Expression of Lhx6 in the adult and developing mouse retina

PurposeTo investigate the expression patterns of LIM Homeobox 6 (Lhx6) in the adult and developing mouse retina.MethodsThe Lhx6-GFP knock-in allele was used to activate constitutive expression of a GFP reporter in Lhx6 expressing cells. Double labeling with GFP and retinal markers in the mouse retina at postnatal day 56 (P56) was performed to identify the cell types expressing Lhx6. To determine the neuronal cell types that express Lhx6, double labeling with GFP and various retinal markers was employed in the differentiating retina at P7 and P15.ResultsGFP + Lhx6 lineage cells were determined in Brn3a + retinal ganglion cells (RGCs), ChAT + amacrine cells (ACs), and Islet-class LIM-homeodomain 1 (Isl1+) ACs in the mouse retina at P56. In the ganglion cell layer (GCL), Lhx6 was expressed in Brn3a + RGCs but not Brn3b + RGCs at P15. Moreover, in the inner nuclear layer (INL), Lhx6 was not expressed in Bhlhb5+ ACs at P15. However, Lhx6 was weakly expressed in Glyt1+ ACs and Pax6+ ACs, and strongly expressed in Isl1+ and ChAT + ACs at P15.ConclusionLhx6 was expressed in RGCs and ACs in both the adult and developing mouse retina.     

The expression of LIM‐homeobox genes,Lhx1 and Lhx5, in the forebrain is essential for neural retina differentiation

Elucidating the mechanisms underlying eye development is essential for advancing the medical treatment of eye‐related disorders. The primordium of the eye is an optic vesicle (OV), which has a dual potential for generation of the developing neural retina and retinal pigment epithelium. However, the factors that regulate the differentiation of the retinal primordium remain unclear. We have previously shown that overexpression of Lhx1 and Lhx5, members of the LIM‐homeobox genes, induced the formation of a second neural retina from the presumptive pigmented retina of the OV. However, the precise timing of Lhx1 expression required for neural retina differentiation has not been clarified. Moreover, RNA interference of Lhx5 has not been previously reported. Here, using a modified electroporation method, we show that, Lhx1 expression in the forebrain around stage 8 is required for neural retina formation. In addition, we have succeeded in the knockdown of Lhx5 expression, resulting in conversion of the neural retina region to a pigment vesicle‐like tissue, which indicates that Lhx5 is also required for neural retina differentiation, which correlates temporally with the activity of Lhx1. These results suggest that Lhx1 and Lhx5 in the forebrain regulate neural retina differentiation by suppressing the development of the retinal pigment epithelium, before the formation of the OV.     

Pou4f2‐GFP knock‐in mouse line: A model for studying retinal ganglion cell development

Pou4f2 acts as a key node in the comprehensive and step‐wise gene regulatory network (GRN) and regulates the development of retinal ganglion cells (RGCs). Accordingly, deletion of Pou4f2 results in RGC axon defects and apoptosis. To investigate the GRN involved in RGC regeneration, we generated a mouse line with a POU4F2‐green fluorescent protein (GFP) fusion protein expressed in RGCs. Co‐localization of POU4F2 and GFP in the retina and brain of Pou4f2‐GFP/+ heterozygote mice was confirmed using immunofluorescence analysis. Compared with those in wild‐type mice, the expression patterns of POU4F2 and POU4F1 and the co‐expression patterns of ISL1 and POU4F2 were unaffected in Pou4f2‐GFP/GFP homozygote mice. Moreover, the quantification of RGCs showed no significant difference between Pou4f2‐GFP/GFP homozygote and wild‐type mice. These results demonstrated that the development of RGCs in Pou4f2‐GFP/GFP homozygote mice was the same as in wild‐type mice. Thus, the present Pou4f2‐GFP knock‐in mouse line is a useful tool for further studies on the differentiation and regeneration of RGCs.     

Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B‐GFP transgenic mice

FGF1 is involved in multiple biological functions and exhibits the importance in neuroprotective effects. Our previous studies indicated that, in human brain and retina, the FGF1B promoter controlled the expression of FGF1. However, the exact function and regulation of FGF1 in brain is still unclear. Here, we generated F1B‐GFP transgenic mice that expressed the GFP reporter gene under the control of human FGF1B promoter (?540 to +31). Using the fresh brain sections of F1B‐GFP transgenic mice, we found that the F1B‐GFP cells expressed strong fluorescent signals in the ventricular system throughout the brain. The results of immunohistochemistry further showed that two distinct populations of F1B‐GFP⁺ cells existed in the brains of F1B‐GFP transgenic mice. We demonstrated that one population of F1B‐GFP⁺ cells was ependymal cells, which distributed along the entire ventricles, and the second population of F1B‐GFP⁺ cells was neuronal cells that projected their long processes into multiple directions in specific areas of the brain. The double labeling of F1B‐GFP⁺ cells and tyrosine hydroxylase indicated that a subpopulation of F1B‐GFP⁺‐neuronal cells was dopaminergic neurons. Importantly, these F1B‐GFP⁺/TH⁺ cells were distributed in the main dopaminergic neuronal groups including hypothalamus, ventral tegmental area, and raphe nuclei. These results suggested that human FGF1B promoter was active in ependymal cells, neurons, and a portion of dopaminergic neurons. Thus, the F1B‐GFP transgenic mice provide an animal model not only for studying FGF1 gene expression in vivo but also for understanding the role of FGF1 contribution in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 232–248, 2015     

Genomic structure, chromosomal localization, and expression pattern of the human LIM-homeobox3 (LHX 3) gene

Lhx3 is a LIM-homeobox protein essential for pituitary development in mice. The human homologue gene spans 7.2 kb and contains 7 exons, including two alternatively spliced first exons. This structure encodes two distinct protein isoforms, LHX3a and LHX3b, that differ exclusively in their amino-terminus. The LHX3 gene was localized at 9q34.2-34.3. The predicted protein sequence is highly homologous to other known Lhx3 proteins, the highest degree of homology being in the conserved domains. The highest expression of LHX3 was found in pituitary gland, spinal cord, and lung. Among different pituitary cell types, corticotrophs appear to express preferentially LHX3b isoform, suggesting a distinct role of the b-form in the development of this cell lineage. Although the human LHX3 gene structure would provide a ground for clarification of the molecular basis of complete anterior pituitary deficiency, we were unable to identify any mutation in the LHX3 gene of 46 such patients.