Table of contents

Other Lim domain proteins of mammals

lim1 is a member of the LIM class homeobox gene family in the mouse. lim1 cDNA encodes a predicted 406 amino acid protein that is 93% identical with the product of the Xenopus LIM class homeobox gene Xlim1. lim1 is expressed both during embryogenesis and in the adult brain. lim1 is expressed in the central nervous system from the telencephalon through the spinal cord and in the developing excretory system including pronephric region, mesonephros, nephric duct, and metanephros. In the metanephros, lim1 is strongly expressed in renal vesicles and S-shaped bodies, and transcripts are also detected in the ureteric branchesm (Fujii, 1994).

cDNAs encoded by the mouse homolog (Lim-1) of the Xenopus LIM-class homeobox gene Xlim-1 have been isolated from an 8.5-day mouse embryo cDNA library. Nucleotide and deduced amino acid sequences show a high degree of identity with Xlim-1 in the LIM and homeodomains, and 85% identity over the whole protein. An interspecific back-cross has been used to show close linkage of Lim-1 to the endogenous proviral marker Mpmv-4 on mouse chromosome 11. In mid- to late-streak stage embryos, Lim-1 is expressed in a restricted region of mesoderm in the primitive streak, with the highest level of signal at the anterior. At 7.5 days, transcripts can be seen in a horseshoe-shaped pattern in the periphery of the node, as well as along both sides of the immediately adjacent notochord. In addition, transcripts are present in presumptive lateral and intermediate mesoderm. Later, expression becomes progressively restricted to intermediate mesoderm, the nephrogenic cords, and eventually mesonephric ducts and tubules. By 10.5 days Lim-1 transcripts also appear in restricted regions of the central nervous system (CNS) that are associated with sensory function. The lateral diencephalon, hindbrain, and presumed commissural neurons in the dorsal spinal cord all show Lim-1 expression. In the adult, Lim-1 is expressed in the cerebellum/medulla and kidney, and at very low levels in the cerebrum. These data suggest that in the mouse embryo Lim-1 plays a role in early mesoderm formation and later specification of a differentiated phenotype in subsets of cells of the mesonephros and sensory neurons of the CNS (Barnes, 1994).

Recent embryological and genetic experiments have suggested that the anterior visceral endoderm and the anterior primitive streak of the early mouse gastrula function as head- and trunk-organizing centers, respectively. HNF3beta and Lim1 are coexpressed in both organizing centers suggesting synergistic roles for these genes in regulating organizer functions and hence axis development in the mouse embryo. To investigate this possibility, compound HNF3beta and Lim1 mutant embryos were generated. An enlarged primitive streak and a lack of axis formation were observed in double mutant but not in single homozygous mutant embryos. Chimera experiments indicate that the primary defect in these double homozygous mutants is due to loss of activity of HNF3beta and Lim1 in the visceral endoderm. Altogether, these data provide evidence that these genes function synergistically to regulate organizer activity of the anterior visceral endoderm. Moreover, double mutant embryos also exhibit defects in mesoderm patterning that are likely due to lack of specification of anterior primitive streak cells (Perea-Gómez, 1999).

The first morphological sign of A-P pattern in the epiblast of the mouse embryo is the site of formation of the primitive streak at the posterior end of the embryo. The genetic pathway that initiates primitive streak formation remains to be elucidated, but expression of T on one side of the epiblast at the onset of gastrulation marks posterior primitive streak cells. In HNF3beta,Lim1 double mutant embryos, T expression in the epiblast is no longer restricted posteriorly, but is instead expressed throughout the epiblast by the mid-streak stage. Thus, A-P polarity of the epiblast is abnormal in HNF3beta,Lim1 embryos and widespread expression of T strongly suggests that mutant epiblast cells are transformed into primitive streak cells. The loss of epiblast cells is confirmed by the absence and reduction of expression of Otx2 and Oct4, respectively. In addition, mid-streak-stage embryos also show ectopic mesoderm formation as demonstrated by the expression of MesP1 and Lefty2. As a consequence of these early patterning defects, ectoderm and neurectoderm cells that are derived from distal and anterior epiblast cells are missing in these embryos at 7.5-7.75 d.p.c. These epiblast defects are not observed in single homozygous HNF3beta and Lim1 mutants. Altogether, these data demonstrate that HNF3beta and Lim1 function synergistically to establish A-P patterning of the epiblast and to restrict primitive streak formation to the posterior side of mouse embryos (Perea-Gómez, 1999).

Murine Lhx3 cDNA isolated from the mouse pituitary cDNA library encodes a LIM-type homeodomain protein that contains two tandemly repeated LIM domains and the homeodomain. The identities of predicted amino acid sequences between the mouse of Lhx3 and Xenopus Xlim-3 genes are 80, 95, and 97% in the LIM domains 1 and 2, and the homeodomain, respectively, and 84% in the entire protein. 5'-RACE procedures and genomic cloning revealed that two distinct N-terminal sequences arise from two different exons 1a and 1b. Exon 1a encodes a sequence similar to that of Xlim-3, whereas exon 1b encodes a different N-terminus. It is likely that there are two transcription initiation sites in the Lhx3 gene. The Lhx3 transcripts were detected by whole mount in situ hybridization as early as day E9.5 post coitum in Rathke's pouch and the closing neural tube. During subsequent development, Lhx3 expression was observed in the anterior and intermediate but not in the posterior lobes of the pituitary, and in the ventral hindbrain and spinal cord. Lhx3 mRNA persists in the adult pituitary. The expression pattern of Lhx3 is well conserved between Xenopus and mouse, underscoring the functional importance of this gene as a regulator of development. A number of established cell lines of pituitary origin express Lhx3 and therefore constitute a useful tool for further study of Lhx3 gene function (Zhadanov, 1995a).

Early expression of mouse Lhx3 in oral ectoderm that is committed to contribute to the anterior and intermediate lobes of the pituitary and its perseverance in the adult gland strongly suggest an involvement of the gene in mediating and maintaining the differentiation program of this important endocrine system. Additional functions are suggested by the fact that Lhx3 is also expressed bilaterally along the spinal cord and the hindbrain at early stages of mouse development. The gene is composed of six exons and five introns. Two different exons, Ia and Ib, appear to be alternatively spliced to exon II. The first LIM domain is encoded by exon II and the second by exon III. The homeobox is shared by exons IV and V. Lhx3 maps to the proximal region of mouse chromosome 2 in a region that shares homology with human chromosomes 9q and 10p (Zhadanov, 1995b).

Targeted gene disruption in mice showed that Lhx3, a LIM homeobox gene expressed in the pituitary throughout development, is essential for differentiation and proliferation of pituitary cell lineages. In mice homozygous for the Lhx3 mutation, Rathke's pouch forms but fails to grow and differentiate; such mice lacked both the anterior and intermediate lobes of the pituitary. The determination of all pituitary cell lineages, except the corticotrophs, was affected, suggesting that a distinct, Lhx3-independent ontogenetic pathway exists for the initial specification of this lineage (Sheng, 1996).

During differentiation of the embryonic anterior pituitary, distinct hormone cell types are generated in a precise temporal and spatial order from an apparently homogenous ectodermal primordium. The anterior pituitary derives from Rathke's pouch (RP), a specialized region of the oral roof ectoderm. The posterior pituitary derives from the infundibulum (INF) an evagination of the ventral diencephalon. Evidence is provided that in RP, the coordinate control of progenitor cell identity, proliferation and differentiation is imposed by spatial and temporal restrictions in FGF- and BMP-mediated signals. These signals derive from adjacent neural and mesenchymal signaling centers: the infundibulum and ventral juxtapituitary mesenchyme (VJM), respectively. The infundibulum appears to have a dual signaling function, serving initially as a source of BMP4 and subsequently of FGF8. The onset of FGF8 expression in the INF coincides with that of Lhx3 expression in RP. The ability of the INF over the period E10.5 to E12.5 to extinguish Isl1 (in the dorsal aspect of the RP) and promote Lhx3 in the same region corresponds more closely to the temporal expression of FGF8 than of BMP4. FGF8 can mimic the ability of the INF to repress Isl1 and maintain Lhx3 expression in explants. In vitro, FGFs promote the proliferation of progenitor cells, prevent their exit from the cell cycle and contribute to the specification of progenitor cell identity. Late FGF8 signaling controls corticotroph differentiation in the dorsal Lhx3+, Isl1- domain. Maintained FGF8 signaling from the INF expands still further the dorsal corticotroph prrogenitor population; as a consequence, the most ventral of these progenitors become located beyond range of FGF signaling. Since these progenitors are also beyond range of, or by this time refractory to, BMP2/7 signals (derived from the VJM), they progress to an ACTH+ definitive corticotroph state (Ericson, 1998).

The ventral domain of RP serves as the origin of thyrotrophs, defined by expression of the alpha-glycoprotein and thyroid stimulating hormone beta subunits. The continued proliferation of progenitor cells in the dorsal domain of RP (stimulated by FGF8 signals derived from the INF) results in the progressive ventral displacement of thyrotroph progenitors such that they come to be located beyond the range of FGF8 signaling. The ventral juxtapituitary mesenchyme appears to serve as a later source of BMP2 and BMP7. BMPs have no apparent effect on cell proliferation but instead appear to act with FGFs to control the initial selection of thyrotroph and corticotroph progenitor identity. BMPs promote prospective thyrotroph differentiation and suppress corticotroph differentiation. BMPs expressed by the VJM promote Isl1 expression in the ventral domain of RP. Ultimately cells in the ventral domain begin to express TSHbeta and alpha-glycoprotein, having become established as definitive thyrotrophs (Ericson, 1998).

mLIM3, a member of the LIM homeobox family, was recently demonstrated to be critical for proliferation and differentiation of the pituitary cell lineage. Using a pool of degenerate oligonucleotides the DNA sequence ANNAGGAAA(T/C)GA(CIG)AA was determined as the set preferentially recognized by mLIM3. A nearly identical sequence is found in the prolactin (PRL) promoter, within a 15-mer stretch [from nucleotides (nts) -218 to -204] that is highly conserved between human, rat, and bovine. PRL mRNA expression was induced in 3 separate stable transfectants of mLIM3 cDNA in AtT20 tumor cells. Gel retardation experiments performed using nuclear extracts isolated from one of the AtT20/mLIM3 stable transfectants revealed affinity of mLIM3 toward the 15-mer element of the PRL promoter. From these results, it is proposed that the PRL promoter element (nts -218 to -204) is functional in vivo. In AtT20 cells prolactin mRNA expression is not induced by the Pit-1/GHF-1 pathway and growth hormone mRNA is not detected concomitantly with prolactin. It is concluded that mLIM3 may play a key role in inducing PRL gene expression in lactotrophs by binding to a conserved motif close to a Pit-1/GHF-1 site within the proximal PRL promoter (Girardin, 1998).

The Ptx1 (pituitary homeobox 1) homeobox transcription factor is a transcription factor of the pituitary POMC gene. In corticotrope cells that express POMC, cell-specific transcription is conferred in part by the synergistic action of Ptx1 with the basic helix-loop-helix factor NeuroD1. Since Ptx1 expression precedes pituitary development and differentiation, its expression and function was examined in other pituitary lineages. Ptx1 is expressed in most pituitary-derived cell lines as is the related Ptx2 (Rieger) gene. However, Ptx1 appears to be the only Ptx protein in corticotropes and the predominant one in gonadotrope cells. Most pituitary hormone-coding gene promoters are activated by Ptx1. Thus, Ptx1 appears to be a general regulator of pituitary-specific transcription. In addition, Ptx1 action is synergized by cell-restricted transcription factors to confer promoter-specific expression. Indeed, in the somatolactotrope lineage, synergism between Ptx1 and Pit1 is observed on the PRL promoter, and strong synergism between Ptx1 and SF-1 is observed in gonadotrope cells on the betaLH promoter but not on the alphaGSU (glycoprotein hormone alpha-subunit gene) and betaFSH promoters. Synergism between these two classes of factors is reminiscent of the interaction between the products of the Drosophila genes ftz (fushi tarazu) and ftz-F1. Antisense RNA experiments performed in alphaT3-1 cells that express the alphaGSU gene show that expression of endogenous alphaGSU is highly dependent on Ptx1, whereas many other genes are not affected. Interestingly, the only other gene found to be highly dependent on Ptx1 for expression is the gene for the Lim3/Lhx3 transcription factor. Thus, these experiments place Ptx1 upstream of Lim3/Lhx3 in a cascade of regulators that appear to work in a combinatorial code to direct pituitary-, lineage-, and promoter-specific transcription (Tremblay, 1998).

The mammalian hippocampus contains the neural circuitry that is crucial for cognitive functions such as learning and memory. The development of such circuitry is dependent on the generation and correct placement of the appropriate number and types of neurons. Mice lacking function of the LIM homeobox gene Lhx5 show a defect in hippocampus development. Hippocampal neural precursor cells are specified and proliferate, but many of them fail to either exit the cell cycle or to differentiate and migrate properly. Histological analysis of the mutant embryos revealsthat the hippocampus is misformed, the choroid plexus of both the lateral ventricle and the third ventricle is missing, and the anterior callosal axons fail to cross the midline. Although these defects may not cause the lethality, the cyanotic appearance of dying animals suggests the existence of additional defects in respiratory control centers. However, morphological defects either in the hindbrain or in other discrete regions of the forebrain (including the neocortex, basal ganglia, thalamus, and hypothalamus), midbrain, and spinal cord (where Lhx5 is normally expressed) could not be detected. It is possible that functional compensation for Lhx5 is rendered, at least in part, by Lhx1, a closely related LIM homeobox gene that is coexpressed with Lhx5 in these regions. At E18.5, the Lhx5 homozygous mutant embryos show histological defects in the hippocampal regions. The ventricular zone is thicker than normal. Numerous cells are clustered in a region ventral to the lateral ventricle, but these cells fail to form the morphologically distinctive structures of Ammon's horn and the dentate gyrus. The fimbria and the hippocampal commissure, two major axon tracts of the hippocampal formation, are entirely missing from the mutant embryos. Consistent with the thickening of the hippocampal ventricular zone, BrdU pulse labeling at E18.5 shows that the number of proliferating cells in the mutant hippocampal ventricular zone is increased as compared to that in the wild-type control. By E18.5, many BrdU-labeled postmitotic cells are observed in the hippocampal region of mutant embryos. These cells migrate out of the ventricular zone, but they fail to position themselves properly to form the distinctive structures of Ammon's horn and the dentate gyrus that are observed in wild-type embryos. Lhx5 is therefore essential for the regulation of precursor cell proliferation and the control of neuronal differentiation and migration during hippocampal development (Zhao, 1999).

LIM-homeobox containing (Lhx) genes encode transcriptional regulators that play critical roles in a variety of developmental processes. Two identifed genes belong to a novel subfamily of mammalian Lhx genes, designated Lhx6 and Lhx7. The overall similarity between Lhx6 and Lhx7 is 75%, with the homeodomain and the LIM domains being the most conserved regions (95% and 74% identity, respectively. Sequence comparison of the Lhx6 and Lhx7 homeodomains to those of other LIM homeodomain proteins shows that they are more similar to each other than to other members of this family, indicating that they constitute a novel subfamily of the Lhx protein family. Whole-mount in situ hybridization shows that Lhx6 and Lhx7 are expressed during mouse embryogenesis in overlapping domains of the first branchial arch and the basal forebrain. More specifically, expression of Lhx6 and Lhx7 is detected prior to initiation of tooth formation in the presumptive oral and odontogenic mesenchyme of the maxillary and mandibular processes. During tooth formation, expression is restricted to the mesenchyme of individual teeth. Using explant cultures, expression of Lhx6 and Lhx7 in mandibular mesenchyme is under the control of signals derived from the overlying epithelium; such signals are absent from the epithelium of the non-odontogenic second branchial arch. Expression studies and bead implantation experiments in vitro provide strong evidence that Fgf8 is primarily responsible for the restricted expression of Lhx6 and Lhx7 in the oral aspect of the maxillary and mandibular processes. In the telencephalon, expression of both genes is predominantly localized in the developing medial ganglionic eminences, flanking a Fgf8-positive midline region. It is suggested that Fgf8, Lhx6 and Lhx7 are all key components of signaling cascades that determine morphogenesis and differentiation in the first branchial arch and the basal forebrain (Grigoriou, 1998).

A novel LIM-homeodomain gene, Lhx9, was isolated by degenerate RT-PCR followed by mouse embryonic library screening. Lhx9 cDNA encodes a protein that is most closely related to Drosophila Apterous and rodent Lhx2 proteins. The Lhx9 spatiotemporal pattern of expression during embryogenesis is similar but distinct from Lhx2. Highest expression levels are found in the diencephalon, telencephalic vesicles, and dorsal mesencephalon. Domains of expression respect the proposed neuromeric boundaries. Lhx9 is also expressed in the spinal cord, forelimb and hindlimb mesenchyme, and urogenital system. Although Lhx9 expression is sustained in diencephalon and mesencephalon from embryonic day 10.5 (E10.5) to postnatal stages, it is transient in the future cerebral cortex, where it is turned off between E14.5 and E16.5. Lhx9 expression is highest if not exclusively located (depending on the region of interest) in the intermediate and mantle zones, as opposed to the mitotic ventricular zone. Lhx9 protein was tested for interaction with the recently discovered cofactors of LIM-homeodomain proteins and was found to interact strongly both with CLIM1 and CLIM2. The expression pattern and structural characteristics of Lhx9 suggest that it encodes a transcription factor that might be involved in the control of cell differentiation of several neural cell types. Furthermore, Lhx9 protein might act in a combinatorial manner with other LIM-homeodomain factors expressed in overlapping patterns (Retaux, 1999).

In order to explain the phenotype observed in Lhx2 mutant embryos, it has been proposed that an Lhx2 related gene might exist. A new LIM/homeobox gene called Lhx9 has been cloned. Lhx9 is closely related to Lhx2 and is expressed in the developing central nervous system (CNS). Lhx9 and Lhx2 have expression patterns that overlap in some areas but are distinct in others. Thus, in some developmental domains these two highly related proteins may be functionally redundant. Lhx9 is expressed in the pioneer neurons of the cerebral cortex, while Lhx2 is expressed throughout the cortical layers. Postnatally, Lhx9 is expressed in the inner nuclei of the cerebellum, at the same time that Lhx2 is expressed in the granular layer. In the developing limbs, both genes are highly expressed in a pattern that is similar to one another. Based on the expression pattern and the developmental regulation of Lhx9, it is proposed that Lhx9 may be involved in the specification or function of the pioneer neurons of the cerebral cortex. Both Lhx9 and Lhx2 can bind the LIM domain binding protein Ldb1/Nli1/Clim2 (Bertuzzi, 1999).

The vertebrate cranial vault, or calvaria, forms during embryonic development from cranial mesenchyme, which has multiple embryonic origins. Inductive interactions are thought to specify the number and location of the calvarial bones, including interactions between the neuroepithelium and cranial mesenchyme. An important feature of calvarial development is the local inhibition of osteogenic potential that occurs between specific bones and results in the formation of the cranial sutures. These sutures allow for postnatal growth of the skull to accommodate postnatal increase in brain size. The molecular genetic mechanisms responsible for the patterning of individual calvarial bones and for the specification of the number and location of sutures are poorly understood at the molecular genetic level. During calvarial development, the LIM-homeodomain gene lmx1b is expressed in the neuroepithelium, in underlying portions of the developing skull and in cranial mesenchym which contributes to portions of the cranial vault. Lmx1b is essential for proper patterning and morphogenesis of the calvaria since the supraoccipital and interparietal bones of lmx1b mutant mice are either missing or severely reduced. Moreover, lmx1b mutant mice have severely abnormal sutures between the frontal, parietal, and interparietal bones. These results indicate that lmx1b is required for multiple events in calvarial development and suggest possible genetic interaction with other genes known to regulate skull development and suture formation (Chen, 1998).

Cells in the caudal mesencephalon and rostral metencephalon become organized by signals emanating from the isthmus organizer (IsO). The IsO is associated with the isthmus, a morphological constriction of the neural tube that eventually defines the mesencephalic/ metencephalic boundary (MMB). The transcription factor Lmx1b is expressed and functions in a distinct region of the IsO. Lmx1b expression is maintained by the glycoprotein Fgf8, a signal capable of mediating IsO signaling. Lmx1b, in turn, maintains the expression of the secreted factor Wnt1. These conclusions are substantiated by the following: (1) Lmx1b mRNA becomes localized to the isthmus immediately after Fgf8 initiation, (2) Wnt1 expression is localized to the Lmx1b expression domain, but with slightly later kinetics, (3) Fgf8-soaked beads generate similar domains of expression for Lmx1b and Wnt1 and (4) retroviral-mediated expression of Lmx1b (Lmx1b/RCAS) maintains Wnt1 expression in the mesencephalon. Ectopic Lmx1b is insufficient to alter the expression of a number of other genes expressed at the IsO, suggesting that it does not generate a new signaling center. Instead, if Lmx1b/RCAS-infected brains are allowed to develop longer, changes in mesencephalic morphology are detected. Since both ectopic and endogenous Lmx1b expression occurs in regions of the isthmus undergoing morphological changes, it could normally play a role in this process. Furthermore, a similar phenotype is not observed in Wnt1/RCAS-infected brains, demonstrating that ectopic Wnt1 is insufficient to mediate the effect of ectopic Lmx1b in this assay. Since Wnt1 function has been linked to the proper segregation of mesencephalic and metencephalic cells, it is suggested that Lmx1b and Wnt1 normally function in concert to affect IsO morphogenesis (Adams, 2000).

Lim homeodomain proteins and axon guidance

Motor neurons extend axons along specific trajectories, but the molecules that control their pathfinding remain poorly defined. Two LIM homeodomain transcription factors, Lim1 and Lmx1b, control the initial trajectory of motor axons in the developing mammalian limb. The expression of Lim1 by a lateral set of lateral motor column (LMC) neurons ensures that their axons select a dorsal trajectory in the limb. In a complementary manner, the expression of Lmx1b by dorsal limb mesenchymal cells controls the dorsal and ventral axonal trajectories of medial and lateral LMC neurons. In the absence of these two proteins, motor axons appear to select dorsal and ventral trajectories at random. Thus, LIM homeodomain proteins act within motor neurons and cells that guide motor axons to establish the fidelity of a binary choice in axonal trajectory (Kania, 2000).

The possibility that LIM HD proteins control the trajectory of vertebrate motor axons emerged initially from an analysis of their patterns of expression within columnar subclasses of motor neurons. Direct evidence in support of this idea has been difficult to obtain, however, because of the earlier activities of Isl1 and Lim1 in the control of embryonic pattern and neuronal fate. In mice lacking Lhx3 and Lhx4, motor neurons are generated and their axons exit the spinal cord at an abnormal dorsal position. This projection defect, however, is likely to be a secondary consequence of a prior perturbation in the specification of motor neuron identity and migratory pattern (Kania, 2000 and references therein).

Lim1 appears to control the trajectory of LMC(l) axons (lateral motor column, lateral division axons) without a prior influence on the specification of LMC neuron subtype identity, or on the pattern of motor neuron migration in the spinal cord. A parallel to the function of Lim1 in vertebrate motor neurons can be found in the activities of the Drosophila LIM homeobox genes Islet and Lim3, which control motor axon pathfinding without affecting neuronal identity. Studies of neural development in C. elegans have also revealed roles for LIM homeobox genes in the specification of neuronal connections, but it remains unclear whether these connectivity defects are a consequence of alterations in axonal guidance (Kania, 2000 and references therein).

How do LIM homeobox genes control the trajectory of vertebrate motor axons? Lim1 is required for LMC(l) axons to invariably select a dorsal trajectory upon entry into the limb. Nevertheless, only about half the normal number of Lim1 mutant LMC(l) neurons continue to select a dorsal trajectory in the limb. Thus Lim1 is not required in an absolute sense for dorsally directed axonal growth. The loss of Lim1 function instead appears to randomize the selection of a dorsal or ventral trajectory by an individual LMC(l) axon as it enters the limb. Thus, Lim1 may control the ability of LMC(l) axons to respond to guidance cue(s) present in the limb mesenchyme that direct their dorsal trajectory. In addition, dorsoventral axonal pathway selection by both LMC(l) axons and LMC(m) axons (lateral motor column, medial division axons) appears to be randomized in Lmx1b mutants, indicating that the expression of Lmx1b by dorsal limb mesenchymal cells controls the ability of LMC axons to establish distinct dorsal and ventral trajectories. Presumably, Lmx1b establishes a molecular distinction in the expression of guidance cues by dorsal and ventral mesenchymal cells that is perceived by motor axons as they enter the limb. Since the axons of LMC(l) and LMC(m) neurons segregate immediately on entry into the limb, the guidance cues controlled by Lmx1b are likely to act at short range (Kania, 2000).

Few cell surface or secreted proteins with differential patterns of expression in dorsal and ventral limb mesenchymal cells have been identified, and none of these have yet been linked directly to motor axon guidance. Thus, the distinct trajectories of LMC(m) and LMC(l) axons could be controlled by the differential dorsoventral expression of attractant or repellant factors. It also remains unclear whether Lmx1b acts to activate or repress the expression of guidance cues. Nevertheless, these results do appear to rule out the possibility that the dorsally restricted trajectory of LMC(l) axons is achieved through ventral expression of an activity that prevents the extension of these axons in an absolute, and context-independent manner (Kania, 2000).

The Eph tyrosine kinase receptors EphA4 and EphA7 are expressed in a proximal domain of the dorsal limb mesenchyme, close to the position at which the axons of LMC neurons segregate into dorsal and ventral nerve branches. Eph signals contribute to the guidance of axons in other neural systems, raising the possibility that they also participate in the regulation of motor axon growth into the limb (Kania, 2000 and references therein).

The axons of distinct classes of motor neurons project to their targets in a stepwise manner, diverging in trajectory at intermediate 'decision regions'. These studies provide evidence that LIM HD proteins coordinate one key pathfinding decision: the dorsoventral choice of motor axon trajectory at the base of the limb. The selectivity of the defects in axonal pathfinding observed in Lim1 and Lmx1b mutants also provides genetic evidence that discrete molecular programs control distinct steps in motor axon guidance. The loss of Lim1 from LMC(l) neurons is without effect on motor axon exit from the spinal cord, or on the extension of motor axons through a mesodermal environment en route to the limb. Moreover, the bifurcation of the motor nerve into dorsal and ventral branches occurs at its normal position in both Lim1 and Lmx1b mutants. The decision to establish dorsal and ventral motor nerve branches is therefore separable from the decision of individual axons to select a dorsal or ventral trajectory. These results also show that the apposition of limb mesenchymal cells of dorsal and ventral identity is not required for the bifurcation of the motor nerve into dorsal and ventral branches. An independent mechanism, perhaps linked to the onset of chondrogenesis at the core of the limb bud, may impose this aspect of motor nerve branching. The decision of motor axons to project distally in the limb is also separable from the selection of dorsal or ventral trajectory, since in Lmx1b mutants both LMC(l) and LMC(m) axons project distally despite dorsoventral projection errors. Nevertheless, Lim1 controls both the selection of dorsoventral trajectory and the projection of LMC(l) axons into the distal limb (Kania, 2000).

Discrete steps in motor axon pathfinding are also controlled by distinct environmental signals. The initial phase of axon extension by distinct sets of motor axons appears to be controlled differentially by netrins, semaphorins, and hepatocyte growth factor. In addition, Eph signaling has been implicated in selective motor axon growth through the anterior half of the somite and in the topographic pattern of muscle innervation by motor axons (Kania, 2000 and references therein).

The role of LIM HD proteins in subdividing LMC neurons and the limb mesenchyme raises the issue of whether the differentiation of these two cell types might be coordinated during development. The generic identity of LMC neurons appears to be imposed by a signal provided by limb level paraxial mesoderm. In parallel, a limb level paraxial mesoderm signal has been suggested to specify the position of formation of the limb field, a process mediated by the downstream activation of an FGF signaling pathway. These observations suggest that positional signals provided by limb level paraxial mesoderm act on neural cells to initiate the specification of LMC neurons and on lateral plate mesoderm cells to initiate the formation of the limb field (Kania, 2000 and references therein).

Might there also be coordination in the specification of the mediolateral subdivision of the LMC and the dorsoventral subdivision of the limb mesenchyme? The establishment of a generic LMC neuronal identity is marked by the expression of the retinoid synthetic enzyme RALDH2. The RALDH2-dependent synthesis of retinoids by early born LMC neurons has been implicated in the induction of LMC(l) neuronal identity. Within the limb, the subdivision of the mesenchyme into dorsal and ventral domains appears to be controlled, at least in part, by Wnt-mediated signals derived from the dorsal limb ectoderm. It may be worth considering whether retinoids have a role in the specification of the signaling properties of the dorsal limb ectoderm, and thus in the emergence of the dorsal subdivision of the limb mesenchyme (Kania, 2000 and references therein).

Further analysis of the molecular basis of motor axon guidance in the limb may help to define two interrelated issues in the patterning of neuronal projections. First, what is the identity of the effector molecules that guide axons at critical binary choice points along their trajectories? Second, how is the specification of neuronal responsivity to guidance cues matched with the regionally restricted expression of such cues (Kania, 2000)?

Combinatorial expression of transcription factors forms transcriptional codes to confer neuronal identities and connectivity. However, how these intrinsic factors orchestrate the spatiotemporal expression of guidance molecules to dictate the responsiveness of axons to guidance cues is less understood. Thalamocortical axons (TCAs) represent the major input to the neocortex and modulate cognitive functions, consciousness and alertness. TCAs travel a long distance and make multiple target choices en route to the cortex. The homeodomain transcription factor Gbx2 is essential for TCA development, as loss of Gbx2 abolishes TCAs in mice. Using a novel TCA-specific reporter, this study has discovered that thalamic axons are mostly misrouted to the ventral midbrain and dorsal midline of the diencephalon in Gbx2-deficient mice. Furthermore, conditionally deleting Gbx2 at different embryonic stages has revealed a sustained role of Gbx2 in regulating TCA navigation and targeting. Using explant culture and mosaic analyses, it was demonstrated that Gbx2 controls the intrinsic responsiveness of TCAs to guidance cues. The guidance defects of Gbx2-deficient TCAs are associated with abnormal expression of guidance receptors Robo1 and Robo2. Finally, Gbx2 was demonstrated to control Robo expression by regulating LIM-domain transcription factors through three different mechanisms: Gbx2 and Lhx2 compete for binding to the Lmo3 promoter and exert opposing effects on its transcription; repressing Lmo3 by Gbx2 is essential for Lhx2 activity to induce Robo2; and Gbx2 represses Lhx9 transcription, which in turn induces Robo1. These findings illustrate the transcriptional control of differential expression of Robo1 and Robo2, which may play an important role in establishing the topography of TCAs (Chatterjee, 2012).

Lhx6 delineates a pathway mediating innate reproductive behaviors from the amygdala to the hypothalamus

In mammals, innate reproductive and defensive behaviors are mediated by anatomically segregated connections between the amygdala and hypothalamus. This anatomic segregation poses the problem of how the brain integrates activity in these circuits when faced with conflicting stimuli eliciting such mutually exclusive behaviors. Using genetically encoded and conventional axonal tracers, it was found, in studies with mice, that the transcription factor Lhx6 delineates the reproductive branch of this pathway. Other Lhx proteins mark neurons in amygdalar nuclei implicated in defense. Parallel projections have been traced from the posterior medial amygdala, activated by reproductive or defensive olfactory stimuli, respectively, to a point of convergence in the ventromedial hypothalamus. The opposite neurotransmitter phenotypes of these convergent projections suggest a 'gate control' mechanism for the inhibition of reproductive behaviors by presentation of a threatening stimulus (in the form of a collar worn by a domestic cat). These data therefore identify a potential neural substrate for integrating the influences of conflicting behavioral cues and a transcription factor family that may contribute to the development of this substrate (Choi, 2005).

Virtually all metazoan organisms exhibit innate reproductive and defensive behaviors that are triggered by signals sensed from conspecifics or predators. Such behaviors are crucial for the survival of each species. The stereotypical nature of these behaviors suggests that their underlying neural circuits are likely to be genetically 'hard-wired.' At the same time, animals are frequently faced with conflicting cues in their natural environment and therefore must make rapid decisions to engage in defensive versus reproductive, or other appetitive, behaviors. The neural mechanisms are poorly understood that determine which of these hard-wired behaviors will predominate when conflicting stimuli are present (Choi, 2005).

The basic neural pathways that mediate reproductive (such as mating or maternal) and defensive (such as aggressive or predator-avoidance) behaviors in rodents have been intensively studied. Olfactory stimuli play an important role in the release of such behaviors. These stimuli activate primary sensory neurons in the main olfactory epithelium or vomeronasal organ, which project to the main or accessory olfactory bulbs (AOB), respectively. While recent genetic evidence increasingly suggests an important role for the main olfactory system in processing reproductive stimuli (provided by female urine for example), a great deal of attention has been focused on a parallel pathway involving the accessory olfactory system, which responds to pheromonal cues. Projection neurons in the AOB develop synapses in the medial amygdalar nucleus (MEA), which, in turn, projects to a series of nuclei in the medial hypothalamus. The MEA also projects indirectly to the hypothalamus through the bed nucleus of the stria terminalis (BST) (Choi, 2005 and references therein).

The posterior portion of the MEA is subdivided into dorsal and ventral subnuclei (MEApd and MEApv). The projections from these two subnuclei to the medial hypothalamus exhibit a striking anatomic segregation, which is thought to reflect their involvement in either reproduction or defense. The dorsal portion (MEApd) is activated by reproductive stimuli and projects to three interconnected hypothalamic nuclei implicated in reproductive behaviors: the medial preoptic nucleus (MPN), ventrolateral part of the ventromedial hypothalamic nucleus (VMHvl), and the ventral premammillary nucleus (PMv). The ventral portion (MEApv) is activated by defensive stimuli such as predator odors and projects to the anterior hypothalamic nucleus (AHN) and the dorsomedial part of the VMH (VMHdm); these regions are involved in defensive behaviors. This striking anatomical and functional segregation suggests that these neural pathways for reproduction and defense are likely genetically determined, but genes that might control their wiring have not yet been identified (Choi, 2005 and references therein).

Such parallel circuit organization brings into question how rapid decisions between competing reproductive and defensive behaviors are made by organisms faced with conflicting cues. Such decision-making would seem to require crosstalk between these subcircuits, but there are very few interconnections between the reproductive and the defensive hypothalamic nuclei. The MEApv projects to the reproductive as well as the defensive hypothalamic nuclei, but the function of this divergent projection is not known. Some evidence suggests that suppression of reproductive behaviors by threatening stimuli may be exerted within the amygdalar-hypothalamic pathway. For example, exposure of virgin female rats to newborn pups promotes defensive behaviors and inhibits maternal behavior. This inhibition can be overcome by lesions of the medial amygdala and involves projections from this structure to VMH. The circuit-level mechanisms that mediate such behavioral inhibition are not understood (Choi, 2005).

The identification of genes that mark amygdalar-hypothalamic circuits might shed further light on both their functional organization and developmental specification. Different LIM homeodomain transcription factors mark neurons in different subnuclei of the medial amygdala. Both genetic and classical neuroanatomical tracing techniques, in conjunction with markers of neuronal activation and neurotransmitter phenotype, were used to determine the relationship of these molecularly identified neurons to the functions and connectivity attributed to the nuclei in which they reside. The results indicate that Lhx6 delineates a reproductive pathway that involves neurons in both the MEApd and the BSTpr and their projections to the three reproductive nuclei in the hypothalamic medial behavioral control column (MPN, VMHvl, and PMv). Further analysis reveals counter-intuitively that VMHvl receives inhibitory projections from this reproductive pathway and a convergent excitatory projection from neurons in the MEApv that are activated by a predator odor. It is suggested that this point of convergence may serve to 'gate' the expression of reproductive behavior under conditions in which animals are exposed to threatening stimuli. Thus, the data identify a potential neural substrate within the hypothalamus for controlling behavioral decisions in the face of conflicting cues and a transcription factor family that may contribute to the development of this substrate (Choi, 2005).

Table of contents

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

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