Beadex

EVOLUTIONARY HOMOLOGS

Hematopoietic stem cells are derived from ventral mesoderm during vertebrate development. Gene targeting experiments in the mouse have demonstrated key roles for the basic helix-loop-helix transcription factor SCL (related protein, Drosophila Helix loop helix protein 3B) and the GATA-binding protein GATA-1 in hematopoiesis. When overexpressed in Xenopus animal cap explants, SCL and GATA-1 are each capable of specifying mesoderm to become blood. Forced expression of either factor in whole embryos, however, does not lead to ectopic blood formation. This apparent paradox between animal cap assays and whole embryo phenotype has led to the hypothesis that additional factors are involved in specifying hematopoietic mesoderm. SCL and GATA-1 interact in a transcriptional complex with the LIM domain protein LMO-2. The Xenopus homolog of LMO-2 has been cloned and it has been shown to be expressed in a pattern similar to SCL during development. LMO-2 can specify hematopoietic mesoderm in animal cap assays. SCL and LMO-2 act synergistically to expand the blood island when overexpressed in whole embryos. Furthermore, co-expression of GATA-1 with SCL and LMO-2 leads to embryos that are ventralized and have blood throughout the dorsal-ventral axis. The synergistic effect of SCL, LMO-2 and GATA-1, taken together with the findings that these factors can form a complex in vitro, suggests that this complex specifies mesoderm to become blood during embryogenesis (Mead, 2001).

Lmo1, Lmo2, and Lmo3 show individually unique but partially overlapping patterns of expression in several regions of the adult mouse forebrain, including hippocampus, caudate putamen, medial habenula, thalamus, amygdala, olfactory bulb, hypothalamus, and cerebral cortex. In the hippocampal formation, Lmo1, Lmo2, and Lmo3 show different combinatorial patterns of expression levels in CA pyramidal and dentate granule neurons, and Lmo1 is present in topographically restricted subpopulations of astrocytes. Kainic acid-induced limbic seizures differentially regulate Lmo1, Lmo2, and Lmo3 mRNA levels in hippocampal pyramidal and granule neurons, such that Lmo1 mRNA increases, whereas Lmo2 and Lmo3 mRNAs decrease significantly, with maximal changes at 6 hr after seizure onset and a return to baseline by 24 hr. These findings show that Lmo1, Lmo2, and Lmo3 continue to be expressed in the adult mammalian CNS in a cell type-specific manner, are differentially regulated by neuronal activity, and may thus be involved in cell phenotype-specific regulatory functions (Hinks, 1997).

LMO4 is a novel member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors. LMO1, LMO2, and LMO4 have distinct expression patterns in adult tissue, and nuclear retention of LMO proteins is enhanced by the nuclear LIM interactor (NLI). In situ hybridization to early mouse embryos of 8-14.5 days reveals a complex pattern of LMO4 expression spatially overlapping with NLI and LHX genes. LMO4 expression in somites is repressed in mice mutant for the segment polarity gene Mesp2 (related to neurogenic HLH transcription factors) and expanded in Splotch (see Drosophila Paired) mutants. During jaw and limb outgrowth, LMO4 and LMO2 expression defines mesenchyme that is uncommitted to regional fates. Although both LMO2 and LMO4 are activated in thymic blast cells, only LMO4 is expressed in mature T cells. Mesenchymal and thymic blast cell expression patterns of LMO4 and LMO2 are consistent with the suggestion that LMO genes inhibit differentiation (Kenny, 1998).

Many vertebrate homologs of Drosophila genes important for wing patterning have been found to play a role in limb development. To determine whether the LMO genes might also be involved, in situ hybridization was performed using the mouse LMO-2 cDNA on E10.5 mouse embryos. LMO-2 RNA is present in the developing limb bud at E10.5. Expression is seen in a band centered on the D-V boundary of the developing limb bud. In situ hybridization to sections of the limb bud reveals that LMO-2 mRNA is present in a broad field of the mesenchyme underlying the apical ectodermal ridge, a structure known to be an important organizing structure of the limb bud. Expression is also seen at the somite boundaries (Zeng, 1998).

The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. A widely expressed protein, Ldb1 (Drosophila homolog: Chip), has been identified whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER proerythroblast cells inhibits cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibits erythroid differentiation. These studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state (Visvader, 1997).

The product of the scl (also called tal-1 or TCL5) gene is a basic domain, helix-loop-helix (bHLH) transcription factor required for the development of hematopoietic cells. Additionally, scl gene disruption and dysregulation, by either chromosomal translocations or a site-specific interstitial deletion whereby 5' regulatory elements of the sil gene become juxtaposed to the body of the scl gene, is associated with T-cell acute lymphoblastic leukemia (ALL) and T-cell lymphoblastic lymphoma. An inappropriately expressed scl protein, driven by sil regulatory elements, can cause aggressive T-cell malignancies in collaboration with a misexpressed LMO1 protein, thus recapitulating the situation seen in a subset of human T-cell ALL. Inappropriately expressed scl can interfere with the development of other tissues derived from mesoderm. An scl construct lacking the scl transactivation domain collaborates with misexpressed LMO1, demonstrating that the scl transactivation domain is dispensable for oncogenesis, and supporting the hypothesis that the scl gene product exerts its oncogenic action through a dominant-negative mechanism (Aplan, 1997).

The LIM-finger protein Lmo2, which is activated in T cell leukemias by chromosomal translocations, is required for yolk sac erythropoiesis. Because Lmo2 null mutant mice die at embryonic day 9-10, it prevents an assessment of a role in other stages of hematopoiesis. The hematopoietic contribution of homozygous mutant Lmo2 -/- mouse embryonic stem cells has been studied and Lmo2 -/- cells are found not to contribute to any hematopoietic lineage in adult chimeric mice, but reintroduction of an Lmo2-expression vector rescues the ability of Lmo2 null embryonic stem cells to contribute to all lineages tested. This disruption of hematopoiesis probably occurs because interaction of Lmo2 protein with factors such as Tal1/Scl is precluded. Thus, Lmo2 is necessary for early stages of hematopoiesis, and the Lmo2 master gene encodes a protein that has a central and crucial role in the hematopoietic development (Yamada, 1998).

The LIM-only protein LMO2 is expressed aberrantly in acute T-cell leukemias as a result of the chromosomal translocations t(11;14) (p13;q11) or t(7;11) (q35;p13). In a transgenic model of tumorigenesis by Lmo2, T-cell acute leukemias arise after an asymptomatic phase in which an accumulation of immature CD4(-) CD8(-) double negative thymocytes occurs. Possible molecular mechanisms underlying these effects have been investigated in T cells from Lmo2 transgenic mice. Isolation of DNA-binding sites by CASTing and band shift assays demonstrates the presence of an oligomeric complex involving Lmo2 that can bind to a bipartite DNA motif comprising two E-box sequences approximately 10 bp apart, which is distinct from that found in erythroid cells. This complex occurs in T-cell tumors and is restricted to the immature CD4(- )CD8(-) thymocyte subset in asymptomatic transgenic mice. Thus, ectopic expression of Lmo2 by transgenesis, or by chromosomal translocations in humans, may result in the aberrant protein interactions causing abnormal regulation of gene expression, resulting in a blockage of T-cell differentiation and providing precursor cells for overt tumour formation (Grutz, 1998).

The LIM-only protein Lmo2, activated by chromosomal translocations in T-cell leukemias, is normally expressed in hematopoiesis. It interacts with TAL1 and GATA-1 proteins, but the function of the interaction is unexplained. In erythroid cells Lmo2 forms a novel DNA-binding complex with GATA-1, TAL1, E2A, and the recently identified LIM-binding protein, Ldb1/NLI. This oligomeric complex binds to a unique, bipartite DNA motif comprising an E-box (CAGGTG), followed approximately 9 bp downstream by a GATA site. In vivo assembly of the DNA-binding complex requires interaction of all five proteins and establishes a transcriptional transactivating complex. These data demonstrate one function for the LIM-binding protein Ldb1 and establish a function for the LIM-only protein Lmo2 as an obligatory component of an oligomeric, DNA-binding complex, which may play a role in hematopoiesis (Wadman, 1997).

Nuclear LIM domains interact with a family of coregulators referred to as Clim/Ldb/Nli. Although one family member, Clim-2/Ldb-1/Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain factor is known to be expressed in epidermis. Therefore, the conserved LIM-interaction domain of Clim coregulators was used to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and a factor was isolated that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in an overlapping manner with Clim-2 in epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because LMO/Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins, LMO-4 was used as a bait to screen for such DNA-binding proteins in epidermis. Identified was the mouse homolog of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element. The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse DEAF-1, distinct from the helix-loop-helix and GATA domains previously shown to interact with LMOs, thus defining an additional LIM-interacting domain (Sugihara, 1998).

It is now widely accepted that hemopoietic cells born intraembryonically are the best candidates for the seeding of definitive hemopoietic organs. To further understand the mechanisms involved in the generation of definitive hemopoietic stem cells, the expression of the hemopoietic-related transcription factors Lmo2 and GATA-3 during the early steps of mouse development (7-12 dpc) was analysed, with a particular emphasis on intraembryonic hemogenic sites. Both Lmo2 and GATA-3 are present in the intraembryonic regions known to give rise to hemopoietic precursors in vitro and in vivo, suggesting that they act together at key points of hemopoietic development. Lmo2 mRNA is observed in all the sites endowed with a hemopoietic potential, where its expression is tightly regulated spatiotemporally. The rapid modifications of Lmo2 expression patterns suggest that it allocates specific combinations of transcription factors during key points of development. The overlapping expressions of Lmo2 and GATA-3 suggests combined functions during specific steps of definitive hemopoietic development, namely: (1) endodermal induction leading to the emergence of hemopoietic precursors in the mesoderm; (2) determination of these cells from the mesoderm, and (3) their production in the aortic region from 9 to 12 dpc as well as their release into the blood stream. Lmo2 and GATA-3 are expressed in the caudal mesoderm during the phase that determines intraembryonic precursors. A highly transient concomitant expression is observed in the caudal intraembryonic definitive endoderm, suggesting that these factors are involved in the specification of intraembryonic hemopoietic precursors. Lmo2 and GATA-3 are expressed within the hemopoietic clusters located in the aortic floor during fetal liver colonization. Furthermore, a strong GATA-3 signal allowed the uncovering of previously unreported mesodermal aggregates beneath the aorta. Combined in situ and immunocytological analysis strongly suggests that ventral mesodermal GATA-3 patches are involved in the process of intraembryonic stem cell generation (Manaia, 2000).

In the stretch-reflex system, proprioceptive sensory neurons make selective synaptic connections with different subsets of motoneurons, according to the peripheral muscles they supply. To examine the molecular mechanisms that may influence the selection of these synaptic targets, single-cell cDNA libraries were constructed from sensory neurons that innervate antagonist muscles. Differential screening of these libraries identified a transcription regulatory co-factor of the LIM homeodomain proteins, the LIM domain only 4 protein Lmo4, expressed in most adductor but few sartorius sensory neurons. Differential patterns of Lmo4 expression were also seen in sensory neurons supplying three other muscles. A subset of motoneurons also expresses Lmo4 but the pattern of expression is not specific for motor pools. Differential expression of Lmo4 occurs early, as neurons develop their characteristic LIM homeodomain protein expression patterns. Moreover, ablation of limb buds does not block Lmo4 expression, suggesting that an intrinsic program controls the early differential expression of Lmo4. LIM homeodomain proteins are known to regulate several aspects of sensory and motor neuronal development. The results suggest that Lmo4 may participate in this differentiation by regulating the transcriptional activity of LIM homeodomain proteins (Chen, 2002).

Lmo4 can regulate the transcriptional activities of LIM homeodomain factors in several ways. Lmo transcriptional regulatory factors lack a DNA binding domain but contain two protein-protein interaction LIM domains. Lmo proteins can compete for NLI with LIM homeodomain transcription factors, and thereby regulate the formation of LIM homeodomain/NLI complexes and their transcriptional activity. Drosophila Lmo can bind to Chip with higher affinity than the LIM homeodomain of Apterous and thereby regulate Apterous activity levels in vivo. Whether there is a differential affinity to NLI between Lmo4 and other LIM homeodomain proteins is not yet known (Chen, 2002).

Lmo4 may also compete for other co-factors besides NLI that are specific for individual LIM homeodomain proteins and could thus regulate the expression of downstream target genes. For example, data based on the expression of chimeric LIM domains derived from different Islet family members (i.e. Isl1, Isl2 and ISL3) in zebrafish, has led to the conclusion that Isl2 probably forms a transcriptional complex with an Isl2-specific co-factor, in addition to NLI. Interaction with an Isl2-specific co-factor could contribute to the role of Isl2 in the differentiation of primary motoneurons, neuronal positioning, peripheral axonal outgrowth and neuronal transmitter expression in zebrafish (Chen, 2002).

Combinatorial interactions of Lmo4 with other transcription factors might provide additional mechanisms for the regulation of transcription during neuronal development. In enkaphalin-producing neurons, Lmo4 interacts with the transcription factor DEAF1 (deformed epidermal autoregulatory factor 1). DEAF1 has been implicated in opioid production by regulating enkaphalin transcription through a retinoic acid-responsive element. Interestingly, in the fetal and adult mouse brain, Lmo4 expression is region specific: high levels of expression are present in the limbic system and in regions involved in autonomic, motor and neuroendocrine regulation. Recently, studies in breast cancer cell lines have demonstrated that Lmo4 expression is upregulated and forms a multiprotein complex with CtIP and BRCA1. A role for BRCA1 in neurons has not been explored (Chen, 2002).

Different LIM homeodomain proteins are known to activate different downstream target genes. The pattern of neuronal generation in the ventral neural tube is achieved primarily by the spatially restricted expression of transcriptional repressors. By modulating the transcriptional activity of LIM homeodomain proteins, Lmo4 is likely to be involved in the specification of motor neuronal identity. Its restricted expression in subsets of muscle sensory neurons suggests that it contributes to the specification of sensory neurons as well (Chen, 2002).

Cysteine-rich LIM-only proteins, CRP1 and CRP2, expressed during cardiovascular development act as bridging molecules that associate with serum response factor and GATA proteins. SRF-CRP-GATA complexes strongly activate smooth muscle gene targets. CRP2 is found in the nucleus during early stages of coronary smooth muscle differentiation from proepicardial cells. A dominant-negative CRP2 mutant blocks proepicardial cells from differentiating into smooth muscle cells. Together with SRF and GATA proteins, CRP1 and CRP2 converts pluripotent 10T1/2 fibroblasts into smooth muscle cells, while muscle LIM protein CRP3 inhibits the conversion. Thus, LIM-only proteins of the CRP family play important roles in organizing multiprotein complexes, both in the cytoplasm, where they participate in cytoskeletal remodeling, and in the nucleus, where they strongly facilitate smooth muscle differentiation (Chang, 2003).

The LIM domain protein Lmo2 and the basic helix-loop-helix transcription factor Scl/Tal1 (distantly related to Drosophila Twist) are expressed in early haematopoietic and endothelial progenitors and interact with each other in haematopoietic cells. While loss-of-function studies have shown that Lmo2 and Scl/Tal1 are essential for haematopoiesis and angiogenic remodelling of the vasculature, gain-of-function studies have suggested an earlier role for Scl/Tal1 in the specification of haemangioblasts, putative bipotential precursors of blood and endothelium. In zebrafish embryos, Scl/Tal1 can induce these progenitors from early mesoderm mainly at the expense of the somitic paraxial mesoderm. This restriction to the somitic paraxial mesoderm correlates well with the ability of Scl/Tal1 to induce ectopic expression of its interaction partner Lmo2. Co-injection of lmo2 mRNA with scl/tal1 dramatically extends its effect to head, heart, pronephros and pronephric duct mesoderm inducing early blood and endothelial genes all along the anteroposterior axis. Erythroid development, however, is expanded only into pronephric mesoderm, remaining excluded from head, heart and somitic paraxial mesoderm territories. This restriction correlates well with activation of gata1 transcription and co-injection of gata1 mRNA along with scl/tal1 and lmo2 induces erythropoiesis more broadly without ventralizing or posteriorizing the embryo. While no ectopic myeloid development from the Scl/Tal1-Lmo2-induced haemangioblasts was observed, a dramatic increase in the number of endothelial cells was found. These results suggest that, in the absence of inducers of erythroid or myeloid haematopoiesis, Scl/Tal1-Lmo2-induced haemangioblasts differentiate into endothelial cells (Gering, 2003).

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