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The timing of cortical neurogenesis has a major effect on the size and organization of the mature cortex. The deletion of the LIM-homeodomain transcription factor Lhx2 in cortical progenitors by Nestin-cre leads to a dramatically smaller cortex. Here we report that Lhx2 regulates the cortex size by maintaining the cortical progenitor proliferation and delaying the initiation of neurogenesis. The loss of Lhx2 in cortical progenitors results in precocious radial glia differentiation and a temporal shift of cortical neurogenesis. We further investigated the underlying mechanisms at play and demonstrated that in the absence of Lhx2, the Wnt/β-catenin pathway failed to maintain progenitor proliferation. We developed and applied a mathematical model that reveals how precocious neurogenesis affected cortical surface and thickness. Thus, we concluded that Lhx2 is required for β-catenin function in maintaining cortical progenitor proliferation and controls the timing of cortical neurogenesis. [ABSTRACT FROM AUTHOR]

Generating engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is an ideal approach for obtaining induced HSCs for cell therapy. However, the path from PSCs to robustly induced HSCs (iHSCs) in vitro remains elusive. We hypothesize that the modification of hematopoietic niche cells by transcription factors facilitates the derivation of induced HSCs from PSCs. The Lhx2 transcription factor is expressed in fetal liver stromal cells but not in fetal blood cells. Knocking out Lhx2 leads to a fetal hematopoietic defect in a cell non-autonomous role. In this study, we demonstrate that the ectopic expression of Lhx2 in OP9 cells (OP9-Lhx2) accelerates the hematopoietic differentiation of PSCs. OP9-Lhx2 significantly increased the yields of hematopoietic progenitor cells via co-culture with PSCs in vitro. Interestingly, the co-injection of OP9-Lhx2 and PSCs into immune deficient mice also increased the proportion of hematopoietic progenitors via the formation of teratomas. The transplantation of phenotypic HSCs from OP9-Lhx2 teratomas but not from the OP9 control supported a transient repopulating capability. The upregulation of Apln gene by Lhx2 is correlated to the hematopoietic commitment property of OP9-Lhx2. Furthermore, the enforced expression of Apln in OP9 cells significantly increased the hematopoietic differentiation of PSCs. These results indicate that OP9-Lhx2 is a good cell line for regeneration of hematopoietic progenitors both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Background: Vertebrate retinal development is a complex process, requiring the specification and maintenance of retinal identity, proliferative expansion of retinal progenitor cells (RPCs), and their differentiation into retinal neurons and glia. The homeobox gene is expressed in RPCs and required for the proper execution of this retinal Vsx2 program. However, our understanding of the mechanisms by which Vsx2 does this is still rudimentary. To define the autonomy requirements for Vsx2 in the regulation of RPC properties, we generated chimeric mouse embryos comprised of wild-type and Vsx2-deficient cells. Results: We show that Vsx2 maintains retinal identity in part through the cell-autonomous repression of the retinal pigment epithelium determinant Mitf, and that Lhx2 is required cell autonomously for the ectopic Mitf expression in Vsx2-deficient cells. We also found significant cell-nonautonomous contributions to Vsx2-mediated regulation of RPC proliferation, pointing to an important role for Vsx2 in establishing a growth-promoting extracellular environment. Additionally, we report a cell-autonomous requirement for Vsx2 in controlling when neurogenesis is initiated, indicating that Vsx2 is an important mediator of neurogenic competence. Finally, the distribution of wild-type cells shifted away from RPCs and toward retinal ganglion cell precursors in patches of high Vsx2-deficient cell density to potentially compensate for the lack of fated precursors in these areas. Conclusions: Through the generation and analysis of genetic chimeras, we demonstrate that Vsx2 utilizes both cell-autonomous and cell-nonautonomous mechanisms to regulate progenitor properties in the embryonic retina. Importantly, Vsx2's role in regulating Mitf is in part separable from its role in promoting proliferation, and proliferation is excluded as the intrinsic timer that determines when neurogenesis is initiated. These findings highlight the complexity of Vsx2 function during retinal development and provide a framework for identifying the molecular mechanisms mediating these functions. [ABSTRACT FROM AUTHOR]

In the murine hair follicle ( HF), the transcription factors LHX2 and SOX9 are implicated in epithelial hair follicle stem cell ( eHFSC) self-renewal and the maintenance of eHFSC niche characteristics. However, the exact expression patterns of LHX2 and SOX9 in the human HF are unclear. Therefore, we have quantitatively mapped the localisation of known human eHFSC markers keratin 15 (K15) and keratin 19 (K19) in the outer root sheath (ORS) of male occipital scalp anagen HFs and related this to the localisation of LHX2 and SOX9 protein expression. As expected, K15+ and K19+ cells represented two distinct progenitor cell populations in the bulge and in the proximal bulb ORS (pb ORS). Interestingly, cell fluorescence for K19 was significantly stronger within the pb ORS versus the bulge, and vice versa for K15, describing a hitherto unrecognised differential expression pattern. LHX2 and SOX9 expressing cells were distributed throughout the ORS, including the bulge, but were not restricted to it. SOX9 expression was most prominent in the ORS immediately below the human bulge, whereas LHX2+ cells were similarly distributed between the sub-bulge and pb ORS, that is compartments not enriched with quiescent eHFSCs. During catagen development, the intensity of LHX2 and SOX9 protein expression increased in the proximal HF epithelium. Double immunostaining showed that the majority of SOX9+ cells in the human anagen HF epithelium did not co-express K15, K19 or LHX2. This expression profile suggests that LHX2 and SOX9 highlight distinct epithelial progenitor cell populations, in addition to K15+ or K19+ cells, that could play an important role in the maintenance of the human HF epithelium. [ABSTRACT FROM AUTHOR]

LIM homeobox transcription factor Lhx2 is known to be an important regulator of neuronal development, homeostasis of hair follicle stem cells, and self-renewal of hematopoietic stem cells; however, its function in skeletal muscle development is poorly understood. In this study, we found that overexpression of Lhx2 completely inhibits the myotube-forming capacity of C2C12 cells and primary myoblasts. The muscle dedifferentiation factors Msx1 and Msx2 were strongly induced by the Lhx2 overexpression. Short interfering RNA-mediated knockdown of Lhx2 in the developing limb buds of mouse embryos resulted in a reduction in Msx1 and Msx2 mRNA levels, suggesting that they are downstream target genes of Lhx2. We found two Lhx2 consensus-binding sites in the −2097 to −1189 genomic region of Msx1 and two additional sites in the −536 to +73 genomic region of Msx2 . These sequences were shown by luciferase reporter assay to be essential for Lhx2-mediated transcriptional activation. Moreover, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that Lhx2 is present in chromatin DNA complexes bound to the enhancer regions of the Msx1 and Msx2 genes. These data demonstrate that Msx1 and Msx2 are direct transcriptional targets of Lhx2. In addition, overexpression of Lhx2 significantly enhanced the mRNA levels of bone morphogenetic protein 4 and transforming growth factor beta family genes. We propose that Lhx2 is involved in the early stage of skeletal muscle development by inducing multiple differentiation inhibitory factors. [ABSTRACT FROM AUTHOR]

The LIM proteins (Lhx1, Lhx2, Lhx3 and Lhx4) have been report to play important roles in human development. The function role of Lhxs have been characterized in various tumor tissues as cancer suppressors or promoters in different can status and types. The aim of present study was to clarify the function role of Lhx proteins in human pancreatic ductal adenocarcinoma (PDA). The gene expression profiles of Lhxs was evaluated using real-time quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) analysis and immunohistochemistry in human PDA tissues compared with normal pancreatic tissues, which identified the gene overexpression of Lhx2 in PDA. Furthermore, we discovered that Lhx2 promoted cancer cell proliferation in vitro/vivo and elevated β-catenin levels correlated with Lhx2 expression in PDA while the Lhx2 simulated β-catenin activation was required for LMO1's oncogenic effects. Mechanistically, Lhx2 facilitate TCF4 to bind to β-catenin and form a stable Lhx2/TCF4/β-catenin complex and trans-active its downstream target gene. Lhx2 mutations that disrupt the Lhx2-β-catenin interaction partially prevent its function in tumor cells. [ABSTRACT FROM AUTHOR]

Lhx2, a member of LIM homeobox transcription factors, plays a key role in normal tissue development. However, the molecular mechanism of Lhx2 gene in the regulation of the secondary hair follicle cycling in cashmere goat remains largely unknown. In the present study, the Lhx2 gene was cloned and characterized in cashmere goat. The cloned cDNA of Lhx2 was 1233 bp in length, encoding for proteins of 406 amino acids which contained all functionally important domains conserved among vertebrate Lhx2 gene. Tissue distribution analysis showed that Lhx2 mRNA was highly expressed in the skin and low expressed in all other tissues. Immunohistochemical localization revealed that Lhx2 was expressed in secondary hair follicles. Analysis of expression profiles of Lhx2 mRNA during different development stages in secondary hair follicles showed that the highest expression was observed at the anagen stage, while the lowest expression was detected at the telogen stage. The expression tendency during the development stages was that it increased from telogen to anagen, decreased from anagen to catagen, and decreased from catagen to telogen. The expression pattern of Lhx2 protein and mRNA was similar. The mRNA and protein expression of Lhx2 were consistent throughout the development cycle in secondary hair follicles. These findings provided a better understanding of the function of Lhx2 and suggested that the cyclic expression of Lhx2 might play important roles during secondary hair follicle development in cashmere goat. [ABSTRACT FROM AUTHOR]

An epithelial‐mesenchymal transition (EMT) is a critical process during embryonic development and the progression of epithelial tumors to metastatic cancers. Gene expression profiling has uncovered the transcription factor LIM homeobox gene 2 (Lhx2) with up‐regulated expression during TGFβ‐induced EMT in normal and cancerous breast epithelial cells. Loss and gain of function experiments in transgenic mouse models of breast cancer and of insulinoma in vivo and in breast cancer cells in vitro indicate that Lhx2 plays a critical role in primary tumor growth and metastasis. Notably, the transgenic expression of Lhx2 during breast carcinogenesis promotes vessel maturation, primary tumor growth, tumor cell intravasation and metastasis by directly inducing the expression of platelet‐derived growth factor (PDGF)‐B in tumor cells and by indirectly increasing the expression of PDGF receptor‐β (PDGFRβ) on tumor cells and pericytes. Pharmacological inhibition of PDGF‐B/PDGFRβ signaling reduces vessel functionality and tumor growth and Lhx2‐induced cell migration and cell invasion. The data indicate a dual role of Lhx2 during EMT and tumor progression: by inducing the expression of PDGF‐B, Lhx2 provokes an autocrine PDGF‐B/PDGFRβ loop required for cell migration, invasion and metastatic dissemination and paracrine PDGF‐B/PDGFRβ signaling to support blood vessel functionality and, thus, primary tumor growth. Highlights: Lhx2 expression is induced during EMT by canonical TGFβ signaling.Lhx2 expression is elevated during both human and mouse carcinogenesis.Lhx2 promotes primary tumor growth by paracrine PDGF‐B/PDGFRβ signaling‐mediated tumor vessel maturation and functionality.Lhx2 promotes metastasis formation via autocrine PDGF‐B/PDGFRβ signaling‐induced tumor cell migration and invasion. [ABSTRACT FROM AUTHOR]

A bstract We previously demonstrated that hematopoietic stem cell (HSC)-like cells are robustly expanded from mouse embryonic stem cells (ESCs) by enforced expression of Lhx2, a LIM-homeobox domain (LIM-HD) transcription factor. In this study, we analyzed the functions of Lhx2 in that process using an ESC line harboring an inducible Lhx2 gene cassette. When ESCs are cultured on OP9 stromal cells, hematopoietic progenitor cells (HPCs) are differentiated and these HPCs are prone to undergo rapid differentiation into mature hematopoietic cells. Lhx2 inhibited differentiation of HPCs into mature hematopoietic cells and this effect would lead to accumulation of HSC-like cells. LIM-HD factors interact with LIM domain binding (Ldb) protein and this interaction abrogates binding of LIM-only (Lmo) protein to Ldb. We found that one of Lmo protein, Lmo2, was unstable due to dissociation of Lmo2 from Ldb1 in the presence of Lhx2. This effect of Lhx2 on the amount of Lmo2 contributed into accumulation of HSC-like cells, since enforced expression of Lmo2 into HSC-like cells inhibited their self-renewal. Expression of Gata3 and Tal1/Scl was increased in HSC-like cells and enforced expression of Lmo2 reduced expression of Gata3 but not Tal1/Scl. Enforced expression of Gata3 into HPCs inhibited mature hematopoietic cell differentiation, whereas Gata3-knockdown abrogated the Lhx2-mediated expansion of HPCs. We propose that multiple transcription factors/cofactors are involved in the Lhx2-mediated expansion of HSC-like cells from ESCs. Lhx2 appears to fine-tune the balance between self-renewal and differentiation of HSC-like cells. S tem C ells 2013;31:2680-2689 [ABSTRACT FROM AUTHOR]

Abstract: The neocortex represents the brain region that has undergone a major increase in its relative size during the course of mammalian evolution. The larger cortex results from a corresponding increase in progenitor cell number. The progenitors giving rise to neocortex are located in the ventricular zone of the dorsal telencephalon and highly express Lhx2, a LIM-homeodomain transcription factor. The neocortex fails to form in the Lhx2 constitutive knockout, indicating a role for Lhx2 in corticogenesis, but mid-embryonic lethality of the Lhx2 knockout requires the use of conditional strategies for further studies. Therefore, to explore Lhx2 function in neocortical progenitors, we generated mice with Lhx2 conditionally deleted from cortical progenitors at the onset of neurogenesis. We find that Lhx2 is critical for maintaining the proliferative state of neocortical progenitors during corticogenesis. In the conditional knockouts, the neocortex is formed but is significantly smaller than wild type. We find that deletion of Lhx2 leads to significantly decreased numbers of cortical progenitors and premature neuronal differentiation. A likely mechanism is indicated by our findings that Lhx2 is required for the expression of Hes1 in cortical progenitors, a key effector in the Notch signaling pathway that maintains the proliferative progenitor state. We conclude that Lhx2 regulates the balance between proliferation and differentiation in cortical progenitors and through this mechanism Lhx2 controls cortical size. [Copyright &y& Elsevier]

Early forebrain patterning entails the correct regional designation of the neuroepithelium, and appropriate specification, generation, and distribution of neural cells during brain development. Specific signaling and transcription factors are known to tightly regulate patterning of the dorsal telencephalon to afford proper structural/functional cortical arealization and morphogenesis. Nevertheless, whether and how changes of the chromatin structure link to the transcriptional program(s) that control cortical patterning remains elusive. Here, we report that the BAF chromatin remodeling complex regulates the spatiotemporal patterning of the mouse dorsal telencephalon. To determine whether and how the BAF complex regulates cortical patterning, we conditionally deleted the BAF complex scaffolding subunits BAF155 and BAF170 in the mouse dorsal telencephalic neuroepithelium. Morphological and cellular changes in the BAF mutant forebrain were examined using immunohistochemistry and in situ hybridization. RNA sequencing, Co-immunoprecipitation, and mass spectrometry were used to investigate the molecular basis of BAF complex involvement in forebrain patterning. We found that conditional ablation of BAF complex in the dorsal telencephalon neuroepithelium caused expansion of the cortical hem and medial cortex beyond their developmental boundaries. Consequently, the hippocampal primordium is not specified, the mediolateral cortical patterning is compromised, and the cortical identity is disturbed in the absence of BAF complex. The BAF complex was found to interact with the cortical hem suppressor LHX2. The BAF complex suppresses cortical hem fate to permit proper forebrain patterning. We provide evidence that BAF complex modulates mediolateral cortical patterning possibly by interacting with the transcription factor LHX2 to drive the LHX2-dependent transcriptional program essential for dorsal telencephalon patterning. Our data suggest a putative mechanistic synergy between BAF chromatin remodeling complex and LHX2 in regulating forebrain patterning and ontogeny., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nguyen, Sokpor, Parichha, Pham, Saikhedkar, Xie, Ulmke, Rosenbusch, Pirouz, Behr, Stoykova, Brand-Saberi, Nguyen, Staiger, Tole and Tuoc.)

Abstract: Lhx2, a member of LIM homeobox transcription factors, plays a key role in central nervous system (CNS) and embryonic tissue development. However, molecular mechanism of Lhx2 gene regulation remains largely unknown. Here, we identified and characterized a regulatory region of Lhx2 gene which mediates responses to two different signals such as inhibition of HDAC3 and stimulation by E2F1. In particular, the promoter region of −229 to −126 was responsible not only for basal expression but also for a inhibitor of histone deacetylase, trichostatin A (TSA)-mediated activation of Lhx2 gene. Intriguingly, transcription factor E2F1 also activates Lhx2 gene via direct binding to the same −229 to −126 region. Based on these observations, we could have demonstrated that E2F1 is necessary for TSA-mediated activation of Lhx2 gene and acetylation of histone 3 is involved in this event. This study provides evidence that the histone modification and E2F1 binding are integral parts of the mechanism for Lhx2 gene expression. [Copyright &y& Elsevier]

LMO1, LMO3 and LMO4 were cloned from the adult porcine pituitary cDNA library. Amino acid sequences of porcine LMO1, LMO3 and LMO4 were highly conserved among mammalian species. Transfection assay of the pituitary-derived cell line LβT2 was carried out using the pituitary αGSU (glycoprotein hormone α-subunit) promoter (-1059/+12 b) fused to pSEAP2-Basic vector as a reporter gene. The results demonstrated that, whereas LMO4 showed no apparent effect, αGSU promoter activity was markedly repressed by LMO1 but activated by LMO3, indicating the different roles of the three highly homologous proteins, LMO1, LMO3 and LMO4. Knockdown assay by LMO siRNAs (small interfering RNAs) confirmed the above results for LMO1 and LMO3, whereas that by LMO4 siRNA increased the expression, indicating different modes of action. RT-PCR (reverse transcription-PCR) for total RNAs of several cell lines showed that LMO1 and LMO4 mRNAs were present ubiquitously in all cell lines, except for LMO1 in L929 cells. In contrast, LMO3 mRNA was abundant only in LβT4 and GH3 cells with only small amounts in LβT2 and MtT/S cells, indicating the cell-type-specific function of this protein. Real-time analyses of porcine pituitary ontogeny revealed that the three LMO genes are expressed during the fetal period and decline immediately afterwards, followed by a remarkably low level of LMO3 and LMO4 after birth. RT-PCR of the porcine tissues examined showed ubiquitous expression of LMO4, whereas LMO1 and LMO3 are expressed tissue specifically. Thus the present study demonstrated that three highly related LIM cofactors, LMO1, LMO3 and LMO4, have different effects on αGSU gene expression in the pituitary glands. [ABSTRACT FROM AUTHOR]

Abstract: The mammalian pituitary gland originates from two separate germinal tissues during embryonic development. The anterior and intermediate lobes of the pituitary are derived from Rathke''s pouch, a pocket formed by an invagination of the oral ectoderm. The posterior lobe is derived from the infundibulum, which is formed by evagination of the neuroectoderm in the ventral diencephalon. Previous studies have shown that development of Rathke''s pouch and the generation of distinct populations of hormone-producing endocrine cell lineages in the anterior/intermediate pituitary lobes is regulated by a number of transcription factors expressed in the pouch and by inductive signals from the ventral diencephalon/infundibulum. However, little is known about factors that regulate the development of the posterior pituitary lobe. In this study, we show that the LIM-homeobox gene Lhx2 is extensively expressed in the developing ventral diencephalon, including the infundibulum and the posterior lobe of the pituitary. Deletion of Lhx2 gene results in persistent cell proliferation, a complete failure of evagination of the neuroectoderm in the ventral diencephalon, and defects in the formation of the distinct morphological features of the infundibulum and the posterior pituitary lobe. Rathke''s pouch is formed and endocrine cell lineages are generated in the anterior/intermediate pituitary lobes of the Lhx2 mutant. However, the shape and organization of the pouch and the anterior/intermediate pituitary lobes are severely altered due to the defects in development of the infundibulum and the posterior lobe. Our study thus reveals an essential role for Lhx2 in the regulation of posterior pituitary development and suggests a mechanism whereby development of the posterior lobe may affect the development of the anterior and intermediate lobes of the pituitary gland. [Copyright &y& Elsevier]

Abstract: It has been reported that miR-124a is abundant in the central nervous system including the eye, and is related to neurogenesis in several species. However, the role of miR-124a in the eye remains unclear. In this study, we show that the expression of miR-124a in Xenopus laevis begins along the neural fold, including the protruding eye anlagen, at a low level at around stage 18; its expression level gradually increases in the neural tube and the eye as embryos develop into later stages and then maintains at a high level in eye to adult stages. Microinjection of a miR-124a precursor at the 8-cell stage leads to malformation of the optic nerve and optic cup, indicating the importance of maintaining low levels of miR-124a during early embryonic development. In addition, miR-124a overexpression markedly down regulates the expression of its predicted targets Lhx2, Hairy2, Gli3, NeuroD1 and Otx2 in/around the eye anlagen, and the interaction of miR-124a with the 3′ UTR of Lhx2 represses gene expression as shown by luciferase assays. Moreover, excess miR-124a inhibits cell proliferation in the eye of Xenopus embryos during retinogenesis. These results indicate that miR-124a acts as a post-transcriptional regulator in the genetic network controlling eye morphogenesis and neurogenesis. The mechanism of miR-124a’s early interaction with the genetic network may also persist in its later role in the maturing and adult eye and brain. [Copyright &y& Elsevier]

Abstract: In mammals, a limited set of homeobox-containing transcription factors are expressed in the presumptive eye field and required to initiate eye development. How these factors interact together at the genetic and molecular level to coordinate this developmental process is poorly understood. We found that the Lhx2 and Pax6 transcription factors operate in a concerted manner during retinal development to promote transcriptional activation of the Six6 homeobox-gene in primitive and mature retinal progenitors. Lhx2 demarcates the presumptive retina field at the neural plate stage and Lhx2 inactivation delays initiation of Rx, Six3 and Pax6 expression in this domain. The later expressed Six6 is properly activated in the pituitary/hypothalamic axis of Lhx2 −/− embryos, but expression fails to be initiated in the optic vesicle. Lhx2 and Pax6 associate with the chromatin at several regions of Six6 in vivo and cooperate for trans-activation of Six6 regulatory elements in vitro. In retinal progenitor/stem cells, both Lhx2 and Pax6 are genetically required for proper Six6 expression and forced co-expression of Lhx2 and Pax6 can synergistically trans-activate the Six6 locus. Our work reveals how two master regulators of eye development coordinate their action to sequentially promote tissue-specific transcriptional initiation and full activation of a retinal determinant gene. [Copyright &y& Elsevier]

Abstract: The homeostasis of both cornea and hair follicles depends on a constant supply of progeny cells produced by populations of keratin (K) 14-expressing stem cells localized in specific niches. To investigate the potential role of Co-factors of LIM domains (Clims) in epithelial tissues, we generated transgenic mice expressing a dominant-negative Clim molecule (DN-Clim) under the control of the K14 promoter. As expected, the K14 promoter directed high level expression of the transgene to the basal cells of cornea and epidermis, as well as the outer root sheath of hair follicles. In corneal epithelium, the transgene expression causes decreased expression of adhesion molecule BP180 and defective hemidesmosomes, leading to detachment of corneal epithelium from the underlying stroma, which in turn causes blisters, wounds and an inflammatory response. After a period of epithelial thinning, the corneal epithelium undergoes differentiation to an epidermis-like structure. The K14-DN-Clim mice also develop progressive hair loss due to dysfunctional hair follicles that fail to generate hair shafts. The number of hair follicle stem cells is decreased by at least 60% in K14-DN-Clim mice, indicating that Clims are required for hair follicle stem cell maintenance. In addition, Clim2 interacts with Lhx2 in vivo, suggesting that Clim2 is an essential co-factor for the LIM homeodomain factor Lhx2, which was previously shown to play a role in hair follicle stem cell maintenance. Together, these data indicate that Clim proteins play important roles in the homeostasis of corneal epithelium and hair follicles. [Copyright &y& Elsevier]

Abstract: The thalamocortical tract is the primary source of sensory information to the cerebral cortex, but the mechanisms regulating its pathfinding are not completely understood. LIM-homeodomain (LIM–HD) gene Lhx2 has been proposed to participate in a combinatorial “code” to regulate dorsal thalamic patterning and also the topography of thalamocortical projections. Here, we report that Lhx2−/− embryos exhibit a gross disruption in the early development of the thalamocortical tract, such that thalamic axons are unable to enter the ventral telencephalon. A possible cause for this deficit is a severe reduction of “pioneer” cells in the mutant ventral telencephalon that constitutes a putative mechanism for guiding the entry of the thalamocortical tract into this structure in vivo. However, in vitro, the thalamocortical tract is able to enter the ventral telencephalon, and this permitted an examination of whether thalamocortical topography is normal in the Lhx2 mutant. Contrary to hypotheses that proposed a cell-autonomous role for Lhx2 in the thalamus, Lhx2−/− thalamic explants generate a normal topography of projections in control ventral telencephalic preparations. This is consistent with our findings of normal patterning of the Lhx2 mutant dorsal thalamus using a wide array of markers. In the reverse experiment, however, control thalamic explants display aberrant topography in Lhx2−/− telencephalic preparations. This perturbation is restricted to projections from caudal thalamic explants, while rostral and middle explants project normally. Thus Lhx2 is required for multiple steps in thalamocortical tract pathfinding, but these functions appear localized in the ventral telencephalon rather than in the dorsal thalamic neurons. Furthermore, the absence of Lhx2 in the ventral telencephalon selectively disrupts a subset of thalamic axon topography, indicating a specific rather than a general perturbation of cues in this structure. [Copyright &y& Elsevier]

Abstract: We have cloned a porcine orthologue of cofactor CLIM2 (Ldb1/NLI) from the porcine pituitary cDNA library by protein–protein interaction with the Yeast Two-Hybrid System using porcine Lhx2 as a bait protein. Porcine CLIM2 shows a high identity (99%) in the dimerization domain, nuclear localization signal and LIM binding domain with those of man and mouse. The expression of CLIM2 gene in the anterior pituitary lobe was detected during the porcine fetal and postnatal period by RT-PCR analysis, suggesting that this protein is constitutively expressing and plays a basic role in the anterior pituitary. Transfection assay to the pituitary tumor derived LβT2 cells, and the Chinese hamster ovary cells demonstrated that CLIM2 acts as a corepressor of the porcine Lhx2 function. Interestingly, CLIM2 alone apparently repressed the high level of αGSU gene expression in LβT2 cells. These data suggest that CLIM2 is a basic factor in the pituitary development and function, and plays the role of repressor to modify the function of Lhx2 on the αGSU gene expression. [Copyright &y& Elsevier]

Abstract: The mouse homeobox gene Barhl1 plays a central role in cerebellum development and its expression is activated by the transcription factor Math1 which is involved in bone morphogenetic protein response pathways. We studied the human ortholog BARHL1 and we found that human, mouse, monkey, rat, and zebrafish orthologs were highly conserved and are members of the BarH homeogene family, containing Drosophila BarH1 and BarH2. The N-terminus of BARHL1 protein presents two FIL domains and an acidic domain rich in serine/threonine and proline, while the C-terminus contains a canonical proline-rich domain. Secondary structure analysis showed that outside the three helixes of the homeodomain, BARHL1 protein has essentially random coil structure. We isolated BARHL1 and defined its expression pattern in human embryonic and fetal central nervous system (CNS) and compared it to the mouse Barhl1 transcription. BARHL1 mRNA was found exclusively in the CNS restricted to p1–p4 prosomeres of the diencephalon, to the dorsal cells of the mesencephalon, to the dorsal dl1 sensory neurons of the spinal cord, and to the rhombic lips yielding the cerebellar anlage. Detailed analysis of BARHL1 expression in fetal cerebellar cell layers using our new optic microscopy technology showed BARHL1 expression in external and internal granular cells and also in mouse adult granular cells, in agreement to Barhl1 null mouse phenotype affecting the differentiation and migration of granular cells. These findings indicate that the regional and cellular specificities of BARHL1 transcriptional control well correspond to the mouse Barhl1 transcription and suggest a potential role of this gene in the differentiation of BARHL1-expressing neuronal progenitors involved in the pattern formation of human cerebral and cerebellar structures. [Copyright &y& Elsevier]

Abstract: The telencephalon shows the greatest degree of size variation in the vertebrate brain. Understanding the genetic cascade that regulates telencephalon growth is crucial to our understanding of how evolution of the normal human brain has supported such a variation in size. Here, we present a simple and quick approach to analyze this cascade that combines caged-mRNA technology and the use of antisense morpholino oligonucleotides in zebrafish embryos. Lhx2, a LIM-homeodomain protein, and Six3s (Six3b and Six3a), another homeodomain proteins, show very similar expression patterns early in forebrain development, and these are known to be involved in the growth of this part of the brain. The telencephalon of six3b and six3a double morphant (six3 morphant) embryos is markedly reduced in size due to impaired cellular proliferation. Head-specific overexpression of Lhx2 by photoactivation of a caged-lhx2 mRNA completely rescued this size reduction, whereas similar head-specific activation of Six3b could not rescue the knockdown effect of lhx2. In the forebrain of medaka embryos, Six3 facilitates cellular proliferation by sequestration of Geminin from Cdt1, a key component in the assembly of the prereplication complex. Our results suggest that Lhx2 may mediate an alternative or parallel pathway for control of cellular proliferation in the developing forebrain via Six3. [Copyright &y& Elsevier]

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

Liver development is based on reciprocal interactions between ventral foregut endoderm and adjacent mesenchymal tissues. Targeted disruption of the LIM-homeobox gene Lhx2 has revealed that it is important for the expansion of the liver during embryonic development, whereas it appears not to be involved in the induction of hepatic fate. It is not known whether Lhx2 is expressed in the endodermal or mesenchymal portion of the liver, or if the cells normally expressing Lhx2 are absent or present in the liver of Lhx2-/- embryos. To address this we have analyzed gene expression from the Lhx2 locus during hepatic development in wild type and Lhx2-/- mice. Lhx2 is expressed in cells of the septum transversum mesenchyme adjacent to the liver bud from embryonic day 9. The hepatic cords subsequently migrate into and intermingle with the Lhx2+ cells of the septum transversum mesenchyme. Lhx2 expression is thereafter maintained in a subpopulation of mesenchymal cells in the liver until adult life. In adult liver the Lhx2+ mesenchymal cells co-express desmin, a marker associated with stellate cells. At embryonic day 10.5, cells expressing the mutant Lhx2 allel are present in Lhx2-/- livers, and expression of Hlx, hepatocyte growth factor, Hex and Prox1, genes known to be important in liver development, is independent of functional Lhx2 expression. Thus, Lhx2 is specifically expressed in the liver-associated septum transversum mesenchyme that subsequently becomes an integral part of the liver and the formation of these mesenchymal cells does not require functional Lhx2. [Copyright &y& Elsevier]

The expression pattern of Lmx1a, a LIM-homeodomain gene disrupted in the dreher mouse neurological mutant, is described during development. Lmx1a is predominantly expressed in the developing nervous system from embryonic day E8.5 to adulthood, in restricted areas. Major expression domains include the dorsal midline (roof plate) of the neural tube, the cortical hem, the otic vesicles, the developing cerebellum and the notochord. The Lmx1a expression pattern is therefore well correlated with the various aspects of the phenotype of the dreher mutant mice. [Copyright &y& Elsevier]

Nasopharyngeal carcinoma (NPC) is a malignant tumor in the nasopharyngeal cavity. LncRNA PTPRG-AS1 is essential in NPC radiosensitivity. This study sought to explore the mechanism of PTPRG-AS1 in NPC radiosensitivity by regulating the miR-124-3p/LHX2 axis. First, NPC-related microarray was analyzed to screen differentially expressed lncRNAs. PTPRG-AS1 and miR-124-3p expression patterns in NPC tissues and adjacent tissues of NPC patients and NPC cell lines were detected by RT-qPCR. PTPRG-AS1 was knocked down in CNE2 and 5-8 F cells by transfection. The radiosensitivity, proliferation and apoptosis before and after radiotherapy (0/6 Gy) were detected by cloning formation assay, CCK-8 assay, and flow cytometry. Bioinformatics, Pearson correlation analysis, RNA pull-down, and luciferase reporter assays were performed to explore the regulatory relationship of the lncRNA PTPRG-AS1/miR-124-3/LHX2 axis. The corresponding functions were verified in the complementation test. The levels of LHX2 and Notch pathway-related proteins were detected by Western blot. PTPRG-AS1 was upregulated in NPC cell lines and tissues. PTPRG-AS1 knockdown decreased NPC cell proliferation and promoted radiotherapy-induced apoptosis and cell radiosensitivity. PTPRG-AS1 upregulated LHX2 as a ceRNA of miR-124-3p. miR-124-3p inhibition partially reversed PTPRG-AS1 silencing-induced NPC cell radiosensitivity. miR-124-3p targeted LHX2. LHX2 overexpression attenuated the miR-124-3p overexpression-induced NPC cell radiosensitivity. LHX2 attenuated NPC cell radiosensitivity by activating the Notch pathway. Briefly, lncRNA PTPRG-AS1 reduced NPC cell radiosensitivity by regulating the miR-124-3p/LHX2 axis through the ceRNA mechanism.

Introduction: Hepatocellular carcinoma and hepatoblastoma are two liver cancers characterized by gene deregulations, chromosomal rearrangements, and mutations in Wnt/beta-catenin (Wnt) pathway-related genes. LHX2, a transcriptional factor member of the LIM homeobox gene family, has important functions in embryogenesis and liver development. LHX2 is oncogenic in many solid tumors and leukemia, but its role in liver cancer is unknown., Methods: We analyzed the expression of LHX2 in hepatocellular carcinoma and hepatoblastoma samples using various transcriptomic datasets and biological samples. The role of LHX2 was studied using lentiviral transduction, in vitro cell-based assays (growth, migration, senescence, and apoptosis), molecular approaches (phosphokinase arrays and RNA-seq), bioinformatics, and two in vivo models in chicken and Xenopus embryos., Results: We found a strong connection between LHX2 downregulation and Wnt activation in these two liver cancers. In hepatoblastoma, LHX2 downregulation correlated with multiple poor outcome parameters including higher patient age, intermediate- and high-risk tumors, and low patient survival. Forced expression of LHX2 reduced the proliferation, migration, and survival of liver cancer cells in vitro through the inactivation of MAPK/ERK and Wnt signals. In vivo, LHX2 impeded the development of tumors in chick embryos and repressed the Wnt pathway in Xenopus embryos. RNA-sequencing data and bioinformatic analyses confirmed the deregulation of many biological functions and molecular processes associated with cell migration, cell survival, and liver carcinogenesis in LHX2-expressing hepatoma cells. At a mechanistic level, LHX2 mediated the disassembling of beta-catenin/T-cell factor 4 complex and induced expression of multiple inhibitors of Wnt (e.g., TLE/Groucho) and MAPK/ERK (e.g., DUSPs) pathways., Conclusion: Collectively, our findings demonstrate a tumor suppressive function of LHX2 in adult and pediatric liver cancers., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 by S. Karger AG, Basel.)

Simple Summary: Cashmere goats are the most important goat breed due to the high yield and fineness of the fibers that they produce. Cashmere fiber is a luxury product since it is soft, light and warm. The development of this fiber depends on the hair follicle (HF) cyclical activity, which is characterized by the succession of growth and regressive phases. In the transition between telogen and anagen phases, many growth factors work to activate the HF stem cells and to allow the growth of a new cashmere fiber. As several factors involved in the stem cell activation, Platelet-Derived Growth Factor A (PDGFA), Bone Morphogenetic Protein 2 (BMP2) and Lim-Homeobox gene 2 (LHX2) were analyzed in this work to evaluate their activity during the cashmere HF cycle. These molecules were studied using different approaches and finally, PDGFA and BMP2 appeared to have higher levels of expression during the cycle activation phase with respect to the LHX2, which suggests that they play a main role in the development of a new cashmere fiber. The obtained data will improve the knowledge of the HF cycle in the cashmere goat and they could be a useful tool for improving cashmere fiber production. The cashmere hair follicle (HF) perpetually goes through cycles of growth, involution and rest. The photoperiod is the main factor in the control of seasonal coat change in cashmere goats while stem cells play a crucial role in the HF growth. Several factors, including Platelet-Derived Growth Factor A (PDGFA), Bone Morphogenetic Protein 2 (BMP2) and Lim-Homeobox gene 2 (LHX2) are implicated in HF morphogenesis and cycle. In this work, the mentioned molecules were investigated to evaluate their role in follicular cycle activation. The study was performed on skin samples collected at different periods of HF cycle and the molecular expression of PDGFA, BMP2 and LHX2 was evaluated by Real-Time PCR (qPCR) at each time point. Since PDGFA showed the most variation, the goat PDGFA gene was sequenced and the protein localization was investigated by immunohistochemistry together with PDGF receptor α (PDGFRα). PDGFA immunostaining was observed in the basal layer of the HF outer root sheath and the immunoreaction appeared stronger in the regressive HFs compared to those in the anagen phase according to qPCR analysis. PDGFRα was observed in the HF epithelium, proving the effect of PDGFA on the follicular structure. The data obtained suggest that PDGFA and BMP2 are both implicated in HF cycle in goat. In particular, PDGFA secreted by the HF is involved in the anagen activation. [ABSTRACT FROM AUTHOR]

The transcript factor LHX2 is dysregulated in many cancers but its role in osteosarcoma (OS) remains unclear. In this study, we confirm that LHX2 is up-regulated in osteosarcoma, and that its silencing inhibits OS malignancy and induces autophagy via mTOR signaling. We further demonstrate that miR-129-5p negatively regulates LHX2 and suppresses the malignant phenotypes of OS. LHX2 overexpression could restore the malignant phenotypes. In conclusion, LHX2 regulates tumorigenesis and autophagy via mTOR in OS and is negatively regulated by miR-129-5p. Targeting the miR-129-5p/LHX2/mTOR axis therefore represents a novel therapeutic strategy for OS treatment.

The terminal nerve ganglion (TNG) is a well-known structure of the peripheral nervous system in cartilaginous and teleost fishes. It derives from the olfactory placode during embryonic development. While the differentiation and migration of gonadotropin releasing hormone (GnRH)-expressing neurons from the olfactory placode has been well documented, the TNG has been neglected in birds and mammals, and its development is less well described. Here we describe the formation of a ganglion-like structure from migratory olfactory placodal cells in chicken. The TNG is surrounded by neural crest cells, but in contrast to other cranial sensory ganglia, we observed no neural crest corridor, and olfactory unsheathing cells appear only after the onset of neuronal migration. We identified Isl1 and Lhx2 as two transcription factors that label neuronal subpopulations in the forming TNG, distinct from GnRH1 + cells, thereby revealing a diversity of cell types during the formation of the TNG. We also provide evidence for extensive apoptosis in the terminal nerve ganglion shortly after its formation, but not in other cranial sensory ganglia. Moreover, at later stages placode-derived neurons expressing GnRH1, Isl1 and/or Lhx2 become incorporated in the telencephalon. The integration of TNG neurons into the telencephalon together with the earlier widespread apoptosis in the TNG might be an explanation why the TNG in mammals and birds is much smaller compared to other vertebrates., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The Wnt- and BMP-rich cortical hem has been demonstrated to be critical for the pattern formation of the telencephalon, and it is particularly important for the induction of the hippocampus. Meanwhile, the cortical hem is one of the sources of Cajal-Retzius cells. Many Cajal-Retzius cells are produced in the hem and populated to the media-caudal surface of the telencephalon. However, the mechanism of the maintenance of the hem remain unclear. In this study, we generated a transgenic mouse line CAG-loxp-stop-loxp-Foxg1-IRES-EGFP. By crossing Fzd10CreERTM with this line, combined with tamoxifen induction, Foxg1 was ectopically expressed in the hem from embryonic day 10.5 (E10.5) onwards. We have found the hem-derived Cajal-Retzius cells were transformed into dentate granule neurons accompanied with ectopic expression of Lhx2. However, the morphology of the hem displayed no obvious changes. The hem specific markers, Wnt3a and Wnt2b, were slightly downregulated. Our results indicate that Foxg1 is sufficient to induce the expression of Lhx2 in the dorsal part of the hem. The ectopic Lhx2 and decreased Wnt signals may both contribute to the cell fate switch. Our study provides new insight into the mechanism underlying the maintenance of the hem. [ABSTRACT FROM AUTHOR]