LHX2 is necessary for the maintenance of optic identity and for the progression of optic morphogenesis

Abstract

Eye formation is regulated by a complex network of eye field transcription factors (EFTFs), including LIM-homeodomain gene LHX2. We disrupted LHX2 function at different stages during this process using a conditional knock-out strategy in mice. We find that LHX2 function is required in an ongoing fashion to maintain optic identity across multiple stages, from the formation of the optic vesicle to the differentiation of the neuroretina. At each stage, loss of Lhx2 led to upregulation of a set of molecular markers that are normally expressed in the thalamic eminence and in the anterodorsal hypothalamus in a portion of the optic vesicle or retina. Furthermore, the longer LHX2 function was maintained, the further optic morphogenesis progressed. Early loss of function caused profound mispatterning of the entire telencephalic-optic-hypothalamic field, such that the optic vesicle became mispositioned and appeared to arise from the diencephalic-telencephalic boundary. At subsequent stages, loss of Lhx2 did not affect optic vesicle position but caused arrest of optic cup formation. If Lhx2 was selectively disrupted in the neuroretina from E11.5, the neuroretina showed gross dysmorphology along with aberrant expression of markers specific to the thalamic eminence and anterodorsal hypothalamus. Our findings indicate a continual requirement for LHX2 throughout the early stages of optic development, not only to maintain optic identity by suppressing alternative fates but also to mediate multiple steps of optic morphogenesis. These findings provide new insight into the anophthalmic phenotype of the Lhx2 mutant and reveal novel roles for this transcription factor in eye development.

Figure 1.

The Lhx2 mutant displays an abnormal optic-like vesicle at the DTB. A–H, In E12.5 coronal sections of control brains, expression of optic transcription factors Rax, Six3, Pax6, Mab21l2, and Otx2 is seen in the neuroretina (A–E, open arrow), whereas Fgf17 and Wnt8b are expressed in the optic stalk (F,G, open arrow). In Lhx2 mutant brains, these markers are expressed in a vesicle at the DTB (A–G, arrowhead). *Six3, Pax6, Mab21l2, and Wnt8b are also expressed in the thalamic eminence. H, Schematic showing morphology of typical control and mutant sections, with the mutant vesicle shown in blue. I, Schematic showing horizontal plane of sectioning through the DTB (J–L). J, Horizontal section of E12.5 control brain shows Wnt8b expressed in the caudomedial telencephalon (open arrowhead). K, L, In the Lhx2 mutant, horizontal sections show the Wnt8b-expressing vesicle positioned at the junction of the caudal telencephalon and diencephalon (arrowheads). Schematics for the control and Lhx2 mutant sections in J and L are shown in M and N, respectively. O–T, Whole-mount in situ hybridization reveals Rax expression in the control eye at E10.5 (O, open arrow), the optic vesicle at E9.25 (Q, open arrow), and in the anterior neural plate at E8.25 (S). In the Lhx2 mutant, a reduced Rax-expressing vesicle is seen at E10.5 and E9.25 (P,R, arrowhead). Rax expression is also seen in the hypothalamus of control and mutant embryos (Q,R, notched arrowhead). Frontal views of E8.25 Lhx2 mutant embryos reveal a reduced domain of Rax expression compared with that in control embryos (S, T). S, T, Yellow dashed lines indicate the margin of the head fold. *Thalamic eminence. Arrowhead indicates Lhx2 mutant vesicle. 3V, Third ventricle; hy, hypothalamus. Scale bars: A–G, J–L, 300 μm; O–T, 200 μm.

Figure 2.

Altering the timing of Lhx2 inactivation partially rescues the mutant phenotype. A–I, Conditional deletion of Lhx2 using a tamoxifen-inducible CreER driver. Tamoxifen administration at E8.25 (A,D,G, T8.25) does not interfere with optic development in control embryos. In floxed (CreER;Lhx2^lox/−) embryos, tamoxifen administration at E8.25 produces a vesicle at the DTB (B,E,H, arrowheads). A probe against Lhx2 exon 2,3 indicates that Lhx2 deletion has occurred throughout the brain (B). Serial sections of the same embryo reveal the expression of optic markers Six3 (E) and Mab21l2 (H) in the mutant vesicle. In floxed embryos, tamoxifen administration at E8.75 (T8.75) produces a vesicle positioned intermediate between the wild-type and null mutant location (C,F,I). Six6 is expressed in the hypothalamus and in the stalk of the mutant vesicle (C, arrowhead), and Six3 (F) and Mab21l2 (I) are seen in the vesicle itself, which does not progress to the optic cup stage. J–Q, Whole-mount preparations of E9.25 brains. J–M, Tamoxifen administration at E8.25 and examination at E9.25 reveals similar territories of expression of optic marker Six6 in the optic vesicle (J, open arrow; K, arrowhead); notched arrowheads indicate Six6 expression in the hypothalamus in both control and Lhx2 conditional mutant brains (J, K). Similarly, a two color in situ reveals comparable expression of optic marker Rax (brown, L, open arrow; M, arrowhead) and anterodorsal hypothalamic marker Sim1 (purple, asterisk) in control and conditional mutant brains. N–Q, Two color in situ hybridization of E9.25 control and Lhx2 standard knock-out (−/−) embryos reveals a reduced Rax-expressing vesicle (brown) in the mutants (arrowheads) compared with controls (open arrows). Six6 expression (N, O, purple) is seen in the hypothalamus (notched arrowhead), and Emx2 expression identifies the dorsal telencephalon (P, Q, purple) in both control and mutant embryos. Scale bars: A–I, 300 μm; J–Q, 200 μm.

Figure 3.

Late removal of Lhx2 permits partial rescue of optic development. A–C, Late-arising optic transcription factors Chx10, Six6, and Mitf are seen in the control neuroretina (A–C, open arrow), but not in the Lhx2 standard knock-out vesicle (A–C, arrowhead). D–I′, Tamoxifen administration at E9.25 (T9.25) does not interfere with optic development in control embryos (D,F,H). In Lhx2 floxed embryos, tamoxifen administration at E9.25 and examination at E12 reveal a normally positioned vesicle that has progressed to forming a partial optic cup (E′, blue dashed lines) with patches of pigmented epithelium (I′, open arrowheads). A probe against Lhx2 exon 2,3 indicates that Lhx2 deletion has occurred throughout the brain and optic vesicle (E,E′). The control neuroretina expresses Six6 and Mitf (F,H, open arrows), and these markers are seen for the first time in the conditional mutant optic cup as well (G,G′,I,I′, arrowheads). J–O, Retinae from control and Chx10-Cre;Lhx2^lox/lox (conditional mutant) embryos, harvested at E13.5 (J–L) and P0 (M–O). Compared with the controls, the mutant retinae are smaller and display profound disorganization by P0. Retinal progenitor markers Chx10 and Rorb and early photoreceptor markers Crx and Prdm1 are expressed in the conditional mutant neuroretina at E13.5 (J–L) and P0 (M–O). L, Open notched arrowheads show normally positioned as well as displaced immature photoreceptors in a disorganized mutant retina. Scale bars: A–I′, 300 μm; J–O, 100 μm.

Figure 4.

Loss of Lhx2 disrupts patterning of the optic vesicle, the thalamic eminence, and the anterior hypothalamus. A–E, AP2α, Lhx1, Lhx5, Lhx9, and Tbr1 are expressed in the control and Lhx2 standard knock-out thalamic eminence (white asterisk) and also in the mutant vesicle (arrowhead), but not in the control neuroretina (open arrows; dark retinal pigment epithelium seen in some control sections should not be confused with marker expression). F, G, Serial coronal sections of control embryos reveal Sim1 and Otp expression in the anterodorsal hypothalamus (notched arrowhead) but not in the neuroretina (open arrow). In Lhx2 mutant brains, Sim1 and Otp expression domain is aberrantly expanded into the CGE (open arrowhead) and also in the mutant vesicle (arrowhead). Sim1 in the normal location of anterodorsal hypothalamus appears to be missing in the Lhx2 mutant (F, black asterisk). H–L, Mab21l2, Zic2, and Lhx1 expression is seen in the thalamic eminence of control and Lhx2 mutant brains and also appears in the Lhx2 mutant vesicle (H–J, arrowheads). Mab21l2 and Zic2 expression is seen in the control anterior hypothalamus (white asterisk) but is not detectable in the Lhx2 mutant (H,I, black asterisk). Lhx1, Vax1, and Arx expression is seen in the intrahypothalamic diagonal/anteroventral hypothalamus (J–L, white asterisk), adjacent to the optic stalk of control brains, but is in a very reduced and aberrantly located domain in the mutant (J–L, open arrowhead). However, some aspects of regional patterning are preserved in the absence of LHX2. Both control and Lhx2 mutant brains show apparently normal expression of Zic2 in the thalamus (I) and Arx expression in the prethalamus of the Lhx2 mutant (L, arrow). Shh and Nkx2.1 expression also appears to mark similar domains in the posteroventral hypothalamus of control and Lhx2 mutant brains (M, N). O, The molecular patterning of the Lhx2 null mutant vesicle (red oval) is compared with wild-type optic structures (green oval) in a Venn diagram. Genes common to both control and mutant structures are in the overlapping yellow region. Genes seen only in the mutant, but not in the control optic structures, are further divided into the pink oval, which contains genes expressed by the wild-type thalamic eminence, and orange oval, which contains genes shared by the anterodorsal hypothalamus. Arrowhead, Lhx2 mutant vesicle; open arrow, control optic cup. Scale bar, 300 μm.

Figure 5.

Ongoing role of LHX2 in the suppression of alternate tissue fates. A–L′, Conditional deletion of Lhx2 using a tamoxifen-inducible CreER driver. Tamoxifen administration at E8.75 (A,C,E, T8.75) or E9.25 (G,I,K, T9.25) does not interfere with optic development in control (CreER;Lhx2^lox/+) embryos. In T8.75 and T9.25 floxed embryos harvested at E12, the ectopic expression of thalamic eminence marker Lhx5 and anterodorsal hypothalamic markers Otp and Sim1 is seen in the mutant vesicle (B,D,F, arrowheads) and in the mutant partial optic cup (H,J,L, arrowheads). H′, J′, L′, High magnification views of the optic cups in H, J, and L, respectively. J, L, Adjacent sections reveal very similar domains of Sim1 and Otp expression in the partial optic cup. Blue dashed lines (H′) outline the partially formed optic cup. Open arrowheads indicate the partially formed pigmented epithelium (J′,L′). Open arrow, control optic cup. Scale bar, 300 μm.

Figure 6.

Distinct molecular domains within the Lhx2 mutant vesicle/optic cup. A–L, Retinae from control (A–C) and Chx10-Cre;Lhx2^lox/lox (D–L) embryos harvested at E13.5 (A–I) and P0 (J–L). A–C, The control retina expresses Rax uniformly but does not express Lhx5 or Otp. The conditional mutant retina displays patches of Rax expression (E,H,K) that show little or no overlap with regions expressing Lhx5 or Otp (D,G,J). M–U, Control and Lhx2 standard knock-out embryos displaying expression of telencephalic and thalamic eminence marker Tbr1, as well as Rax and Otp. Tbr1 and Otp expression is absent in the Rax-positive control optic cup (M–O, open arrow). Serial sections of Lhx2 mutant embryos reveal aberrant expression of Tbr1 and Otp in the mutant vesicle in domains that exclude the Rax expression domain. F, I, L, R, U, False-color overlays of the adjacent pairs of sections. Scale bars: A–L, 100 μm; M–U, 300 μm.

Figure 7.

The Pax6 mutant optic vesicle does not display aberrant molecular identity or position. The Pax6 mutant optic vesicle, identified in coronal sections of four different E12.5 embryos, arises from a position similar to that in control brains, adjacent to the hypothalamus. Control optic cups and Pax6 mutant optic vesicles express optic transcription factors Rax, Six3, Mab21l2, and Six6 (compare open arrows and arrowheads, A–D). However, neither structure expresses thalamic eminence markers Ap2α, Lhx5, Lhx9, or Tbr1 (compare open arrows and arrowheads, E–H). Open arrow indicates normal optic cup; arrowhead, Pax6 mutant optic vesicle. Scale bar, 300 μm.

Figure 8.

Multiple roles of LHX2 in optic development. A summary of the temporally dynamic roles of LHX2 in optic development. The x-axis represents the timing of Lhx2 deletion: from E0.0 (standard knock-out), or conditionally using tamoxifen-inducible CreER from E8.25/E8.75/E9.25, or using the retina-specific Chx10-Cre, which acts from E11.5. a, The position of the mutant vesicle is ectopic, intermediate, or normal depending on the timing of Lhx2 deletion. b, The optic vesicle arrests without forming an optic cup if Lhx2 is deleted before or at E8.75. c, Late optic markers, including retinal pigmented epithelium, are seen if LHX2 function is allowed to persist upto E9.25 d, However, LHX2 is continuously required to suppress alternate tissue fates even after the retina has formed, and its deletion at all stages examined causes aberrant marker expression in the optic structures.