Table of contents

Lim domain proteins of mammals: Lhx2 an ortholog of Apterous

The gene Lhx2 is considered to be the only known ortholog (related by descent from a common ancestor) of Drosophila apterous. All the other genes described in this section are to be considered paralogs, that is structurally related but of diverged sequence and/or function. The function of Lhx2, a LIM homeobox gene expressed in developing B-cells, forebrain and neural retina, was analyzed by using embryos deficient in functional Lhx2 protein. Lhx2 mutant embryos are anophthalmic, have malformations of the cerebral cortex, and die in utero due to severe anemia. In Lhx2-/- embryos specification of the optic vesicle occurs, however, development of the eye arrests prior to formation of an optic cup. Pax-6 (Drosophila homolog: Eyeless) expression in the optic vesicle is normal in knockout mice, but Pax-6 expression in the ectoderm overlying the optic vesicle is deficient, suggesting the Lhx2 function in the optic vesicle is necessary for either induction or maintence of Pax6 expression in the presumptive lens ectoderm. Deficient cellular proliferation in the forebrain results in hypoplasia of the neocortex and aplasia of the hippocampal anlagen. In addition to the central nervous system malformations, a cell non-autonomous defect of definitive erythropoiesis causes severe anemia in Lhx2-/- embryos. It is thought that the cell non-autonomous defect is due to a defect in the fetal hepatic microenvironment. Thus Lhx2 is necessary for normal development of the eye, cerebral cortex, and efficient definitive erythropoiesis (Porter, 1997).

Msx genes encode a family of homeoproteins that function as transcription repressors through protein-protein interactions. Lhx2, a LIM-type homeoprotein, is a protein partner for Msx1 in vitro and in cellular extracts. The interaction between Msx1 and Lhx2 is mediated through the homeodomain-containing regions of both proteins. Interestingly, the LIM domains, which serve as protein interaction domains for other partners of Lhx2, are not required for the Msx1-Lhx2 association. Msx1 and Lhx2 form a protein complex in the absence of DNA, and DNA binding by either protein alone can occur at the expense of protein complex formation. The significance of this protein-protein interaction is underscored by the expression patterns of Msx1 and Lhx2, which are partially overlapping during murine embryogenesis. The description of Lhx2 as a protein partner for Msx1 suggests that the functional specificity of homeoproteins in vivo is determined by a balance between their association with DNA and their protein partners (Bendall, 1998).

The Drosophila apterous (ap) gene encodes a protein of the LIM-homeodomain family. Many transcription factors of this class have been conserved during evolution; however, the functional significance of their structural conservation is generally not known. ap is best known for its fundamental role as a dorsal selector gene required for patterning and growth of the wing, but it also has other important functions required for neuronal fasciculation, fertility, and normal viability. The mouse (mLhx2) and human (hLhx2) ap orthologs have been isolated. MLHX2 and HLHX2 differ only in four amino acids, except for an extended amino terminus of the mouse protein. The fly and mouse/human proteins show three major domains of sequence similarity that correspond to the LIM domain 1 (57% identity), LIM domain 2 (56% identity), and homeodomain (93% identity). However, conserved amino acids are found also outside these domains. The MLHX2 protein is 90% identical to a rat protein known as rLH2, probably the rat ortholog of mLhx2. Mouse genes related to the mLhx2 gene have been identifed, but lack of sequence data prevents their comparison. The human Ap protein (HLHX2) is 92% identical to hLH2, a protein aberrantly expressed in chronic myelogenous leukemia. hLhx2 maps to chromosomal region 9q33-34.1 by fluorescent in situ hybridization, the same chromosomal region where hLH2 maps (Rincon-Limas, 1999).

The differentiation of areas of the mammalian neocortex has been hypothesized to be controlled by intrinsic genetic programs and extrinsic influences such as those mediated by thalamocortical afferents (TCAs). To address the interplay between these intrinsic and extrinsic mechanisms in the process of arealization, the requirement of TCAs in establishing or maintaining graded or areal patterns of gene expression in the developing mouse neocortex has been analyzed. The differential expression of Lhx2, SCIP, and Emx1, representatives of three different classes of transcription factors, is described as well as the type II classical cadherins Cad6, Cad8, and Cad11, which are expressed in the cortical plate in graded or areal patterns, as well as layer-specific patterns. The differential expression of Lhx2, SCIP, Emx1, and Cad8 in the cortical plate is not evident until after TCAs reach the cortex, whereas Cad6 and Cad11 show subtle graded patterns of expression before the arrival of TCAs, which later become stronger. These genes exhibit normal-appearing graded or areal expression patterns in Mash-1 mutant mice that fail to develop a TCA projection. These findings show that TCAs are not required for the establishment or maintenance of the graded and areal expression patterns of these genes and strongly suggest that their regulation is intrinsic to the developing neocortex (Nakagawa, 1999).

There are striking similarities in the expression patterns of the Drosophila and murine genes. Both mLhx2 and ap are expressed in the respective nerve cords, eyes, olfactory organs, brain, and limbs. The patterns of mLhx2 are reminiscent of ap expression in the embryonic and adult brain, optic lobe, antenna, maxillary palpus (the fly olfactory organs) and ventral nerve cord (VNC). The cells that express ap in the Drosophila VNC are interneurons, as revealed by driving expression of the tau-GFP reporter gene from the ap VNC enhancer. Thus the identity of the cells expressing mLhx2 in the mouse neural tube has been investigated. mLhx2 expression is detected in a dorso-lateral domain where dorsal commissural neurons are located. Interestingly, these are a subset of interneurons that, like Drosophila ap interneurons, send axons along longitudinal ascending tracts. In the Drosophila VNC, ap is expressed in a small number of cells per hemisegment. The activation of the tau-GFP reporter gene by the ap-GAL4MD544 driver allows the visualization of the projections of these interneurons. Interestingly, in situ hybridization on mouse neural tube sections shows that the cells expressing mLhx2 precisely colocalize with a subset of interneurons that, like Drosophila ap interneurons, project along ascending longitudinal tracts to anterior segments and/or to the brain. Drosophila ap mutants show neuronal pathfinding defects; thus these observations suggest a conserved role for ap in interneuron identity or pathfinding. Other regions of mLhx2 expression include the liver, the infundibulum of the pituitary, and a small region of the branchial arches in E9.5 embryos. In addition, mLhx2 expression has been detected in the limb buds, specifically in the mesenchyme of the progress zone, but excluded from the apical ectodermal ridge. Sections through the limb buds show that mLhx2 is expressed both dorsally and ventrally in contrast to the dorsal-specific expression of Drosophila ap. Also, unlike ap, no mLhx2 expression is detected in the somatic mesoderm (Rincon-Limas, 1999).

Transgenic animals and rescue assays have been used to investigate the conservation of the Ap protein during evolution. The human protein LHX2 is able to correctly regulate ap target genes in the fly, causes the same phenotypes as Ap when ectopically produced, and most importantly rescues ap mutant phenotypes as efficiently as the fly protein. UAS:hLhx2 and UAS:ap were expressed within the wing ventral compartment along the antero-posterior compartment boundary. fng expression in the wild-type wing disc is almost completely restricted to the dorsal compartment. hLhx2, like ap, activates fng expression in the ventral compartment. The Ser wild-type expression pattern is also restricted to the dorsal compartment of the disc. Ectopic expression of ap and hLhx2 produce similar activation of Ser expression along the antero-posterior boundary. vg and wg are expressed along the dorsal-ventral compartment boundary, respectively. When ap is ectopically expressed, vg and wg are ectopically activated as two parallel stripes within the ventral compartment. These regulatory interactions are mimicked by hLhx2 ectopic expression. As expected, the adult wings resulting from these crosses exhibit an ectopic wing margin along the ventral compartment. These results demonstrate the conservation of Ap protein function across phyla and argue that aspects of its expression pattern have also been conserved from a common ancestor of insects and vertebrates (Rincon-Limas, 1999).

Human LH-2 is a putative transcription factor containing two cysteine-rich regions (LIM domains) and a homeobox (Hox) DNA-binding domain. High levels of hLH-2 expression have been observed in all cases of chronic myelogenous leukemia (CML) tested, regardless of disease status. hLH-2 maps to chromosome 9Q33-34.1, in the same region as the reciprocal translocation that creates the BCR-ABL chimera of the Philadelphia chromosome (Ph'), the hallmark of CML; hLH-2 is retained on the derivative 9 chromosome and is therefore centromeric to c-ABL. The proximity of hLH-2 to the breakpoint on chromosome 9 raises the possibility of cis-activation by the t(9;22)(q34;q11) translocation. In addition to finding hLH-2 expression in all cases of CML, expression is observed in lymphoid malignancies and myeloid cell lines, but not in primary cases of acute myelogenous leukemia. The role of hLH-2 in the development or progression of leukemia is not known. However, hLH-2 may prove useful as a marker of CML for monitoring residual disease (Wu, 1992).

A screen for early markers of B-lymphocyte differentiation has identified a homeobox gene, denoted LH-2, that has a pattern of expression distinct from that of other related genes. The LH-2 cDNA sequence encodes a polypeptide of 426 amino acids that contains a homeodomain and two repeats of a cysteine-rich domain referred to as a LIM domain. The homeodomain of the LH-2 protein is related to that of other LIM/homeodomain proteins, most strikingly, to that of the Drosophila Apterous protein. Expression of LH-2 is found in B- and T-lymphoid cell lines. Expression in B-cell lines is highest in lines that represent early stages of differentiation, whereas in T-cell lines there is no clear correlation with the stage of differentiation. In embryonic and adult tissues, the highest level of LH-2 expression is found in discrete regions of the developing central nervous system, primarily in diencephalic and telencephalic structures, and in a subset of lymphoid tissues. The expression pattern and structural characteristics of the LH-2 gene suggest that it encodes a transcriptional regulatory protein involved in the control of cell differentiation in developing lymphoid and neural cell types (Xu, 1993).

The genes controlling self-renewal and differentiation in the hematopoietic system are largely unknown. The LIM-homeobox genes are known to be important for asymmetric cell divisions and differentiation of specific cell types and organs. One member of this family, LH2, is expressed in fetal liver at the time of active hematopoiesis. Therefore, the function of LH2 during the formation and initial expansion of the hematopoietic system was assessed by differentiating LH2-transduced embryonic stem (ES) cells in vitro. This procedure generated multipotent hematopoietic precursor cell (HPC) lines that require Steel factor for growth. HPC lines have been maintained in an undifferentiated state in culture for >7 months. Other growth factors tested efficiently induce terminal differentiation of HPCs into various mature myeloid lineages. Steel factor is also required and acts synergistically with the other growth factors to generate multilineage colonies from the HPCs. These HPC lines express transcription factors that are consistent with an immature progenitor, and the pattern of cell surface marker expression is similar to that of early fetal multipotent hematopoietic progenitors. Collectively, these data suggest that the HPC lines represent an early fetal multipotent hematopoietic progenitor, and suggest a role for LH2 in the control of cell fate decision and/or proliferation in the hematopoietic system. It is concluded that expression of the LIM-homeobox gene LH2 generates immortalized Steel factor-dependent multipotent hematopoietic precursors (Pinto do Oa, 1998).

LIM domains are required for both inhibitory effects on LIM homeodomain transcription factors and synergistic transcriptional activation events. The inhibitory actions of the LIM domain can often be overcome by the LIM co-regulator known as CLIM2, LDB1 and NLI (referred to hereafter as CLIM2). The association of the CLIM cofactors with LIM domains does not, however, improve the DNA-binding ability of LIM homeodomain proteins, suggesting the action of a LIM-associated inhibitor factor. Evidence is presented that LIM domains are capable of binding a novel RING-H2 zinc-finger protein, Rlim (for RING finger LIM domain-binding protein), which acts as a negative co-regulator via the recruitment of the Sin3A/histone deacetylase corepressor complex. A corepressor function of RLIM is also suggested by in vivo studies of chick wing development. Overexpression of the gene Rnf12, encoding Rlim, results in phenotypes similar to those observed after inhibition of the LIM homeodomain factor LHX2, which is required for the formation of distal structures along the proximodistal axis, or by overexpression of dominant-negative CLIM1. It is concluded that Rlim is a novel corepressor that recruits histone deacetylase-containing complexes to the LIM domain (Bach, 1999).

Tissue-specific expression of the alpha-subunit gene of glycoprotein hormones involves an enhancer element designated the pituitary glycoprotein basal element, which interacts with the LIM homeodomain transcription factor, Lhx2. In the present studies the function of the LIM domain of Lhx2 in stimulating alpha-subunit transcription has been explored. When fused to the GAL4 DNA-binding domain, the LIM domain of Lhx2 has been shown to contain a transcriptional activation domain. Furthermore, in the context of an alpha-subunit reporter gene in which a GAL4-binding site replaces the pituitary glycoprotein basal element, the LIM domain enhances both basal and Ras-mediated transcription. In addition, a synergistic response to Ras activation was observed when the Lhx2 LIM domain and the transactivation domain of Elk1 are directed to a minimal reporter gene. A yeast two-hybrid screen has identified the recently described melanocyte-specific gene-related gene 1 (MRG1) as an Lhx2 LIM-interacting protein. MRG1 binds Lhx2 in vitro, and a co-immunoprecipitation assay has provided evidence that endogenous MRG1 forms a complex with Lhx2 in alphaT3-1 cells. Expression of MRG1 in alphaT3-1 cells enhances alpha-subunit reporter gene activity. MRG1 was also shown to bind in vitro to the TATA-binding protein and the transcriptional coactivator, p300. These data suggest a model in which the Lhx2 LIM domain activates transcription through interaction with MRG1 leading to recruitment of p300/CBP and the TATA-binding protein (Glenn, 1999).

DNA methylation plays an important role in gene regulation. A human LIM-HOX gene, hLH-2, is highly expressed in chronic myelogenous leukemia (CML) and located on chromosome 9q33-34.1, in the same region as the reciprocal translocation that creates the Bcr-Abl chimera of Philadelphia chromosome. To elucidate the mechanism of hLH-2 transcriptional activation, the methylation status of hLH-2 was studied in normal bone marrow and CML cells. When blots containing genomic DNA digested with Hpa II or Msp I are hybridized with a full-length cDNA probe, hLH-2 is methylated in normal bone marrow cells in which hLH-2 is not expressed; in contrast, both alleles of hLH-2 locus in CML cells are heavily hypomethylated. Using the sensitive RT-PCR technique, the expression of LH-2 was examined in mouse x human hybrids and a wide array of mouse cell lines containing Abl or Bcr-Abl: a consistent expression pattern in the cell lines tested could not be demonstrated. These results suggest that the transcriptional activation of hLH-2 in CML is likely due to a cis-acting effect, but not a trans-acting effect of the Bcr-Abl fusion protein. Because hypomethylated genes generally are transcribed more efficiently than hypermethylated genes, the high level of hLH-2 mRNA in CML cells probably is a consequence of the low level of methylation of the gene in the leukemic cells (Wu, 1997).

Two of the vertebrates genes that specify limb DV identity, Wnt-7a and En-1, do not appear to have functional equivalents in Drosophila. A third gene, Lmx-1, has been proposed to be a homolog of the Drosophila apterous gene, since not only do these genes share sequence similarity but also both genes are sufficient to specify dorsal cell fate. Importantly, besides determining dorsality, apterous is also necessary for wing outgrowth and contributes to the positioning of the wing margin at the dorsoventral boundary through regulating the expression of the fringe and Serrate genes. It appears that Lmx-1 does not share this activity since mis-expression of the Lmx-1 gene has no effect on limb outgrowth, or on apical ectodermal ridge (AER, a key structure required for limb outgrowth) formation. Lmx-1 is not only sufficient, but also necessary, to specify dorsal cell fate, since down-regulation of Lmx-1 activity results in limbs lacking dorsal-specific structures. Lmx-1 is neither able to induce R-fng when mis-expressed in the limb or the flank of the embryo, nor Drosophila fringe, when ectopically expressed in flies. Further, another LIM-homeodomain gene has been cloned, Lhx2, that shows higher sequence conservation with apterous; unlike Lmx-1, when mis-expressed in the flank of the embryo, it induces ectopic R-fringe expression. Lhx2 is also able to induce fringe and Wingless (downstream targets of apterous) expression in Drosophila. Consistent with these data, down-regulation of Lhx2 activity causes a down-regulation of genes required for the outgrowth of the limb along its proximodistal axis and consequently results in arrested limb outgrowth. Finally, unlike Lmx-1, Lhx2 does not specify dorsal cell fate (Rodriguez-Esteban, 1998).

These data suggest that Lhx2 could be a bona fide vertebrate homolog of apterous, yet in vertebrates, it does not specify dorsal cell fate. This raises the question of whether in vertebrates, unlike in Drosophila, limb outgrowth can be dissociated from the establishment of the DV axis and brings into focus the questions of whether or not vertebrate and Drosophila limbs can be considered to be homologous structures and how limbs in flies and vertebrates have evolved to be so different (Rodriguez-Esteban, 1998).

Apterous plays several roles during Drosophila wing development. Its role in wing margin formation and wing outgrowth is thought to be realized through the activation of fringe and Serrate expression. Indeed, it has been suggested that Apterous may serve directly as a transcriptional regulator for the fringe gene. Additionally, apterous acts as a selector gene specifying the dorsal wing disk compartment, although the exact molecular pathway is not yet established. The data presented in this paper indicate that in vertebrates these functions are executed by at least two proteins. Expression of Lmx-1 in the dorsal mesoderm is both necessary and sufficient to define dorsal cell fate, but it plays no role in regulating gene expression required for limb outgrowth. Lhx2 on the other hand, which based on sequence comparison, is more similar to Drosophila apterous, does not appear to specify dorsal cell fate and, moreover, is expressed with no dorsoventral asymmetry. However, Lhx2 does appear to perform the second role of Apterous, i.e. it regulates gene expression involved in AER formation and hence limb outgrowth. Furthermore, mis-expression of vertebrate Lmx-1 in Drosophila wing imaginal discs is neither able to rescue apterous mutants (flies lacking wings), nor to induce fringe expression in the imaginal disc. Taken together with the fact that vertebrate Lhx2 is able to rescue apterous mutant flies [i.e. to restore normal fringe and wingless (downstream targets of apterous) expression, wing morphology and other phenotypes due to lack of apterous], it would appear that Lhx2 is functionally closer to Drosophila apterous than Lmx-1 (Rodriguez-Esteban, 1998 and references).

Not only does the appearance of Lhx2 transcripts precede AER formation, but they are also maintained throughout limb bud development. Several lines of evidence suggest that Lhx2 is involved in both induction and maintenance of the AER, and hence in limb outgrowth. (1) Ectopic Lhx2 expression induces R-fng expression, Fgf-8 and Wnt-3a, genes involved in ridge formation. (2) Surgical removal of the AER results in loss of Lhx2 expression. (3) Fgf-8 expression maintains Lhx2 expression. (4) Down-regulation of Lhx2 activity perturbs AER formation and results in arrested limb outgrowth at different stages of development. Thus, Lhx2 may not only be required for continuous limb outgrowth, but may also have a role in positioning the AER at the dorsoventral limb boundary. Like apterous in Drosophila, the role of Lhx2 in AER formation could be mediated through R-fng. It is important to note, however, that while in Drosophila apterous is expressed in the same tissue as fringe, in vertebrates Lhx2 is expressed in the mesoderm. Since it is unlikely that Lhx2 directly regulates R-fringe transcription in the ectoderm, there must be an indirect mechanism that allows Lhx2 to activate R-fringe expression on the ectoderm (Rodriguez-Esteban, 1998).

The early mechanisms that initiate regional patterning in the dorsal telencephalon, which gives rise to cerebral cortex, have been investigated. Members of the LIM-homeodomain (LIM-HD) family of transcription factors are implicated in patterning and cell fate specification in several systems including the mammalian forebrain. Mice in which Lhx2 is disrupted were reported to have reduced telencephalic development, and the hippocampal primordium appear to be missing, by morphological observation. It was hypothesized that this may be due to a defect in the cortical hem, a Wnt- and Bmp-rich putative signaling center in the medial telencephalon, a source of regulatory signals for hippocampal development. The hem intervenes between presumptive cortical tissue and the non-neuronal choroid plexus. The cortical hem in the telencephalon appears to be the counterpart of the roof plate of the spinal cord, where dorso-ventral patterning is regulated by opposing signaling centers, the notochord and floor plate at the ventral midline, and the roof plate at the dorsal midline. It was asked if the expression of any known hem-specific signaling molecule is deficient in Lhx2-/- mice. Unexpectedly, at embryonic day (E)12.5, what appears to be some spared 'lateral' cortex is instead an expanded cortical hem. Normally restricted to the extreme medial edge of the telencephalon, the hem covers almost the entire dorsal telencephalon in Lhx2-/- mice. This indicates a role for Lhx2 in the regulation of the extent of the cortical hem. In spite of an expanded, mislocated hem in the Lhx2-/-telencephalon, a potential source of ectopic dorsalizing cues, no hippocampal differentiation is detected in tissue adjacent to the mutant hem, nor does the overall dorsoventral patterning appear perturbed. It is proposed that Lhx2 is involved at a crucial early step in patterning the telencephalon, where the neuroepithelium is first divided into presumptive cortical tissue, and the cortical hem. The defect in the Lhx2-/- telencephalon appears to be at this step (Bulchand, 2001).

It is proposed that Lhx2 participates in a mechanism by which precursors of the cortex are distinguished from those of the hem. In this model, the hem, a signaling center in the dorsal telencephalon, would be created first, by the allocation of a group of cells specified to a 'hem-precursor' fate; this would be followed by the proliferation of hem- and cortex-precursors, and the subsequent patterning of the latter, potentially involving cues from the hem. Lhx2 expression, normally present in cortical neuroepithelium but excluded from the hem, would serve to restrict the hem to its normal size and location: Lhx2 would participate in specifying 'non-hem' fate in cortical precursors. In the absence of Lhx2, more neuroepithelial precursors would take on a cortical hem fate (Bulchand, 2001).

The anatomical and functional organization of dorsal thalamus (dTh) and ventral thalamus (vTh), two major regions of the diencephalon, is characterized by their parcellation into distinct cell groups, or nuclei, that can be histologically defined in postnatal animals. However, because of the complexity of dTh and vTh and difficulties in histologically defining nuclei at early developmental stages, understanding of the mechanisms that control the parcellation of dTh and vTh and the differentiation of nuclei is limited. A set of regulatory genes, which include five LIM-homeodomain transcription factors (Isl1, Lhx1, Lhx2, Lhx5, and Lhx9) and three other genes (Gbx2, Ngn2, and Pax6), have been defined that are differentially expressed in dTh and vTh of early postnatal mice in distinct but overlapping patterns that mark nuclei or subsets of nuclei. These genes exhibit differential expression patterns in dTh and vTh as early as embryonic day 10.5, when neurogenesis begins; the expression of most of them is detected as progenitor cells exit the cell cycle. Soon thereafter, their expression patterns are very similar to those observed postnatally, indicating that unique combinations of these genes mark specific cell groups from the time they are generated to their later differentiation into nuclei. These findings suggest that these genes act in a combinatorial manner to control the specification of nuclei-specific properties of thalamic cells and the differentiation of nuclei within dTh and vTh. These genes may also influence the pathfinding and targeting of thalamocortical axons through both cell-autonomous and non-autonomous mechanisms (Nakagawa, 2001).

The dTh is parcellated into over one dozen nuclei. The principal sensory nuclei, dorsal lateral geniculate (dLG), ventroposterior (VP), and ventral medial geniculate (MGv), relay sensory information from the periphery to primary sensory areas of the neocortex, visual, somatosensory, and auditory, respectively, via thalamocortical axons (TCAs). Other nuclei, such as posterior (Po) and lateral posterior (LP), project broadly to cortex. The vTh has three major nuclei: reticular (RT), zona incerta (ZI), and ventral lateral geniculate (vLG). Different domains of embryonic vTh are required for TCA pathfinding (Nakagawa, 2001 and references therein).

The vTh and dTh have been defined as adjacent domains of the embryonic diencephalic alar plate based on expression of the homeodomain transcription factors Dlx2 and Gbx2, respectively, and restrictions in cell movement. However, little is known about the organization of embryonic dTh and vTh into discrete cell groups that presage their differentiation into nuclei, because the morphology and connections that define nuclei emerge late in development. The LIM-homeodomain (LIM-HD) family of transcription factors, as well as Gbx2, Pax6, and Neurogenin2, are candidates to be differentially expressed within dTh and vTh and control their parcellation. The LIM-HD genes Lhx1 and Lhx5 are expressed in early embryonic diencephalon, Lhx2 and Lhx9 in embryonic dTh, and Isl1 in adult RT. LIM-HD genes are intriguing because their unique combinations mark subsets of spinal neurons and specify their phenotypes, including axonal projections. Gbx2 is expressed broadly early in dTh and later in a subset of nuclei that require it for their differentiation, as well as for the development of the TCA projection. Pax6, a paired-box transcription factor, is expressed broadly early in vTh, later more discretely, and is required for development of RT, ZI, and vLG and TCA pathfinding. Ngn2, a basic helix-loop-helix transcription factor expressed in a subset of progenitor cells in dTh, is required for sensory neuron differentiation and dorsoventral patterning of the telencephalon. These regulatory genes are expressed in distinct yet often overlapping patterns, suggesting that they cooperate to control the specification and differentiation of thalamic nuclei and cell types (Nakagawa, 2001 and references therein).

The sets of genes that are expressed in dTh and vTh are distinct from one another and similar to those expressed in dorsal and ventral spinal cord, respectively. This similarity suggests that the expression patterns in thalamus might be established by mechanisms similar to those in spinal cord. In spinal cord, inductive signals from the roof plate and floor plate control neuronal fate along the dorsoventral axis. Signals from the roof plate, such as TGFß family members, are required in dorsal spinal cord for the induction of Lhx2 and Lhx9, which define D1A and D1B interneurons, respectively. In ventral spinal cord, distinct classes of motor neurons and ventral interneurons are generated by a graded signaling activity of Shh. Shh controls these neural fates by establishing different progenitor cell populations defined by their expression of Pax6 and Nkx2.2. Pax6 establishes distinct populations of ventral progenitor cells and controls the identity of motor neurons and V1 and V2 interneurons, whereas Nkx2.2 specifies the identity of V3 interneurons at a more ventral location. These genes appear to be essential intermediaries for Shh to regulate the differential expression of LIM-HD proteins, including Lhx1, Lhx3, Lhx4, Lhx5, Isl1, and Isl2. In diencephalon, Shh is transiently expressed as early as E9.5 in the zona limitans intrathalamica , which at this stage is a narrow cell domain interposed between prospective dTh and vTh. Similar to ventral spinal cord, Nkx2.2 and Pax6 are also expressed in progenitor cells in vTh. Shh induces in vitro the expression of Isl1 in chick forebrain explants and neuroepithelial cells from rat forebrain. Therefore, ZLI-derived Shh may specify progenitor cell types in vTh to produce different neuronal subtypes, which are determined by the subset of LIM-HD and other transcription factors expressed by these neurons. Interestingly, dTh, which is adjacent to the ZLI, does not express any of the LIM-HD genes induced by Shh and expressed in vTh. Ngn2, which is expressed by progenitor cells of dTh but not vTh, could act to limit the responsiveness of dTh to an Shh-mediated induction of vTh-type LIM-HD genes, which may be a crucial step in regionalization of the diencephalon (Nakagawa, 2001 and references therein).

Progenitor cells in the mouse olfactory epithelium generate over a thousand subpopulations of neurons, each expressing a unique odorant receptor (OR) gene. This event is under the control of spatial cues, since neurons in different epithelial regions are restricted to express region-specific subsets of OR genes. Progenitors and neurons express the LIM-homeobox gene Lhx2, and neurons in Lhx2-null mutant embryos do not diversify into subpopulations expressing different OR genes and other region-restricted genes such as Nqo1 and Ncam2. Lhx2-/- embryos have, however, a normal distribution of Mash1-positive and neurogenin 1-positive neuronal progenitors that leave the cell cycle, acquire pan-neuronal traits and form axon bundles. Increased cell death in combination with increased expression of the early differentiation marker Neurod1, as well as reduced expression of late differentiation markers (Galphaolf and Omp), suggests that neuronal differentiation in the absence of Lhx2 is primarily inhibited at, or immediately prior to, onset of OR expression. Aberrant regional expression of early and late differentiation markers, taken together with unaltered region-restricted expression of the Msx1 homeobox gene in the progenitor cell layer of Lhx2-/- embryos, shows that Lhx2 function is not required for all aspects of regional specification of progenitors and neurons. Thus, these results indicate that a cell-autonomous function of Lhx2 is required for differentiation of progenitors into a heterogeneous population of individually and regionally specified mature olfactory sensory neurons (Kolterud, 2004).

Lhx2 links the intrinsic and extrinsic factors that control optic cup formation

A crucial step in eye organogenesis is the transition of the optic vesicle into the optic cup. Several transcription factors and extracellular signals mediate this transition, but whether a single factor links them into a common genetic network is unclear. This study provides evidence that the LIM homeobox gene Lhx2, which is expressed in the optic neuroepithelium, fulfils such a role. In Lhx2-/- mouse embryos, eye field specification and optic vesicle morphogenesis occur, but development arrests prior to optic cup formation in both the optic neuroepithelium and lens ectoderm. This is accompanied by failure to maintain or initiate the expression patterns of optic-vesicle-patterning and lens-inducing determinants. Of the signaling pathways examined, only BMP signaling is noticeably altered and Bmp4 and Bmp7 mRNAs are undetectable. Lhx2-/- optic vesicles and lens ectoderm upregulate Pax2, Fgf15 and Sox2 in response to BMP treatments, and Lhx2 genetic mosaics reveal that transcription factors, including Vsx2 and Mitf, require Lhx2 cell-autonomously for their expression. These data indicate that Lhx2 is required for optic vesicle patterning and lens formation in part by regulating BMP signaling in an autocrine manner in the optic neuroepithelium and in a paracrine manner in the lens ectoderm. A model is proposed in which Lhx2 is a central link in a genetic network that coordinates the multiple pathways leading to optic cup formation (Yun, 2009).

Loss of function of factor Sall3 eliminates expression of the horizontal cell-specific transcription factor Lhx1

The mammalian retina is a tractable model system for analyzing transcriptional networks that guide neural development. Spalt family zinc-finger transcription factors play a crucial role in photoreceptor specification in Drosophila, but their role in mammalian retinal development has not been investigated. This study shows that that the spalt homolog Sall3 is prominently expressed in developing cone photoreceptors and horizontal interneurons of the mouse retina and in a subset of cone bipolar cells. Sall3 is both necessary and sufficient to activate the expression of multiple cone-specific genes, and Sall3 protein is selectively bound to the promoter regions of these genes. Notably, Sall3 shows more prominent expression in short wavelength-sensitive cones than in medium wavelength-sensitive cones, and Sall3 selectively activates expression of the short but not the medium wavelength-sensitive cone opsin gene. It was further observed that Sall3 regulates the differentiation of horizontal interneurons, which form direct synaptic contacts with cone photoreceptors. Loss of function of Sall3 eliminates expression of the horizontal cell-specific transcription factor Lhx1, resulting in a radial displacement of horizontal cells that partially phenocopies the loss of function of Lhx1. These findings not only demonstrate that Spalt family transcription factors play a conserved role in regulating photoreceptor development in insects and mammals, but also identify Sall3 as a factor that regulates terminal differentiation of both cone photoreceptors and their postsynaptic partners (de Melo, 2011).

These findings demonstrate that Sall3 plays a crucial role in regulating the development of both cone photoreceptors and horizontal interneurons (see Role of Sall3 in horizontal cell and S-cone photoreceptor development). Sall3 is necessary for the expression of a range of cone-specific genes, with only a very small number of short wavelength-sensitive cone opsin (Sop) positive and cone Arrestin (Arr3) positive cells detectable in Sall3-/- retinas. Microarray experiments revealed that many other cone-enriched transcripts are selectively downregulated in Sall3-/- retinas, including Pde6c, Pde6h and Crb1, whereas some, such as Gnat2 and Gnb3, show little or no change. Strikingly, overexpression of Sall3 by electroporation was sufficient to induce the expression of both Sop and Arr3, not only in photoreceptors but also in a subset of cells in the inner nuclear layer (INL), without affecting expression of medium wavelength-sensitive cone opsin (Mop). Rod photoreceptors overexpressing Sall3 in electroporated retinas were morphologically indistinguishable from those of controls. These rod photoreceptors that overexpressed Sall3 retained their normal morphology, laminar position and pattern of expression of cell-specific markers (de Melo, 2011).

These data imply that Sall3 is activated in Sop-expressing photoreceptors undergoing terminal differentiation, and that Sall3 itself might directly activate a subset of cone-specific genes in a coordinated manner, analogous to the orphan nuclear hormone receptor Errβ (Esrrb) in rod photoreceptors. Several different nuclear hormone receptors, along with the transcriptional co-regulator Pias3, have been shown to be required for medium wavelength-sensitive cones to activate the expression of Mop while simultaneously repressing the expression of Sop. Sall3, however, represents the first cone-expressed transcription factor that selectively actives the expression of Sop (de Melo, 2011).

The strong activation of S-cone-specific transcripts by Sall3 implies a surprising functional homology with the Spalt gene complex of Drosophila in the regulation of photoreceptor differentiation. In Drosophila, Spalt genes are necessary for specification of the inner R7 and R8 photoreceptors, which are responsible for color discrimination and in many respects form a cone-like photoreceptor class in the compound eye. Strikingly, loss of function of Spalt genes results in the ectopic expression of Rh1 opsin (NinaE), which is normally expressed in the outer R1-R6 photoreceptors, in the R7 and R8 cells, whereas expression of Rh3-Rh6 is lost. However, axonal projections of inner photoreceptors are unaltered in Drosophila sal mutants. This partial shift in photoreceptor identity resembles that seen following Sall3 overexpression in rod photoreceptors. These cells appear morphologically normal and continue to express rhodopsin, but now robustly express cone-specific genes. Notably, Spalt genes are selectively expressed in blue-sensitive Rh5-positive R8 photoreceptors and are required for expression of Rh5 opsin (de Melo, 2011).

In mice, it was observed that Sall3 is both necessary and sufficient for the expression of blue-sensitive cone opsin but not green-sensitive cone opsin. Such a direct conservation of gene function in photoreceptor development is unusual, and even more surprising because the blue-sensitive visual opsins of insects and vertebrates evolved independently (Shichida, 2009). Although this observation might represent evolutionary convergence, it could alternatively imply that ancestral bilateria possessed a dedicated short wavelength-sensitive photoreceptor cell type, the differentiation of which was guided by a Spalt family gene, with the blue-sensitive opsin gene expressed by this cell having changed in different lineages; photoreceptors might, at one point, have coexpressed both blue-sensitive ciliary and rhabdomeric opsins. This possibility is not without precedent, as both vertebrate and invertebrate photoreceptors are known to coexpress different opsin genes with similar spectral sensitivities. In some cases, opsin genes with similar spectral sensitivity but which are nonetheless highly divergent at the molecular level are coexpressed, such as the blue-sensitive melanopsin and retinal cone opsins of the chick retina. Analysis of Spalt family gene expression in invertebrates from multiple phyla with well-characterized color vision should further clarify this finding (de Melo, 2011).

Sall3 also plays a pivotal role in regulating the differentiation of horizontal cells, one of two cell types directly postsynaptic to cone photoreceptors, and might do so in part through maintenance of Lhx1 expression. No defects were observed in the expression of other previously reported horizontal cell-expressed transcription factors in Sall3-/- retinas, including Pax6, Foxn4 and Ptf1a, although cell counts did reveal a reduction in the number of Prox1-positive cells in the dorsal retina at P0. However, the final laminar position of Sall3-/- horizontal cells closely resembles that seen in Lhx1 mutants. Horizontal cells of Chx10-Cre; Lhx1lox/lox mutants fail to undergo initial outward radial migration, resulting in ectopic localization to the inner INL and extension of their dendritic arbor within the IPL. Horizontal cells of Sall3-/- retinas appear to initiate Lhx1 expression and undergo outward radial migration normally. However, by P0, Lhx1 expression is drastically reduced, and at later ages the majority of horizontal cell bodies are found at the scleral border of the IPL, eventually taking up residence with amacrine cells and extending dendrites into the IPL, phenocopying the Lhx1 mutants. The phenomenon of ectopic inner nuclear/plexiform layer-associated horizontal cells was also seen in Sall3 overexpression experiments in which Sall3 appeared to be sufficient to at least partially specify horizontal cells, including activating Prox1 and NF165. The lack of coexpression with markers specific to AII amacrine cells indicated that the Prox1-expressing wide-field cells generated in Sall3 electroporation experiments were not a dysmorphic AII population. This does not exclude the possibility that Sall3 overexpression results in the generation of a rare Sall3+ Prox1+ wide-field amacrine subclass. Notably, Spalt genes also regulate prospero expression in developing Drosophila photoreceptors, which then acts to guide differentiation of the R7 photoreceptor subtype. Overexpression of Sall3 in neonatal retinas was not sufficient to induce expression of Lhx1, and, consequently, the horizontal-like cells demonstrated the same ectopic positioning seen in Lhx1 and Sall3 mutants (de Melo, 2011).

The crucial role of Sall3 in regulating Lhx1 expression is further underlined by microarray analysis, which indicates that Lhx1 is one of the most strongly downregulated genes in Sall3-/- retinas. Taken together, these data suggest that sustained Lhx1 expression might regulate multiple stages of horizontal cell migration, and that Sall3 is necessary to maintain Lhx1 expression during the postnatal differentiation of horizontal cells. A limited number of Sall3+ NF165+ and Sall3+ Prox1+ horizontal cells are present in Six3-Cre; Lhx1lox/lox retinas, implying that Sall3 regulates aspects of horizontal cell development at least in part through an Lhx1-independent pathway and that expression of Sall3 itself might not require Lhx1 (de Melo, 2011).

A subset of bipolar interneurons selectively expressed Sall3. Dedicated S-cone-selective bipolar cells in the mouse retina that are analogous to primate S-cone-selective midget bipolar cells have been identified. The tantalizing possibility exists that Sall3 might regulate the differentiation of S-cones and their dedicated bipolar interneuron. However, the microarray analysis did not reveal any significant changes in known mouse bipolar-expressed genes, and the role of Sall3 in bipolar interneuron development remains to be resolved (de Melo, 2011).

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apterous: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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