulting from bmp4 induction, including a defect in the hypothalamic subduction. (C): brief summary of findings.