Dlx1, Dlx2, Pax6, Brn3b, and Chx10 homeobox gene expression defines the retinal ganglion and inner nuclear layers of the developing and adult mouse retina

Abstract

Distal-less homeobox genes are expressed in the developing forebrain. We assessed Dlx gene expression in the developing and adult mouse retina. Dlx1 and Dlx2 are detected in retinal neuroprogenitors by embryonic day (E) 12.5 (Eisenstat et al. [1999] J. Comp. Neurol. 217-237). At E13.5, the expression of four homeodomain proteins, DLX2, BRN3b, PAX6, and CHX10, define distinct yet overlapping domains in the retinal neuroepithelium. By postnatal day (P) 0, DLX2 is expressed in the neuroblastic layer and the ganglion cell layer (GCL) consisting of ganglion and displaced amacrine cells. DLX1 expression resembles DLX2 to P0 but decreases postnatally. In the adult, DLX2 is localized to ganglion, amacrine, and horizontal cells as determined by coexpression with retinal cell-specific markers. There is coincident expression of DLX2 with gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD)65, and GAD67 in the inner nuclear layer (INL) and GCL. In the adult, DLX2 is coexpressed with BRN3b in ganglion cells; PAX6 in amacrine, horizontal, and ganglion cells; and Chx10 in some bipolar cells. We predict that a combinatorial code of these homeobox genes and others specify retinal cell fate. Our results support a possible role for Dlx1 and Dlx2 in inner retinal development and in the terminal differentiation and/or maintenance of INL interneurons and ganglion cells in the adult. The correlation of DLX2 with GABA expression in the mouse retina closely mirrors the relationship of DLX2 to GABAergic neuronal differentiation in the embryonic forebrain, including neocortex, olfactory bulb and hippocampus, signifying a conservation of function of Dlx genes in the developing central nervous system.

GAD isoforms exhibit distinct spatiotemporal expression patterns in the developing mouse lens: Correlation with Dlx2 and Dlx5

“…Staining in the retina was as previously reported and was monitored as control. In all cases, it was identical to the previously reported pattern (Haverkamp and Wassle, 2000;Dkhissi et al, 2001;de Melo et al, 2003). Based on the reverse transcriptase-polymerase chain reaction and Western blot results, most, if not all GAD67-related immunoreactivity during embryonic stages is due to EGAD (GAD25 and GAD44); only adult GAD67 is expressed beyond P7.…”

“…The absence of Dlx1 and Dlx2 in mice results in neonatal lethality, skeletal abnormalities, and a considerable reduction of forebrain GABAergic inhibitory neurons. This finding is accompanied by a parallel down-regulation of Dlx5, Dlx6, and GAD67 expression (Qiu et al, 1995(Qiu et al, , 1997Anderson et al, 1997a,b;Panganiban and Rubenstein, 2002;de Melo et al, 2003de Melo et al, , 2005Masland, 2005). Because no data are currently available on the possible impact of this mutation on GAD expression in the lens, we sought to determine the expression of different GAD isoforms derived from the GAD67 locus in the Dlx1/Dlx2 mutant lens.…”

“…1A,B). It should be noted that both adult and embryonic GAD forms (M. Schwirtlich, unpublished data) and Dlx1/2 (de Melo et al, 2003) are abundantly expressed in the retina during all developmental stages, including the adult; thus, the lens-specific profile cannot be attributed to contamination from retinal tissue.…”

“…The embryonic tissue was immersed in 20% sucrose-PBS solution at 4°C until it sunk, then embedded in Cryo-gel (Instrumedics, Inc., West Chester, PA), frozen in dry ice, and sectioned at 25 m on a cryostat (Microm HM550). Tissues obtained from Dlx1/ Dlx2 mutants and litter age-matched controls were cryopreserved following fixation in paraformaldehyde and immersion in sucrose gradients as previously described (de Melo et al, 2003).…”

“…DLX2 and GAD immunofluorescence was performed on cryosections from wild-type and Dlx1/Dlx2 knockout mice as described (de Melo et al, 2003(de Melo et al, , 2005. Primary antibodies used were as follows: rabbit anti-DLX2 (1: 400), rabbit anti-GAD25 (rabbit serum #8876, 1:1,000); rabbit anti-GAD44 (rabbit serum #3910, 1:500 raised in the Szabo lab [Szabo et al, 1994]); rabbit serum #6799, 1:500, recognizing GAD25, GAD44, and GAD67 (Katarova et al, 1990); rabbit anti-PROX1 (1:500, courtesy of Dr. M.. Nakafuku).…”