LIM Homeobox Gene Research Articles

Accelerated Loss of Germ Cells in LIM-homeobox Gene, Lhx8, Likely Occurs Due to Misexpression of Kit, Kitl, and a Multitude of Germ Cell-Specific Genes.

Accelerated Loss of Germ Cells in LIM-homeobox Gene, Lhx8, Likely Occurs Due to Misexpression of Kit, Kitl, and a Multitude of Germ Cell-Specific Genes.

Lhx2 Expression Promotes Self-Renewal of a Distinct Multipotential Hematopoietic Progenitor Cell in Embryonic Stem Cell-Derived Embryoid Bodies

The molecular mechanisms regulating the expansion of the hematopoietic system including hematopoietic stem cells (HSCs) in the fetal liver during embryonic development are largely unknown. The LIM-homeobox gene Lhx2 is a candidate regulator of fetal hematopoiesis since it is expressed in the fetal liver and Lhx2−/− mice die in utero due to severe anemia. Moreover, expression of Lhx2 in embryonic stem (ES) cell-derived embryoid bodies (EBs) can lead to the generation of HSC-like cell lines. To further define the role of this transcription factor in hematopoietic regulation, we generated ES cell lines that enabled tet-inducible expression of Lhx2. Using this approach we observed that Lhx2 expression synergises with specific signalling pathways, resulting in increased frequency of colony forming cells in developing EB cells. The increase in growth factor-responsive progenitor cells directly correlates to the efficiency in generating HSC-like cell lines, suggesting that Lhx2 expression induce self-renewal of a distinct multipotential hematopoietic progenitor cell in EBs. Signalling via the c-kit tyrosine kinase receptor and the gp130 signal transducer by IL-6 is necessary and sufficient for the Lhx2 induced self-renewal. While inducing self-renewal of multipotential progenitor cells, expression of Lhx2 inhibited proliferation of primitive erythroid precursor cells and interfered with early ES cell commitment, indicating striking lineage specificity of this effect.

The LIM-Homeobox GeneIslet-1Is Required for the Development of Restricted Forebrain Cholinergic Neurons

Forebrain cholinergic neurons modulate complex mammalian behaviors such as reward-related learning and cognitive functions. Although their dysfunction is implicated in various psychiatric and neurodegenerative diseases, the factors governing cholinergic neuron differentiation and diversity are mostly unknown. We tested the role of the LIM-homeobox gene Isl1 in the development of forebrain cholinergic neurons by conditionally deleting Isl1 using a Six3-cre transgene. A depletion of cholinergic interneurons in the dorsal and ventral striatum, and cholinergic projection neurons in the nucleus basalis is observed and is ascribed to an early and persistent defect in cholinergic neuron differentiation. Notably, cholinergic innervation to the neocortex is abolished, whereas that to the hippocampus is unaltered. The unique pattern of cholinergic hypoinnervation encountered is supported by the presence of cholinergic projection neurons in the medial septum, the magnocellular preoptic area, and the substantia innominata. Together, these results demonstrate the requirement for Isl1 in the development of restricted telencephalic cholinergic neurons and link the development of cholinergic neurons in anatomically disparate sites to Isl1 function.

NKX2.1 specifies cortical interneuron fate by activatingLhx6

In the ventral telencephalon, the medial ganglionic eminence (MGE) is a major source of cortical interneurons. Expression of the transcription factor NKX2.1 in the MGE is required for the specification of two major subgroups of cortical interneurons - those that express parvalbumin (PV) or somatostatin (SST) - but direct targets of NKX2.1 remain to be established. We find that electroporation of Nkx2.1 cDNA into the ventral telencephalon of slice cultures from Nkx2.1-/- mouse embryos, followed by transplantation into neonatal cortex to permit postnatal analysis of their fate, rescues the loss of PV- and SST-expressing cells. The LIM-homeobox gene Lhx6 is induced by this rescue experiment, and gain- and loss-of-function studies suggest that Lhx6 is necessary and sufficient to rescue these and other interneuron phenotypes in cells transplanted from Nkx2.1-/- slices. Finally, NKX2.1 protein binds a highly conserved sequence in the Lhx6 promoter, and this sequence appears to mediate the direct activation of Lhx6 by NKX2.1. The slice transfection and transplantation methods employed here are beginning to uncover embryonic mechanisms for specifying neuronal fates that only become definable postnatally.

Islet-1: A Potentially Important Role for an Islet Cell Gene in Visceral Fat

To examine differences in gene expression between visceral (VF) and subcutaneous fat (SF) to identity genes of potential importance in regulation of VF. We compared gene expression (by DNA array and quantitative PCR (qPCR)) in paired VF and SF adipose biopsies from 36 subjects (age 54 +/- 15 years, 15 men/21 women) with varying degrees of adiposity and insulin resistance, in chow and fat fed mice (+/- rosiglitazone treatment) and in c-Cbl(-/-) mice. Gene expression was also examined in 3T3-L1 preadipocytes during differentiation. A twofold difference or more was found between VF and SF in 1,343 probe sets, especially for genes related to development, cell differentiation, signal transduction, and receptor activity. Islet-1 (ISL1), a LIM-homeobox gene with important developmental and regulatory function in islet, neural, and cardiac tissue, not previously recognized in adipose tissue was virtually absent in SF but substantially expressed in VF. ISL1 expression correlated negatively with BMI (r = -0.37, P = 0.03), abdominal fat (by dual energy X-ray absorptiometry, r = -0.44, P = 0.02), and positively with circulating adiponectin (r = 0.33, P = 0.04). In diet-induced obese mice, expression was reduced in the presence or absence of rosiglitazone. Correspondingly, expression was increased in the c-Cbl(-/-) mouse, which is lean and insulin sensitive (IS). ISL1 expression was increased sevenfold in 3T3-L1 preadipocytes during early (day 1) differentiation and was reduced by day 2 differentiation. An important developmental and regulatory gene ISL1 is uniquely expressed in VF, probably in the preadipocyte. Our data suggest that ISL1 may be regulated by adiposity and its role in metabolic regulation merits further study.

Expression dynamics of the LIM-homeobox genes, Lhx1 and Lhx9, in the diencephalon during chick development

The diencephalon is the caudal part of the developing forebrain, which corresponds to prosomeres 1 to 3. The mature diencephalon is functionally and anatomically divided into well-defined nuclei. Previous researches have shown that LIM-homeobox genes are important transcription factors during diencephalon regionalization in mice. Here we examined expression patterns of several chick orthologs of LIM-homeobox genes. Lhx1 and Lhx9 were expressed in the diencephalon from early stages and their expression in the diencephalon became restricted to prosomeres 1 and 2 in distinct fashions. Then we also studied the regulatory effects of possible upstream signals by in ovo electroporation. Lhx1 was found to be up-regulated by Shh signaling. Whereas Lhx9 was up-regulated by Wnt3a and Fgf15, it was down-regulated by Shh. Our data suggest that the LIM-homeobox genes, Lhx1 and Lhx9, regulated by ventral and/or dorsal signals, may play important roles in controlling regionalization of the diencephalon during chick development.

Knockdown of the L3/Lhx8 gene suppresses cholinergic differentiation of murine embryonic stem cell-derived spheres

L3/Lhx8, a member of the Lim-homeobox gene family, is selectively and specifically expressed in the murine embryonic medial ganglionic eminence (MGE). Our previous study demonstrated that L3/Lhx8-deficient mice specifically lack cholinergic neurons in the basal forebrain. In this manuscript, we report the in vitro effects of reduced L3/Lhx8 gene expression on cholinergic differentiation in murine embryonic stem (ES) cell-derived spheres without dissociation. The knockdown of L3/Lhx8 gene expression dramatically decreased the cholinergic phenotype of spheres without altering other known phenotypes (TuJ1, GABA and GFAP). These results strongly suggest that L3/Lhx8 is a key factor for cholinergic differentiation of murine ES cell-derived spheres and is involved in basal forebrain development.

Islet-1 Controls the Differentiation of Retinal Bipolar and Cholinergic Amacrine Cells

Whereas the mammalian retina possesses a repertoire of factors known to establish general retinal cell types, these factors alone cannot explain the vast diversity of neuronal subtypes. In other CNS regions, the differentiation of diverse neuronal pools is governed by coordinately acting LIM-homeodomain proteins including the Islet-class factor Islet-1 (Isl1). We report that deletion of Isl1 profoundly disrupts retinal function as assessed by electroretinograms and vision as assessed by optomotor behavior. These deficits are coupled with marked reductions in mature ON- and OFF-bipolar (>76%), cholinergic amacrine (93%), and ganglion (71%) cells. Mosaic deletion of Isl1 permitted a chimeric analysis of "wild-type" cells in a predominantly Isl1-null environment, demonstrating a cell-autonomous role for Isl1 in rod bipolar and cholinergic amacrine development. Furthermore, the effects on bipolar cell development appear to be dissociable from the preceding retinal ganglion cell loss, because Pou4f2-null mice are devoid of similar defects in bipolar cell marker expression. Expression of the ON- and OFF-bipolar cell differentiation factors Bhlhb4 and Vsx1, respectively, requires the presence of Isl1, whereas the early bipolar cell marker Prox1 initially did not. Thus, Isl1 is required for engaging bipolar differentiation pathways but not for general bipolar cell specification. Spatiotemporal expression analysis of additional LIM-homeobox genes identifies a LIM-homeobox gene network during bipolar cell development that includes Lhx3 and Lhx4. We conclude that Isl1 has an indispensable role in retinal neuron differentiation within restricted cell populations and this function may reflect a broader role for other LIM-homeobox genes in retinal development, and perhaps in establishing neuronal subtypes.

LIM-homeodomain proteins Lhx1 and Lhx5, and their cofactor Ldb1, control Purkinje cell differentiation in the developing cerebellum

Purkinje cells are one of the major types of neurons that form the neural circuitry in the cerebellum essential for fine control of movement and posture. During development, Purkinje cells also are critically involved in the regulation of proliferation of progenitors of granule cells, the other major type of neurons in the cerebellum. The process that controls differentiation of Purkinje cells from their early precursors is poorly understood. Here we report that two closely related LIM-homeobox genes, Lhx1 and Lhx5, were expressed in the developing Purkinje cells soon after they exited the cell cycle and migrated out of the cerebellar ventricular zone. Double-mutant mice lacking function of both Lhx1 and Lhx5 showed a severe reduction in the number of Purkinje cells. In addition, targeted inactivation of Ldb1, which encodes a cofactor for all LIM-homeodomain proteins, resulted in a similar phenotype. Our studies thus provide evidence that these transcription regulators are essential for controlling Purkinje cell differentiation in the developing mammalian cerebellum.

Analysis of LHX8 mutation in premature ovarian failure

The LHX8 (LIM homeobox 8) gene encodes a LIM homeodomain transcriptional regulator that is preferentially expressed in germ cells and critical for mammalian oogenesis. The authors investigated whether nucleotide changes were present in the LHX8 gene of Caucasian women with premature ovarian failure (POF), as compared with control women. When the authors sequenced 95 Caucasian women with POF, they discovered two novel single-nucleotide polymorphisms (SNPs) in intron 3 (c.769+10G>T) and 3' untranslated region (c.1787A>G) of the LHX8 gene. These polymorphisms also were found in controls (n = 94), at frequencies that were not statistically different from those in POF women. Mutations in the LHX8 exons are uncommon in Caucasian women with POF.

Local and cis Effects of the H Element on Expression of Odorant Receptor Genes in Mouse

From the approximately 1,200 odorant receptor (OR) genes in the mouse genome, an olfactory sensory neuron is thought to express only one gene. The mechanisms of OR gene choice are not understood. A 2.1 kilobase region (the H element) adjacent to a cluster of seven OR genes has been proposed as a trans- and pan-enhancer for OR gene expression. Here, we deleted the H element by gene targeting in mice. The deletion abolishes expression of a family of three OR genes proximal to H, and H operates in cis on these genes. Deletion of H has a graded effect on expression of a distal group of four OR genes, commensurate with genomic distance. There is no demonstrable effect on expression of OR genes located outside the cluster. Our findings are not consistent with the hypothesis of H as an essential trans-acting enhancer for genome-wide regulation of OR gene expression.

Ldb1, in conjunction with transcriptional regulators of the LIM-homeobox gene family, orchestrates limb patterning and outgrowth during mouse embryonic development

Ldb1, in conjunction with transcriptional regulators of the LIM-homeobox gene family, orchestrates limb patterning and outgrowth during mouse embryonic development

L3/Lhx8 is a pivotal factor for cholinergic differentiation of murine embryonic stem cells

L3/Lhx8 is a member of the LIM-homeobox gene family. Previously, we demonstrated that L3/Lhx8-null mice specifically lacked cholinergic neurons in the basal forebrain. In the present study, we conditionally suppressed L3/Lhx8 function during retinoic acid-induced neural differentiation of a murine embryonic stem (ES) cell line using an L3/Lhx8-targeted small interfering RNA (siRNA) produced by an H1.2 promoter-driven vector. Our culture conditions induced efficient differentiation of the ES cells into neurons and astrocytes, but far less efficient differentiation into oligodendrocytes. Suppression of L3/Lhx8 expression by siRNA led to a dramatic decrease in the number of cells positive for the cholinergic marker ChAT, and overexpression of L3/Lhx8 recovered this effect. However, no significant changes were observed in the number of Tuj1+ neurons and GABA+ cells. These results strongly suggest that L3/Lhx8 is a key factor in the cholinergic differentiation of murine ES cells and is involved in basal forebrain development.

Lmx1bis essential forFgf8andWnt1expression in the isthmic organizer during tectum and cerebellum development in mice

Secreted factors FGF8 and WNT1 are essential either for the inductive activity of the isthmus organizer or for the regionalization of the midbrain-hindbrain boundary (MHB). However, transcriptional regulation of these secreted factors during development remains to be elucidated. Here we show that the LIM homeobox gene Lmx1b is expressed in the anterior embryo as early as E7.5 and its expression becomes progressively restricted to the isthmus at E9.0. Analysis of gene expression in the MHB of the mutant embryos showed that many genes were lost by E9.5. In the MHB of Lmx1b-/- embryos, the expression of Fgf8, which normally occurs at the 4-somite stage, was completely absent, whereas Wnt1 was downregulated before the 4-somite stage. Moreover, transcription factors En1 and Pax2 were also downregulated prior to the 4-somite stage, whereas Gbx2 downregulation occurred at the 4-somite stage. By contrast, Otx2 and Pax6 expression was not affected in Lmx1b-/- embryos. The requirement of specific Lmx1b expression in the MHB was further confirmed by Wnt1-Cre-mediated region-specific conditional knockout of Lmx1b. As a result of these molecular defects, the development of the tectum and cerebellum was severely impaired in Lmx1b-/- mice. Taken together, our results indicate that Lmx1b plays an essential role in the development of the tectum and cerebellum by regulating expression of Fgf8, Wnt1 and several isthmus-related transcription factors in the MHB, and is a crucial component of a cross-regulatory network required for the induction activity of the isthmic organizer in the MHB.

Transcriptional regulation of stellate cell activation

The identification of activated hepatic stellate cells and related cell types as key fibrogenic effectors during liver injury has led to intense evaluation of transcriptional events underlying their behavior. While initial studies focused on characterizing interactions between transcription factors and regulatory regions within gene promoters, epigenetic mechanisms have emerged as major determinants of gene activation and repression, in particular histone acetylation and promoter methylation, as well as other complex conditional interactions that underlie global changes in gene expression. Three examples are provided that illustrate how stellate cell activation may be controlled by widely divergent regulatory pathways, including alternative splicing of a growth inhibitory transcription factor (Kruppel-like factor-6), epigenetic regulation of a factor regulating stellate cell survival (nuclear factor kappaB), and regulation of a transcription factor whose expression maintains stellate cell quiescence (LIM homeobox gene 2 [Lhx2]). These complex cascades illustrate how clarifying the finely tuned interdependent layers of transcriptional, translational, post-translational and epigenetic gene regulation in stellate cells is raising new prospects for therapy of hepatic fibrosis.

An unusual Zn-finger/FH2 domain protein controls a left/right asymmetric neuronal fate decision in C. elegans

Gene regulatory networks that control the terminally differentiated state of a cell are, by and large, only superficially understood. In a mutant screen aimed at identifying regulators of gene batteries that define the differentiated state of two left/right asymmetric C. elegans gustatory neurons, ASEL and ASER, we have isolated a mutant, fozi-1, with a novel mixed-fate phenotype, characterized by de-repression of ASEL fate in ASER. fozi-1 codes for a protein that functions in the nucleus of ASER to inhibit the expression of the LIM homeobox gene lim-6, neuropeptide-encoding genes and putative chemoreceptors of the GCY gene family. The FOZI-1 protein displays a highly unusual domain architecture, that combines two functionally essential C2H2 zinc-finger domains, which are probably involved in transcriptional regulation, with a formin homology 2 (FH2) domain, normally found only in cytosolic regulators of the actin cytoskeleton. We demonstrate that the FH2 domain of FOZI-1 has lost its actin polymerization function but maintains its phylogenetically ancient ability to homodimerize. fozi-1 genetically interacts with several transcription factors and micro RNAs in the context of specific regulatory network motifs. These network motifs endow the system with properties that provide insights into how cells adopt their stable terminally differentiated states.

Reduced expression of the LIM-homeobox gene Lhx3 impairs growth and differentiation of Rathke’s pouch and increases cell apoptosis during mouse pituitary development

The formation of the anterior and intermediate lobes of the pituitary gland is a multi-step process regulated by cell–cell interactions involving a number of signaling pathways and by cascades of cell-intrinsic transcription factors. The LIM-homeodoamin protein Lhx3 has previously been shown to play an essential role in the growth of Rathke’s pouch, a primordium of the anterior and intermediate lobes of the pituitary. However, the mechanisms underlying the function and regulation of Lhx3 remain to be elucidated. Here we report that a targeted insertion of a DNA fragment in the 3′-untranslated region of the Lhx3 gene reduces the expression of both Lhx3 mRNA and protein in Rathke’s pouch. Mutant mice homozygous for this Lhx3 allele show severe hypoplasia of the pouch, a defect identical to that observed in Lhx3-null mutants. To gain insights into the mechanism of Lhx3 function in pituitary development, we further analyzed the Lhx3 deficient mutants by examination of early pituitary marker expression, cell proliferation, and cell apoptosis. Our results revealed an increase in cell apoptosis and a loss of Islet1 and Calbindin marker expression in Rathke’s pouch of these mutants. Recently, increased cell apoptosis in Rathke’s pouch has been described in mutant mice impaired in the function of the bicoid-like homeodomain proteins Pitx1 and Pitx2. In those mutants, the expression of Lhx3 is absent. Our results thus underscore the view that Lhx3 functions downstream of the Pitx factors in the same transcriptional cascade that controls growth and early cell differentiation of the developing pituitary gland.

Islet1 and Islet2 have equivalent abilities to promote motoneuron formation and to specify motoneuron subtype identity

The expression of LIM homeobox genes islet1 and islet2 is tightly regulated during development of zebrafish primary motoneurons. All primary motoneurons express islet1 around the time they exit the cell cycle. By the time primary motoneurons undergo axogenesis, specific subtypes express islet1, whereas other subtypes express islet2, suggesting that these two genes have different functions. Here, we show that Islet1 is required for formation of zebrafish primary motoneurons; in the absence of Islet1, primary motoneurons are missing and there is an apparent increase in some types of ventral interneurons. We also provide evidence that Islet2 can substitute for Islet1 during primary motoneuron formation. Surprisingly, our results demonstrate that despite the motoneuron subtype-specific expression patterns of Islet1 and Islet2, the differences between the Islet1 and Islet2 proteins are not important for specification of the different primary motoneuron subtypes. Thus, primary motoneuron subtypes are likely to be specified by factors that act in parallel to or upstream of islet1 and islet2.

Sohlh1 is essential for spermatogonial differentiation

Spermatogonia are adult germline stem cells that can both self-renew and differentiate into spermatocytes. Little is known about factors necessary for spermatogonial differentiation. We identified a novel germ-cell-specific transcription factor that we named Sohlh1 (spermatogenesis and oogenesis specific basic helix–loop–helix (bHLH) transcription factor). In males, Sohlh1 is preferentially expressed in prespermatogonia and Type A spermatogonia. Loss of Sohlh1 causes infertility by disrupting spermatogonial differentiation into spermatocytes. Seven-day-old testes without Sohlh1 still express the testis-specific transcription factors Etv5, Taf4b, Zfp148, and Plzf, overexpress a novel Sohlh2 bHLH transcription factor, but lack LIM homeobox gene Lhx8 and show reduced expression of Ngn3. Sohlh1 represents the first testis-specific bHLH transcription factor that is essential for spermatogonial differentiation.

Developmental Regulation of Whole Cell Capacitance and Membrane Current in Identified Interneurons inC. elegans

Postembryonic developmental changes in electrophysiological properties of the AIY interneuron class were investigated using whole cell voltage clamp. AIY interneurons displayed an increase in cell capacitance during larval development, whereas steady-state current amplitude did not increase. The time course of the outward membrane current, carried at least in part by K+ ions, matured, from a slowly activating, sustained current to a rapidly activating, decaying current. We also investigated how the development of capacitance and outward current was altered by loss-of-function mutations in genes expressed in AIY. One such gene, the LIM homeobox gene ttx-3, is known to be involved in the specification of the AIY neuronal subtype. In ttx-3 mutants, capacitance and outward current matured precociously. In mutants of the gene wrk-1, an immunoglobulin superfamily (IgSF) member whose expression is regulated by ttx-3, capacitance matured normally, whereas outward current matured precociously. We conclude that AIY interneurons contain distinct pathways for regulating capacitance and membrane current.