apterous

EVOLUTIONARY HOMOLOGS

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Mammalian Lim domain proteins and early development

Recent experiments have implicated the visceral endoderm in anterior neural induction in mouse. The visceral endoderm is an extraembryonic tissue that surrounds the epiblast of the egg cylinder stage embryo. During gastrulation the visceral endoderm is replaced by definitive endoderm that derives from the anterior portion of the primitive streak. Although no morphological asymmetries are apparent in the visceral endoderm, molecular studies have shown that a distinct anterior-posterior pattern exists in the visceral endoderm prior to the formation of the primitive streak. These studies reveal that the VE-1 antigen and the homeobox genes Hesx1/Rpx and Hex are expressed in the anterior visceral endoderm that underlies the ectoderm fated to form the anterior portion of the neural plate. Ablation experiments have shown that, if the anterior visceral endoderm is removed at the early streak stage, expression of Hesx1/Rpx in the anterior neuroectoderm in late streak/headfold stage embryos is absent or greatly reduced (Shawlot, 1999 and references therein).

Lim1 is a homeobox gene expressed in the extraembryonic anterior visceral endoderm and in primitive streak-derived tissues of early mouse embryos. Mice homozygous for a targeted mutation of Lim1 lack head structures anterior to rhombomere 3 in the hindbrain. To determine in which tissues Lim1 is required for head formation and its mode of action, chimeric mouse embryos were generated and tissue layer recombination explant assays were performed. In chimeric embryos in which the visceral endoderm is composed of predominantly wild-type cells, Lim1 -/- cells are able to contribute to the anterior mesendoderm of embryonic day 7.5 chimeric embryos but embryonic day 9.5 chimeric embryos display a range of head defects. In addition, early somite stage chimeras generated by injecting Lim1 -/- embryonic stem cells into wild-type tetraploid blastocysts lack forebrain and midbrain neural tissue. Furthermore, in explant recombination assays, anterior mesendoderm from Lim1 -/- embryos is unable to maintain the expression of the anterior neural marker gene Otx2 in wild-type ectoderm. In complementary experiments, embryonic day 9.5 chimeric embryos in which the visceral endoderm is composed of predominantly Lim1 -/- cells and the embryo proper of largely wild-type cells, also phenocopies the Lim1 -/- headless phenotype. These results indicate that Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation and that its inactivation in these tissues produces cell non-autonomous defects. A double assurance model in which Lim1 regulates sequential signaling events required for head formation in the mouse is discussed (Shawlot, 1999).

Mammalian Lim domain proteins and subdivision of the spinal cord

Motor neurons located at different positions in the embryonic spinal cord innervate distinct targets in the periphery, establishing a topographic neural map. The topographic organization of motor projections depends on the generation of subclasses of motor neurons that select specific paths to their targets. A family of LIM homeobox genes has been cloned in the chick. The combinatorial expression of four of these genes, Islet-1, Islet-2 (homologs of Drosophila Islet, Lim-1, and Lim-3, defines subclasses of motor neurons that segregate into columns in the spinal cord and select distinct axonal pathways. Thus the combination of LIM domain proteins serve to code motor neuron identity in the spinal cord (Tsuchida, 1994).

These LIM homeodomain proteins are expressed prior to the formation of distinct motor axon pathways and before motor columns appear. Depending on their arrangement in columns and eventual synaptic targets, motor neurons of the chick brain stem are designated as belonging to somatic motor (sm) visceral motor (vm), or branchiomotor (bm) classes or to the ipsilateral or contralateral vestibuloacoustic effect neuronal population. Sm neurons innervate muscle derived from the paraxial mesoderm and prechordal plate mesoderm. Bm, vm and vestibuloacoustic axons extend dorsolaterally for some distance through the neuroepithelium before converging on large single exit points within the dorsal neural tube (alar plate). Bm neurons innervate muscle derived from paraxial mesoderm within the branchial arches, while vm neurons innervate parasympathetic ganglia associated with lacrimal and salivary glands or neuronal plexuses that innervate smooth muscle; vestibuloacoustic efferent neurons innervate the hair cells of the inner ear. Subpopulations of spinal motor neurons within specific locations in the spinal cord and distinct targets in the periphery express different combinations of LIM homeobox genes. Sm neurons of the medial division of the median motor column express Islet-1, Isl-2, and Lim-3, while those of the lateral division of the median motor column and the medial division of the later motor column express Isl-1 and Isl-2. Sm neurons of the lateral division of the lateral motor column express Lim-1 and Isl-2. Since the lateral motor column is present only at limb levels, Lim-1 expression is restricted to these levels of the neuraxis. At early stages, visceral motor neurons express both Isl-1 and Isl-2, but after their migration to form the column of Terni, only a subset of these neurons continues to express Isl-1. These genes are good candidates to confer target specificity upon motor neuron classes, since they are expressed at times before the motor columns have fully segregated and before axons have reached their targets (Tsuchida, 1994).

The generation of distinct classes of motor neurons is an early step in the control of vertebrate motor behavior. To study the interactions that control the generation of motor neuron subclasses in the developing avian spinal cord in vivo grafting studies were performed in which either the neural tube or flanking mesoderm were displaced between thoracic and brachial levels. The positional identity of neural tube cells and motor neuron (MN) subtype identity was assessed by Hox and LIM homeodomain protein expression. Brachial (B) levels of the median motor column (MMC) are organized into three columns: neurons of the medial MMC (MMCM) co-express Isl1, Isl2 and Lim3, neurons of the medial lateral motor column (LMCM) co-express Isl1 and Isl2, and motoneurons of the lateral LMC (LMCL) coexpress Isl2 and Lim1. At thoracic (T) levels motoneurons are also organized into three columns: MMCM neurons; lateral MMC neurons that coexpress Isl1 and Isl2 but not Lim3, and dorsomedially positioned Column of Terni (CT) neurons that express only Isl1. Grafts of 13-15 segment quail T neural tube were placed rostrally at the B level of 12-15 segment chick hosts. Marker and morphological analysis reveals that grafted neural cells divert their normal T fates and their neuronal progeny acquire the molecular properties of B MNs. These changes in the neural tube are restricted to a limited time frame. The rostrocaudal identity of neural cells is plastic at the time of neural tube closure and is sensitive to positionally restricted signals from the paraxial mesoderm. Such paraxial mesodermal signals appear to control the rostrocaudal identity of neural tube cells and the columnar subtype identity of motor neurons. Analysis of neural Hoxc8 expression provides evidence that the change in cell identity after neural tube displacement is not restricted to the MNs; the change occurs in a graded manner along the rostrocaudal axos of the spinal cord, and is associated with both a rostral and caudal respecification in cell fate. In contrast, neural tube grafts between B and T levels do not change the pattern of Hoxc8 expression in the flanking paraxial mesodem. These results suggest that the generation of motor neuron subtypes in the developing spinal cord involves the integration of distinct rostrocaudal and dorsoventral patterning signals that derive, respectively, from paraxial and axial mesodermal cell groups (Ensini, 1998).

The diversification of neuronal cell types in the vertebrate central nervous system depends on inductive signals provided by local organizing cell groups of both neural and nonneural origin. The link between neuronal birth date, migratory pattern, and identity is also evident in the generation of motor neurons in the spinal cord. These conserved features are particularly apparent for motor neurons of the lateral motor column (LMC). This class of motor neurons is generated selectively at brachial and lumbar levels of the spinal cord, and their axons innervate target muscles in the limb. Within the LMC, motor neurons can be further divided into two subclasses: medial LMC neurons that project to ventrally derived limb muscles, and lateral LMC neurons that project to dorsally derived limb muscles. Motor neurons destined to form the medial LMC leave the cell cycle before lateral LMC neurons; as a consequence, prospective lateral LMC neurons emerge from the ventricular zone and migrate past medial LMC neurons to their final position. The time of generation and the distinct migratory environment represent two prominent differences between the development of lateral LMC neurons and other motor neurons. In addition, the total number of motor neurons generated at limb levels of the spinal cord is greater than that at nonlimb levels, presumably to accommodate the formation of the LMC (Sockanathan, 1998 and references).

All somatic motor neurons initially express Isl1 and Isl2, and most maintain the expression of these genes. Lateral LMC neurons, however, extinguish Isl1 and initiate Lim1 expression as they begin to migrate past medial LMC neurons, thus acquiring a unique LIM homeobox gene code. Studies of LIM homeobox gene function in vertebrates and invertebrates have provided evidence that this gene family has a role in motor neuron differentiation and axon pathfinding. The diversification of motor neuron subtypes is initiated by inductive signals from the axial and paraxial mesoderm that operate along the dorsoventral and rostrocaudal axes of the neural tube. However, medial and lateral LMC motor neurons are generated from progenitor cells that occupy the same dorsoventral and rostrocaudal positions, and thus it is unlikely that mesodermal signals impose this distinction. The late birth date of lateral LMC neurons and their migration past early-born LMC neurons prompted a consideration of whether the fate of lateral LMC neurons might be directed by signals provided by early-born LMC neurons. This hypothesis invokes the idea that LMC motor neurons generated at early stages express a local but non-cell-autonomous signal that induces the lateral LMC phenotype in late-born LMC neurons. A retinoid-mediated signal provided by one subset of early-born spinal motor neurons (the medial) imposes a local variation in the number of motor neurons generated at different axial levels and also specifies the identity of a later-born subset of motor neurons (the lateral). Thus, in the vertebrate central nervous system the distinct fates of late-born neurons may be acquired in response to signals provided by early-born neurons (Sockanathan, 1998).

To begin to define the contribution of retinoid signaling to motor neuron differentiation, the pattern of expression of retinaldehyde dehydrogenase 2 (RALDH2) in the developing spinal cord was examined. At brachial levels, RALDH2 expression is first detected at stage 19, and at this and subsequent stages, expression in the ventral spinal cord appears to be restricted to motor neurons. By stage 27, when the medial motor column (MMC) and LMC have segregated, expression of RALDH2 is restricted to LMC neurons. Within the LMC, RALDH2 is expressed by both medial and lateral LMC neurons. A similar LMC-specific pattern of RALDH2 expression is detected at lumbar levels. Consistent with the restriction of RALDH2 expression to LMC neurons, no expression of the gene is detected in motor neurons at thoracic levels. The expression of RALDH2 in motor neurons at brachial and lumbar levels persists until at least stage 35, although from stage 29 onward, expression gradually becomes restricted to specific motor neuron pools. The only other site of RALDH2 expression in the spinal cord is in the roof plate, both at limb and nonlimb levels. These selective results show that (1) RALDH2 expression is initiated during the early phase of motor neuron generation at brachial levels of the spinal cord; (2) RALDH2 expression distinguishes developing LMC neurons from other somatic or visceral motor neurons, and (3) RALDH2 expression precedes the appearance of Isl2+, Lim1+ lateral LMC neurons (Sockanathan, 1998).

The number of Isl+ motor neurons was counted in brachial ventral/floor plate (vf) explants grown either alone or with retinol (Rol), a metabolic precursor of retinoic acid, or with all-trans retinoic acid (RA). The number of Isl+ motor neurons in [vf] explants grown in the presence of either Rol or RA is increased by 60%. The detection of an increase in motor neuron number with Rol, as well as with RA, indicates that explants grown in medium with no added retinoid are deprived of the metabolic substrate required for synthesis of RA by RALDH2. To examine further the involvement of RALDH2 activity in the control of motor neuron number, thoracic [vf] explants, which do not express RALDH2, were exposed to Rol or RA and the number of Isl+ motor neurons measured. In contrast to results obtained with brachial level explants, exposure of thoracic [vf] explants to Rol does not increase motor neuron number, whereas RA similarly induces a 60% increase in Isl+ motor neurons. Taken together, these results provide evidence that (1) retinoids increase the number of motor neurons; (2) the increase in motor neuron number detected after exposure of brachial [vf] explants to Rol is correlated with the synthesis of active retinoids by RALDH2 activity, and (3) the apparent requirement for RALDH2-generated retinoids can be overcome by exogenous RA. The retinoid-induced increase in motor neuron number at brachial levels appears to result from an increase in the number of progenitor cells. These experiments suggest that, at limb levels, a RALDH2-generated LMC source of retinoids acts non-cell-autonomously to increase the number of motor neuron progenitors and, consequently, postmitotic motor neurons. Studies using an RAR antagonist show that retinoid receptor activation is required for the generation of lateral LMC neurons and for the control of motor neuron number. Maintenance of the lateral LMC phenotype appears to require ongoing retinoid signaling over the period that these neurons are migrating to their lateral position (Sockanathan, 1998).

RALDH2-dependent induction of lateral LMC neurons requires non-autonomous RA signaling. The onset of RALDH2 and Lim1 expression by lateral LMC neurons was examined. At stage 23, many Isl2+, Lim1+ lateral LMC neurons are still located medial to Isl1+, Isl2+ medial LMC neurons. These Isl2+, Lim1+ neurons do not express RALDH2, suggesting that their lateral LMC phenotype has not been acquired through cell-intrinsic RALDH2 activity. Many of the motor neurons that are located in an even more medial position, distant from RALDH2+ neurons, will populate the lateral LMC, but at this stage these neurons express Isl1/2 but not Lim1. These observations support the idea that the lateral phenotype of LMC neurons is acquired by virtue of the proximity of the neurons to a RALDH2-dependent signal provided by earlier-born LMC neurons. The late birth date of lateral LMC neurons requires that they migrate past early-born neurons to reach their final position. What role might this inside-out program of neuronal migration have in the establishment of the lateral LMC phenotype? The detection of late-born Isl2+, Lim1+ lateral LMC neurons in positions adjacent but medial to early-born RALDH2+ medial LMC neurons provides evidence that proximity to early-born neurons is sufficient to achieve a lateral LMC identity. The failure of late-born LMC neurons to migrate past medial LMC neurons might, however, have the consequence that some LMC neurons fail to be exposed to retinoid signals before they lose competence to respond. In this view, the migration of prospective lateral LMC neurons through early-born LMC neurons would achieve a rapid intermixing of inductive and responsive neurons and ensure that the entire population of late born LMC neurons efficiently encounters a local source of retinoid signals (Sockanathan, 1998).

The circuits that control movement are comprised of discrete subtypes of motor neurons. How motor neuron subclasses develop and extend axons to their correct targets is still poorly understood. LIM homeodomain factors Lhx3 and Lhx4 are expressed transiently in motor neurons whose axons emerge ventrally from the neural tube (v-MN). Motor neurons develop in embryos deficient in both Lhx3 and Lhx4, but v-MN cells switch their subclass identity to become motor neurons that extend axons dorsally from the neural tube (d-MN). Conversely, the misexpression of Lhx3 in dorsal-exiting motor neurons is sufficient to reorient their axonal projections ventrally. Thus, Lhx3 and Lhx4 act in a binary fashion during a brief period in development to specify the trajectory of motor axons from the neural tube (Sharma, 1998).

Lim domain proteins and limb patterning

It appears that the interaction between Wingless and Apterous in limb compartmentalization is evolutionarily conserved. During vertebrate limb development, the ectoderm directs the dorsoventral patterning of the underlying mesoderm. To define the molecular events involved in this process, an analysis has been made of the function of WNT7a (Drosophila homolog: Wingless), a secreted factor expressed in the dorsal ectoderm, and LMX1, a LIM homeodomain transcription factor expressed in the dorsal mesenchyme. Ectopic expression of Wnt7a is sufficient to induce and maintain Lmx1 expression in limb mesenchyme, both in vivo and in vitro. Ectopic expression of Lmx1 in the ventral mesenchyme is sufficient to generate double-dorsal limbs. Thus, the dorsalization of limb mesoderm appears to involve the WNT7a-mediated induction of Lmx1 in limb mesenchymal cells (Riddle, 1995).

The positional cues that govern the fate of cells along the dorsoventral axis of the developing vertebrate limb are established in the mesoderm before outgrowth of limb buds. apterous, a Drosophila LIM/-homeodomain gene expressed in the dorsal compartment of the wing disc, specifies dorsal cell fate. A vertebrate LIM-homeodomain containing gene, Chick Lmx1 (C-Lmx1), is expressed in the presumptive dorsal limb mesoderm and is restricted thereafter to the dorsal mesoderm of the developing chick bud. C-Lmx1 expression is regulated by the overlying ectoderm where Wnt7a messenger RNA is localized. Wnt7a, required for normal development of the dorsoventral axis in mouse limbs, can induce ectopic expression of C-Lmx1 in ventral mesoderm. Misexpression of C-Lmx1 during limb outgrowth causes ventral to dorsal transformations of limb mesoderm. This paper proposes that C-Lmx1 specifies dorsal cell fate during chick limb development (Vogel, 1995).

Classical embryological experiments have demonstrated that dorsal-ventral patterning of the vertebrate limb is dependent on ectodermal signals. One such factor is Wnt-7a, a member of the Wnt family of secreted proteins, which is expressed in the dorsal ectoderm. Loss of Wnt-7a results in the appearance of ventral characteristics in the dorsal half of the distal limb. Conversely, En-1 (Drosophila homolog: Engrailed) is expressed exclusively in the ventral ectoderm, where it represses Wnt-7a. En-1 mutants have dorsal characteristics in the ventral half of the distal limb. Experiments in the chick suggest that the dorsalizing activity of Wnt-7a in the mesenchyme is mediated through the regulation of the LIM-homeodomain transcription factor Lmx-1. The relationship between Wnt-7a, En-1 and Lmx-1b, a mouse homolog of chick Lmx-1, is examined in the patterning of the mammalian limb. Wnt-7a is required for Lmx-1b expression in distal limb mesenchyme; Lmx-1b activation in the ventral mesenchyme of En-1 mutants requires Wnt-7a. Consistent with Lmx-1b playing a primary role in dorsalization of the limb, a direct correlation is found between regions of the anterior distal limb in which Lmx-lb is misregulated during limb development and the localization of dorsal-ventral patterning defects in Wnt-7a and En-1 mutant adults. Thus, ectopic Wnt-7a expression and Lmx-1b activation underlie the dorsalized En-1 phenotype, although this analysis also reveals a Wnt-7a-independent activity for En-1 in the repression of pigmentation in the ventral epidermis. Ectopic expression of Wnt-7a in the ventral limb ectoderm of En-1 mutants results in the formation of a second, ventral apical ectodermal ridge (AER) at the junction between Wnt-7a-expressing and nonexpressing ectoderm. Unlike the normal AER, ectopic AER formation is dependent upon Wnt-7a activity, indicating that distinct genetic mechanisms may be involved in primary and secondary AER formation (Cygan, 1997).

The mesenchymal cells that contribute to oral and facial hard tissues are derived from cranial neural crest cells, whereas limb mesenchyme cells are derived from axial mesoderm. The outgrowth of facial processes has been compared with limb bud outgrowth; the tooth bud enamel knot has been identified as a signaling center with similarities to both the limb ZPA and AER signaling centers. In addition, several homeobox-containing genes have been implicated in both branchial arch and limb development, such as members of the Msx, Dlx and Lim-homeobox families. The expression of the Lim-domain gene Lhx-6, and its closely associated family member Lhx-7 are largely restricted to anterior mesenchymal cells of the mandibular and maxillary arches. Clim-2 (NLI, Lbd1) is one of two related mouse proteins that interact with Lim-domain homeoproteins. In the mouse, embryonic expression of Clim-2 is particularly pronounced in facial ectomesenchyme and limb bud mesenchyme in association with Lim genes Lhx-6 and Lmx-1, respectively. In common with both these Lim genes, Clim-2 expression is regulated by signals from overlying epithelium. In both the developing face and the limb buds Fgf-8 is identified as the likely candidate signaling molecule that regulates Clim-2 expression. In the mandibular arch, as in the limb, Fgf-8 functions in combination with CD44, a cell surface binding protein that has been considered to be a hyaluronan receptor. Blocking CD44 binding results in inhibition of Fgf8-induced expression of Clim-2 and Lhx-6. CD44 has been shown to be required for presentation of Fgf-8 to its receptor, rather than as a hyaluronan receptor. Regulation of gene expression by Fgf8 in association with CD44 is thus conserved between limb and mandibular arch development (Tucker, 1999).

Interaction of LIM homeodomain proteins with LIM-only proteins (see Drosophila Muscle LIM protein at 60A)

LIM homeodomain and LIM-only (LMO) transcription factors contain two tandemly arranged Zn2+-binding LIM domains capable of mediating protein-protein interactions. These factors have restricted patterns of expression, are found in invertebrates as well as vertebrates, and are required for cell type specification in a variety of developing tissues. A recently identified, widely expressed protein, NLI, binds with high affinity to the LIM domains of LIM homeodomain and LMO proteins in vitro and in vivo. A 38-amino-acid fragment of NLI is sufficient for the association of NLI with nuclear LIM domains. In addition, NLI forms high affinity homodimers through the amino-terminal 200 amino acids, but dimerization of NLI is not required for association with the LIM homeodomain protein Lmxl. Chemical cross-linking analysis reveals higher-order complexes containing multiple NLI molecules bound to Lmx1, indicating that dimerization of NLI does not interfere with LIM domain interactions. NLI formed complexes with Lmx1 on the rat insulin I promoter and inhibits LIM domain-dependent synergistic transcriptional activation by means of Lmx1 and the basic helix-loop-helix protein E47 from the rat insulin I mini-enhancer. These studies indicate that NLI contains at least two functionally independent domains and may serve as a negative regulator of synergistic transcriptional responses that require direct interaction via LIM domains. Thus, NLI may regulate the transcriptional activity of LIM homeodomain proteins by determining specific partner interactions (Jurata, 1997).

Interaction of LIM-homeodomain proteins with LBd1, a LIM Domain-binding protein

LIM domains mediate protein-protein interactions. Within LIM-homeodomain proteins, the LIM domains act as negative regulators of the transcriptional activation function of the protein. The recently described protein Ldb1 (also known as NLI, or LIM domain-binding protein) binds LIM domains in vitro and synergizes with the LIM-homeodomain protein Xlim-1 in frog embryo microinjection experiments. The transcriptional activation domain of Xlim-1 has been localized to its carboxyl-terminal region; the interactions of the amino-terminally located LIM domains with Ldb1 have been characterized. Ldb1 binds LIM domains through its carboxyl-terminal region, and can form homodimers via its amino-terminal region. Optimal binding to Ldb1 requires tandem LIM domains, while single domains can bind with lower, yet clearly measurable efficiencies. In animal explant experiments, synergism of Ldb1 with Xlim-1 in the activation of downstream genes requires both the region containing the dimerization domain of Ldb1 and the region containing the LIM-binding domain. The role of Ldb1 may be to recruit other transcriptional activators depending on the promoter context and LIM-homeodomain partner involved (Breen, 1998).

Coactivators for Lim domain proteins

Two highly homologous proteins specifically interact with the LIM domains of P-Lim/Lhx3 and several other LIM homeodomain factors. Transcripts encoding these factors can be detected as early as mouse E8.5, with maximal expression observed in regions of the embryo in which the LIM homeodomain factors P-Lim/Lhx3, Isl-1, and LH-2 are selectively expressed. These proteins can potentiate transactivation by P-Lim/Lhx-3 and are required for a synergistic activation of the glycoprotein hormone alpha-subunit promoter by P-Lim/Lhx3 and a pituitary Otx class homeodomain transcription factor (P-OTX/Ptx1), with which they also specifically associate. The two new genes are referred to as CLIM-1 and CLIM-2 (cofactor of LIM-homeodomain proteins). The CLIM proteins are required for a transcriptional synergy between P-Lim/Lhx3 and P-OTX/Ptx1. The fact that CLIM-encoded mRNAs show a widely overlapping expression pattern with Otx1 and Otx2 in the developing mouse brain suggests that the CLIM protein family may play critical roles in the functional relationships of LIM homeoproteins and additional Otx factors (Bach, 1997).

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