Expression of LIM-homeodomain transcription factors in the developing and mature mouse retina

Abstract

LIM-homeodomain (LIM-HD) transcription factors have been extensively studied for their role in the development of the central nervous system. Their function is key to several developmental events like cell proliferation, differentiation and subtype specification. However, their roles in retinal neurogenesis remain largely unknown. Here we report a detailed expression study of LIM-HD transcription factors LHX9 and LHX2, LHX3 and LHX4, and LHX6 in the developing and mature mouse retina using immunohistochemistry and in situ hybridization techniques. We show that LHX9 is expressed during the early stages of development in the retinal ganglion cell layer and the inner nuclear layer. We also show that LHX9 is expressed in a subset of amacrine cells in the adult retina. LHX2 is known to be expressed in retinal progenitor cells during development and in Müller glial cells and a subset of amacrine cells in the adult retina. We found that the LHX2 subset of amacrine cells is not cholinergic and that a very few of LHX2 amacrine cells express calretinin. LHX3 and LHX4 are expressed in a subset of bipolar cells in the adult retina. LHX6 is expressed in cells in the ganglion cell layer and the neuroblast layer starting at embryonic stage 13.5 (E13.5) and continues to be expressed in cells in the ganglion cell layer and inner nuclear layer, postnatally, suggesting its likely expression in amacrine cells or a subset thereof. Taken together, our comprehensive assay of expression patterns of LIM-HD transcription factors during mouse retinal development will help further studies elucidating their biological functions in the differentiation of retinal cell subtypes.

Keywords: LIM-homeodomain; Lhx genes; Retinal development; Retinogenesis; Transcription factors.

Figure 1

Expression of Lhx9 mRNA transcript in the developing retina. A: Expression of Lhx9 is faint and mostly undetectable at E13.5 in retinal progenitor cells. B: Strong expression of Lhx9 beings at E15.5 in the GCL and developing NBL. C-D: Expression of Lhx9 continues (E17.5, P5) in the GCL and developing NBL. E-F: Expression of Lhx9 is in cells in the GCL and cells in the INL postnatally (P7, P15). All scale bars are 50 μm.

Figure 2

Characterization of LHX9 expressing cells in the adult retina (P30). A: Co-expression of LHX9 with BRN3A indicates that LHX9 might not be expressed in retinal ganglion cells. B: Co-localization of all LHX9 cells with syntaxin indicates that LHX9 expressing cells are amacrine cells. C: Co-expression of LHX9 with SOX2 does not reveal any LHX9 expressing Müller glial cells. D: Co-expression of LHX9 with CHX10 does not reveal any LHX9 expressing bipolar cells. All scale bars are 50 μm.

Figure 3

Characterization of LHX2 expressing amacrine cells in the adult retina (P30). A: Co-localization of LHX2 with syntaxin indicates the presence of LHX2 expressing amacrine cells (Arrows). B: Co-expression of LHX2 with ChAT shows that LHX2 expressing amacrine cells are not cholinergic. C: Co-expression of LHX2 with calretinin reveals that few LHX2 expressing amacrine cells co-localize calretinin (Arrow). D: LHX2 expressing amacrine cells do not co-localize with TH expressing dopaminergic amacrine cells. E: LHX2 expressing amacrine cells do not co-localize with GAD65, a GABAergic amacrine cell marker. All scale bars are 50μm.

Figure 4

Characterization of LHX3 expressing cells in the adult retina (P30). A: Co-localization of LHX3 with CHX10 indicates that all LHX3 expressing cells are bipolar cells. B: Co-expression of LHX3 is seen with “weakly” ISL1 expressing cells (Arrows). C: Co-expression of a few LHX3 cells is seen with Goα expressing bipolar cells (Arrows). D: LHX3 cells do not co-localize with PKCα expressing bipolar cells. All scale bars are 50 μm.

Figure 5

Characterization of LHX4 expressing cells in the adult retina (P30). A: Co-localization of LHX4 with CHX10 indicates that all LHX4 expressing cells are bipolar cells. B: Co-expression of LHX4 is seen with “weakly” ISL1 expressing cells (Arrows). C: Co-expression of some LHX4 cells is seen with Goα expressing bipolar cells (Arrows). D: LHX4 cells do not co-localize with PKCα expressing bipolar cells. All scale bars are 50 μm.

Figure 6

Expression of Lhx6 mRNA transcript in the developing retina. A: Expression of Lhx6 is not detected at E11.5. B: Expression of Lhx6 is seen in retinal progenitor cells starting at E13.5. C-D: Lhx6 expression is detected in cells in the GCL and the developing NBL (E17.5, P5). E-F: Lhx6 expression is detected in cells in the GCL and cells in the INL postnatally (P7, P15). All scale bars are 50 μm.