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.

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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 knockout 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 (DTB). 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.

Müller glia are the primary glial subtype in the retina and perform a wide range of physiological tasks in support of retinal function, but little is known about the transcriptional network that maintains these cells in their differentiated state. We report that selective deletion of the LIM homeodomain transcription factor Lhx2 from mature Müller glia leads to the induction of reactive retinal gliosis in the absence of injury. Furthermore, Lhx2 expression is also down-regulated in Prph2 Rd2/Rd2 animals immediately before the onset of reactive gliosis. Analysis of conditional Lhx2 knockouts showed that gliosis was hypertrophic but not proliferative. Aging of experimental animals demonstrated that constitutive reactive gliosis induced by deletion of Lhx2 reduced rates of ongoing apoptosis and compromised both rod and cone photoreceptor function. Additionally, these animals showed a dramatically reduced ability to induce expression of secreted neuroprotective factors and displayed enhanced rates of apoptosis in light-damage assays. We provide in vivo evidence that Lhx2 actively maintains mature Müller glia in a nonreactive state, with loss of function initiating a specific program of nonproliferative hypertrophic gliosis.development | neurodegeneration M üller glia are the primary glial cell type in the vertebrate retina (1). The radial morphology of Müller glia creates a columnar matrix that maintains the laminar structure of the retina. Müller glia also perform a wide range of physiological tasks, including reuptake and metabolism of GABA and glutamate, water and ion homeostasis, and energetic support of photoreceptor cells (1, 2). Müller glia also participate in regeneration of the 11-cis retinal chromophore used by cone opsins (3, 4). Although the genetic regulation of Müller glial development has been extensively studied (5-8), virtually nothing is known about the intrinsic transcriptional network that maintains these cells, or any other astroglial subtype, in their terminally differentiated state.Like other astroglia, Müller glia can be induced to undergo reactive gliosis in response to a broad range of physiological stresses and insults (9). Although the molecular signature of reactive gliosis can vary considerably among injury paradigms, reactive astroglia show a set of common features, including cellular hypertrophy, up-regulation of intermediate filament proteins, and, in most cases, down-regulation of glutamine synthetase (GS). Although induction of reactive gliosis is ubiquitous after diverse types of retinal injuries, its function remains ambiguous. The discovery that preconditioning by a wide range of stressors also induces reactive gliosis has led to the suggestion that, at least in its early stages, reactive gliosis represents an coordinated response aimed at mitigating damage to nearby neurons (9). Severe and prolonged reactive gliosis, however, is usually associated with reduced neuronal viability, and it is unclear what regulates this transition between protective and destructive gliosis. It is ...

Understanding homeobox gene specificity and function has been hampered by the lack of proven direct transcriptional targets during development. Dlx genes are expressed in the developing forebrain, retina, craniofacial structures and limbs. Dlx1/Dlx2 double knockout mice die at birth with multiple defects including abnormal forebrain development and decreased Dlx5 and Dlx6 expression. We have successfully applied chromatin immunoprecipitation (ChIP) to identify a direct transcriptional target of DLX homeoproteins from embryonic tissues in vivo. We optimized cross-linking conditions to enrich for protein-DNA complexes, then using specific high affinity DLX antibodies captured immunoenriched DLX genomic DNA transcriptional targets. DLX homeobox proteins bind differentially to the Dlx5/Dlx6 intergenic enhancer in newborn retina (DLX2) and embryonic striatum (DLX1, DLX2) in situ. Reporter gene assays demonstrated the functional significance of the binding of DLX proteins to this regulatory element, confirmed in vitro by electrophoretic mobility shift assays, using tissue extracts or recombinant DLX proteins. ChIP provides the best approach to identify direct Dlx homeoprotein targets from developing tissues in situ. The use of this technology will advance our understanding of Dlx gene function in the vertebrate in vivo and can be applied to examine targets of other homeobox genes and other classes of transcription factors.

Dlx homeobox genes, the vertebrate homologs of Distal-less, play important roles in the development of the vertebrate forebrain, craniofacial structures and limbs. Members of the Dlx gene family are also expressed in retinal ganglion cells (RGC), amacrine and horizontal cells of the developing and postnatal retina. Expression begins at embryonic day 12.5 and is maintained until late embryogenesis for Dlx1, while Dlx2expression extends to adulthood. We have assessed the retinal phenotype of the Dlx1/Dlx2 double knockout mouse, which dies at birth. The Dlx1/2 null retina displays a reduced ganglion cell layer (GCL), with loss of differentiated RGCs due to increased apoptosis, and corresponding thinning of the optic nerve. Ectopic expression of Crx, the cone and rod photoreceptor homeobox gene, in the GCL and neuroblastic layers of the mutants may signify altered cell fate of uncommitted RGC progenitors. However,amacrine and horizontal cell differentiation is relatively unaffected in the Dlx1/2 null retina. Herein, we propose a model whereby early-born RGCs are Dlx1 and Dlx2 independent, but Dlx function is necessary for terminal differentiation of late-born RGC progenitors.

Hypothalamic tanycytes, a radial glial-like ependymal cell population that expresses numerous genes selectively enriched in embryonic hypothalamic progenitors and adult neural stem cells, have recently been observed to serve as a source of adult-born neurons in the mammalian brain. The genetic mechanisms that regulate the specification and maintenance of tanycyte identity are unknown, but are critical for understanding how these cells can act as adult neural progenitor cells. We observe that LIM (Lin-11, Isl-1, Mec-3)-homeodomain gene Lhx2 is selectively expressed in hypothalamic progenitor cells and tanycytes. To test the function of Lhx2 in tanycyte development, we used an intersectional genetic strategy to conditionally delete Lhx2 in posteroventral hypothalamic neuroepithelium, both embryonically and postnatally. We observed that tanycyte development was severely disrupted when Lhx2 function was ablated during embryonic development. Lhx2-deficient tanycytes lost expression of tanycyte-specific genes, such as Rax, while also displaying ectopic expression of genes specific to cuboid ependymal cells, such as Rarres2. Ultrastructural analysis revealed that mutant tanycytes exhibited a hybrid identity, retaining radial morphology while becoming multiciliated. In contrast, postnatal loss of function of Lhx2 resulted only in loss of expression of tanycyte-specific genes. Using chromatin immunoprecipitation, we further showed that Lhx2 directly regulated expression of Rax, an essential homeodomain factor for tanycyte development. This study identifies Lhx2 as a key intrinsic regulator of tanycyte differentiation, sustaining Rax-dependent activation of tanycyte-specific genes while also inhibiting expression of ependymal cell-specific genes. These findings provide key insights into the transcriptional regulatory network specifying this still poorly characterized cell type.

A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.