LIM Homeodomain Research Articles

MiRNA29a-3p negatively regulates ISL1–Integrin β1 axis to suppress gastric cancer progression

Insulin gene enhancer protein 1 (ISL1) belongs to the LIM homeodomain transcription factor family, which is closely related to the development of several cancers. We previously found that abnormally high ISL1 expression is involved in gastric cancer (GC) metastasis. However, the specific role of ISL1 and its regulatory mechanisms in GC metastasis warrant elucidation. In this study, we found that ISL1 is highly expressed in GC tissues and positively correlated with GC development, promoting cell migration and invasion in vivo and in vitro. Moreover, miRNA29a-3p can target ISL1 and thus inhibit GC cell migration. Furthermore, ISL1 upregulates ITGB1 by binding to its enhancer; nevertheless, ISL1–ITGB1 axis expression can be regulated using miRNA29a-3p. In GC cell nuclei, ISL1 and annexin A2 (ANXA2) form a transcriptional activator complex at the ITGB1 enhancer, thus promoting ITGB1 expression. In GC cell cytoplasm, the ISL1–ANXA2 complex synergistically activates matrix metalloproteinases, thus promoting cell migration. In conclusion, ISL1 is a potential therapeutic target for GC.

ETMR-24. SOX2 INFLUENCES IDENTITY OF GLIA-LIKE PROGENITORS AND TUMORIGENESIS IN CHOROID PLEXUS THROUGH LIM HOMEODOMAIN TRANSCRIPTION FACTORS

Abstract Choroid plexus (CP) tumors encompass a spectrum of rare brain neoplasms ranging from benign papilloma (CPP) to aggressive carcinoma (CPC) that collectively affect the pediatric population. Poor outcomes for high-grade lesions and side effects of current treatment paradigms underscore a critical need to develop safer and more efficacious therapies guided by biological understanding of these tumors. We developed mouse models from common molecular defects found in human disease. These models exploit sustained NOTCH signaling activation in the hindbrain upper rhombic lip to generate CPP and CPC. To delineate cellular composition of CP tumors, we conducted snRNA-seq in murine CP tumors. Similar to adult CP, cells in NOTCH-driven CPP partitioned into clusters identified as epithelial, mesenchymal, endothelial, immune, neuronal, and glial cells. Though tumor cells are confined to epithelial compartment based on inferCNV analysis of snRNA-seq data, they are derived from glia-like progenitors. Tumor cells are in a distinct differentiation state characterized by a lack of epithelial maturation, and aberrant expression of markers of neural progenitor/stem-like cells including SRY-Box Transcription Factor 2 (SOX2). Consistently, SOX2 inactivation decreased tumor cell proliferation and blocked NOTCH-driven CP tumor formation. ChIP-seq studies revealed SOX2 binding to promoters of LIM homeodomain transcription factors Lmx1a and Lmx1b. Enforced SOX2 expression activated the expression Lmx1a and Lmx1b, whereas Sox2 loss reduced their expressions in tumor cells. Importantly, knockdown of Lmx1a or Lmx1b decreased tumor cell proliferation, whereas overexpression of Lmx1a/b rescued proliferation of tumor cells subjected to SOX2 suppression. Contextualizing these results in CP development, SOX2 critically maintains progenitor cell identity through regulating Lmx1a/b expression in the rhombic lip. Finally, analysis of human CP tumor samples revealed abnormal expressions of SOX2 and LMX1A. Together, these results indicate that oncogenic signaling subverts SOX2 transcriptional programs that specify normal progenitor identity and lock tumor cells in unique differentiation state to facilitate CP tumor growth.

Structural insights into the recognition of the A/T-rich motif in target gene promoters by the LMX1a homeobox domain.

LIM homeodomain transcription factor 1-alpha (LMX1a) is a neuronal lineage-specific transcription activator that plays an essential role during the development of midbrain dopaminergic (mDA) neurons. LMX1a induces the expression of multiple key genes, which ultimately determine the morphology, physiology, and functional identity of mDA neurons. This function of LMX1a is dependent on its homeobox domain. Here, we determined the structures of the LMX1a homeobox domain in complex with the promoter sequences of the Wnt family member 1 (WNT1) or paired like homeodomain 3 (Pitx3) gene, respectively. The complex structures revealed that the LMX1a homeobox domain employed its α3 helix and an N-terminal loop to achieve specific target recognition. The N-terminal loop (loop1) interacted with the minor groove of the double-stranded DNA (dsDNA), whereas the third α-helix (α3) was tightly packed into the major groove of the dsDNA. Structure-based mutations in the α3 helix of the homeobox domain significantly reduced the binding affinity of LMX1a to dsDNA. Moreover, we identified a nonsyndromic hearing loss (NSHL)-related mutation, R199, which yielded a more flexible loop and disturbed the recognition in the minor groove of dsDNA, consistent with the molecular dynamics (MD) simulations. Furthermore, overexpression of Lmx1a promoted the differentiation of SH-SY5Y cells and upregulated the transcription of WNT1 and PITX3 genes. Hence, our work provides a detailed elucidation of the specific recognition between the LMX1a homeobox domain and its specific dsDNA targets, which represents valuable information for future investigations of the functional pathways that are controlled by LMX1a during mDA neuron development.

LIM-HD transcription factors control axial patterning and specify distinct neuronal and intestinal cell identities in planarians.

Adult planarians can regenerate the gut, eyes and even a functional brain. Proper identity and patterning of the newly formed structures require signals that guide and commit their adult stem cells. During embryogenesis, LIM-homeodomain (LIM-HD) transcription factors act in a combinatorial 'LIM code' to control cell fate determination and differentiation. However, our understanding about the role these genes play during regeneration and homeostasis is limited. Here, we report the full repertoire of LIM-HD genes in Schmidtea mediterranea. We found that lim homeobox (lhx) genes appear expressed in complementary patterns along the cephalic ganglia and digestive system of the planarian, with some of them being co-expressed in the same cell types. We have identified that Smed-islet1, -lhx1/5-1, -lhx2/9-3, -lhx6/8, -lmx1a/b-2 and -lmx1a/b-3 are essential to pattern and size the planarian brain as well as for correct regeneration of specific subpopulations of dopaminergic, serotonergic, GABAergic and cholinergic neurons, while Smed-lhx1/5.2 and -lhx2/9.2 are required for the proper expression of intestinal cell type markers, specifically the goblet subtype. LIM-HD are also involved in controlling axonal pathfinding (lhx6/8), axial patterning (islet1, lhx1/5-1, lmx1a/b-3), head/body proportions (islet2) and stem cell proliferation (lhx3/4, lhx2/9-3, lmx1a/b-2, lmx1a/b-3). Altogether, our results suggest that planarians might present a combinatorial LIM code that controls axial patterning and axonal growing and specifies distinct neuronal and intestinal cell identities.

Regulation of circulating thyroid hormone levels by hypothalamic tanycytes and hypophysial pars tuberalis-specific cells and their morphological and gene- and protein-expression changes under different photoperiods.

Thyroid hormone in the hypothalamus acts as a key determinant of seasonal transitions. Thyroid hormone-levels in the brain are mainly regulated by the hypothalamic tanycytes and pituitary pars tuberalis (PT)-specific cells. TSHβ produced by the PT-specific cells stimulates Dio2 expression and decreases Dio3 expression of the tanycytes. Both tanycytes and PT-specific cells in photosensitive animals exhibit remarkable changes of morphological appearance and expressions of genes and proteins under different photoperiods. Long photoperiods induce increased gene- and protein-expressions and active features. Short photoperiods cause the decreased gene- and protein-expressions and inactive features. In the PT, expressions of TSHβ, common α-subunit of glycoprotein hormones (α-GSU), and MT1 receptor of melatonin receptors and eyes absent 3 change under different photoperiods. Diurnal rhythms of α-GSU mRNA expression are observed in the PT of Djungarian hamsters. Hes1, Nkx2.1, and LIM homeodomain gene 2 (Lhx2) are involved in the differentiation of PT. In the hypothalamic tanycytes, expressions of Dio2, Dio3, vimentin, serine/threonine kinase 33, GPR50, Nestin, Retinoid signaling genes (retinaldehyde dehydrogenase 1, cellular retinol binding protein 1, and Stra6), monocarboxylate transporter 8, and neural cell adhesion molecule change under different photoperiods. Rax, Lhx2, Nfia/b/x, and fibroblast growth factor 10 are involved in the differentiation of tanycytes.

Impact of Drosophila LIM homeodomain protein Apterous on the morphology of the adult mushroom body

A LIM homeodomain transcription factor Apterous (Ap) regulates embryonic and larval neurodevelopment in Drosophila. Although Ap is still expressed in the adult brain, it remains elusive whether Ap is involved in neurodevelopmental events in the adult brain because flies homozygous for ap mutations are usually lethal before they reach the adult stage. In this study, using adult escapers of ap knockout (KO) homozygotes, we examined whether the complete lack of ap expression affects the morphology of the mushroom body (MB) neurons and Pigment-dispersing factor (Pdf)-positive clock neurons in the adult brain. Although ap KO escapers showed severe structural defects of MB neurons, no clear morphological defects were found in Pdf-positive clock neurons. These results suggest that Ap in the adult brain is essential for the neurodevelopment of specific ap-positive neurons, but it is not necessarily involved in the development of all ap-positive neurons.

MBL-1 and EEL-1 affect the splicing and protein levels of MEC-3 to control dendrite complexity.

Transcription factors (TFs) play critical roles in specifying many aspects of neuronal cell fate including dendritic morphology. How TFs are accurately regulated during neuronal morphogenesis is not fully understood. Here, we show that LIM homeodomain protein MEC-3, the key TF for C. elegans PVD dendrite morphogenesis, is regulated by both alternative splicing and an E3 ubiquitin ligase. The mec-3 gene generates several transcripts by alternative splicing. We find that mbl-1, the orthologue of the muscular dystrophy disease gene muscleblind-like (MBNL), is required for PVD dendrite arbor formation. Our data suggest mbl-1 regulates the alternative splicing of mec-3 to produce its long isoform. Deleting the long isoform of mec-3(deExon2) causes reduction of dendrite complexity. Through a genetic modifier screen, we find that mutation in the E3 ubiquitin ligase EEL-1 suppresses mbl-1 phenotype. eel-1 mutants also suppress mec-3(deExon2) mutant but not the mec-3 null phenotype. Loss of EEL-1 alone leads to excessive dendrite branches. Together, these results indicate that MEC-3 is fine-tuned by alternative splicing and the ubiquitin system to produce the optimal level of dendrite branches.

The Roles of Histamine Receptor 1 (hrh1) in Neurotransmitter System Regulation, Behavior, and Neurogenesis in Zebrafish

Histamine receptors mediate important physiological processes and take part in the pathophysiology of different brain disorders. Histamine receptor 1 (HRH1) is involved in the development of neurotransmitter systems, and its role in neurogenesis has been proposed. Altered HRH1 binding and expression have been detected in the brains of patients with schizophrenia, depression, and autism. Our goal was to assess the role of hrh1 in zebrafish development and neurotransmitter system regulation through the characterization of hrh1−/− fish generated by the CRISPR/Cas9 system. Quantitative PCR, in situ hybridization, and immunocytochemistry were used to study neurotransmitter systems and genes essential for brain development. Additionally, we wanted to reveal the role of this histamine receptor in larval and adult fish behavior using several quantitative behavioral methods including locomotion, thigmotaxis, dark flash and startle response, novel tank diving, and shoaling behavior. Hrh1−/− larvae displayed normal behavior in comparison with hrh1+/+ siblings. Interestingly, a transient abnormal expression of important neurodevelopmental markers was evident in these larvae, as well as a reduction in the number of tyrosine hydroxylase 1 (Th1)–positive cells, th1 mRNA, and hypocretin (hcrt)-positive cells. These abnormalities were not detected in adulthood. In summary, we verified that zebrafish lacking hrh1 present deficits in the dopaminergic and hypocretin systems during early development, but those are compensated by the time fish reach adulthood. However, impaired sociability and anxious-like behavior, along with downregulation of choline O-acetyltransferase a and LIM homeodomain transcription factor Islet1, were displayed by adult fish.

ISL1 regulates lung branching morphogenesis via Shh signaling pathway

Lung branching morphogenesis relies on a complex coordination of multiple signaling pathways and transcription factors. Here, we found that ablation of the LIM homeodomain transcription factor Islet1 (Isl1) in lung epithelium resulted in defective branching morphogenesis and incomplete formation of five lobes. A reduction in mesenchymal cell proliferation was observed in Isl1ShhCre lungs. There was no difference in apoptosis between the wild-type (ShhCre) and Isl1ShhCre embryos. RNA-Seq and in situ hybridization analysis showed that Shh, Ptch1, Sox9, Irx1, Irx2, Tbx2, and Tbx3 were downregulated in the lungs of Isl1ShhCre embryos. ChIP assay implied the Shh gene served as a direct target of ISL1, since the transcription factor ISL1 could bind to the Shh epithelial enhancer sequence (MACS1). Also, activation of the Hedgehog pathway via ectopic gene expression rescued the defects caused by Isl1 ablation, confirming the genetic integration of Hedgehog signaling. In conclusion, our works suggest that epithelial Isl1 regulates lung branching morphogenesis through administrating the Shh signaling mediated epithelial-mesenchymal communications.

RARE-06. DISSECTING CELLULAR DIFFERENTIATION HETEROGENEITY IN CHOROID PLEXUS CARCINOMA PATHOGENESIS

Abstract Choroid plexus (CP) neoplasms are rare, predominantly pediatric, brain tumors ranging from benign CP papilloma to aggressive CP carcinoma (CPC) associated with poor survival. Survivors often experience devastating side effects from current treatment strategies. There is an urgent need for safer, more effective therapies for CPC. A subset of human CPC exhibits abnormal NOTCH activity, whereas mutations of tumor suppressor gene TP53 are detected in >50% of CPC and are associated with increased genetic tumor instability and worse prognosis. CPC is one hallmark of Li-Fraumeni syndrome (LFS), a rare genetic disorder associated with germline TP53 mutations. Human CP tumors also frequently display aberrant expression of transcriptional regulators of roof plate/CP differentiation, including LIM homeodomain transcription factors LMX1A, and SRY-Box Transcription Factor 2 (SOX2). To investigate the role of transcriptional regulation in CP tumorigenesis, animal models were developed driven by molecular defects commonly found in CP tumors in humans. Accordingly, sustained NOTCH and Sonic Hedgehog (SHH) activation or combined deletion of Rb1 and Trp53 tumor suppressors led to malignant CP tumors with characteristics of CPC in humans. NOTCH-driven CP tumors arose from monociliated roof plate progenitors and exhibited increased levels of Lmx1a, Lmx1b, and Sox2. In contrast, their expression was significantly reduced, while OTX2 levels were markedly increased in Rb1/Trp53-deficient CPC. Consistently, single-cell transcriptomics data show a proliferative Otx2+ tumor bulk and a small Lmx1a+/Sox2+ population in Rb1/Trp53-deficient CPC. Importantly, deletion of Sox2 in Lmx1a+ progenitors blocked NOTCH-driven CP tumor and reduced Lmx1a/b expression. Thus, LMX1A and SOX2 expression marks tumor-initiating cells, while OTX2 labels differentiated tumor cells in CPC. Together, our studies revealed intra- and inter-tumoral heterogeneity in CPC driven by distinct oncogenic signals. Knowledge of the molecular and cellular basis of heterogeneity in CP tumor development may facilitate the development of innovative diagnostic and therapeutic strategies in CPC.

LC3B is a cofactor for LMX1B-mediated transcription of autophagy genes in dopaminergic neurons.

It is becoming increasingly clear that the Atg8 family of autophagy proteins have roles not only in the cytoplasm, but also in the cell nucleus. In this issue, Jiménez-Moreno et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.201910133) report that nuclear LC3B binds to the LIM homeodomain transcription factor LMX1B and acts as a cofactor for LMX1B-mediated transcription of autophagy genes, providing stress protection and ensuring survival of midbrain dopaminergic neurons.

ATG8-dependent LMX1B-autophagy crosstalk shapes human midbrain dopaminergic neuronal resilience.

The LIM homeodomain transcription factors LMX1A and LMX1B are essential mediators of midbrain dopaminergic neuronal (mDAN) differentiation and survival. Here we show that LMX1A and LMX1B are autophagy transcription factors that provide cellular stress protection. Their suppression dampens the autophagy response, lowers mitochondrial respiration, and elevates mitochondrial ROS, and their inducible overexpression protects against rotenone toxicity in human iPSC-derived mDANs in vitro. Significantly, we show that LMX1A and LMX1B stability is in part regulated by autophagy, and that these transcription factors bind to multiple ATG8 proteins. Binding is dependent on subcellular localization and nutrient status, with LMX1B interacting with LC3B in the nucleus under basal conditions and associating with both cytosolic and nuclear LC3B during nutrient starvation. Crucially, ATG8 binding stimulates LMX1B-mediated transcription for efficient autophagy and cell stress protection, thereby establishing a novel LMX1B-autophagy regulatory axis that contributes to mDAN maintenance and survival in the adult brain.

Role of miR-128/216a Regulating Isl1 Expression during Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Insulin-Producing Cells

Islet-1 (Isl1), a LIM homeodomain protein, is expressed in the embryonic pancreatic epithelium. As a key transcription factor, Isl1 can not only regulate insulin gene expression in normal glucose condition but also maintain β-cell function and impact pancreatic β-cell target genes. Some experiments have suggested that MicroRNA (miRNA) can play a critical role during the induction of insulin-producing cells (IPCs). However, it is unclear whether miRNA may regulate Isl1 expression during differentiation of human umbilical cord mesenchymal stem cells (HUMSCs) into IPCs. In this investigation, we induced HUMSCs into IPCs with a modified two-step protocol, activin A, retinoic acid (step1) and conophylline, nicotinamide (step2). To find the miRNA regulating Isl1 expression, we respectively used TargetScan, miRDB and RNAhybrid to predict and got the result, miR-128 and miR-216a. The miRNAs can inhibit Isl1 expression by dual luciferase assay. The results of real-time Polymerase Chain Reaction (PCR) showed that Isl1 expression level was almost reciprocal to that of miR-128 and miR-216a during differentiation of HUMSCs into IPCs. Furthermore, over-expression of miR-128 or miR-216a down-regulated expression levels of Isl1 and MafA. Therefore, miR-128 or miR-216a may regulate expression of islet-specific transcription factors to control differentiation of HUMSCs into IPCs.

Neurog2 regulates Isl1 to modulate horizontal cell number.

The population sizes of different retinal cell types vary between different strains of mice, and that variation can be mapped to genomic loci in order to identify its polygenic origin. In some cases, controlling genes act independently, whereas in other instances, they exhibit epistasis. Here, we identify an epistatic interaction revealed through the mapping of quantitative trait loci from a panel of recombinant inbred strains of mice. The population of retinal horizontal cells exhibits a twofold variation in number, mapping to quantitative trait loci on chromosomes 3 and 13, where these loci are shown to interact epistatically. We identify a prospective genetic interaction underlying this, mediated by the bHLH transcription factor Neurog2, at the chromosome 3 locus, functioning to repress the LIM homeodomain transcription factor Isl1, at the chromosome 13 locus. Using single and double conditional knockout mice, we confirm the countervailing actions of each gene, and validate in vitro a crucial role for two single nucleotide polymorphisms in the 5'UTR of Isl1, one of which yields a novel E-box, mediating the repressive action of Neurog2.

Intracellular Ca2+ dynamics in the ALA neuron reflect sleep pressure and regulate sleep in Caenorhabditis elegans

SummaryThe mechanisms underlying sleep homeostasis are poorly understood. The nematode Caenorhabditis elegans exhibits 2 types of sleep: lethargus, or developmentally timed, and stress-induced sleep. Lethargus is characterized by alternating cycles of sleep and motion bouts. Sleep bouts are homeostatically regulated, i.e., prolonged active bouts lead to prolonged sleep bouts. Here we reveal that the interneuron ALA is crucial for homeostatic regulation during lethargus. Intracellular Ca2+ in ALA gradually increased during active bouts and rapidly decayed upon transitions to sleep bouts. Longer active bouts were accompanied by higher intracellular Ca2+ peaks. Optogenetic activation of ALA during active bouts caused transitions to sleep bouts. Dysfunction of CEH-17, which is an LIM homeodomain transcription factor selectively expressed in ALA, impaired the characteristic patterns of ALA intracellular Ca2+ and abolished the homeostatic regulation of sleep bouts. These findings indicate that ALA encodes sleep pressure and contributes to sleep homeostasis.

Activation of PI3K/p110α in the Lung Mesenchyme Affects Branching Morphogenesis and Club Cell Differentiation.

Epithelial–mesenchymal interaction is required for normal growth, morphogenetic patterning, and cellular differentiation in developing lungs. Various signaling pathways have been defined in establishing the patterning of this branched organ. The phosphoinositide-3-kinase (PI3K) signaling plays an important role in disease pathogenesis but remains largely uncharacterized in embryonic development. In this study, we activated a specific catalytic subunit of PI3K catalytic enzymes, Class IA p110α (p110α), in the embryonic lung mesenchyme using the Dermo1-Cre mouse. Activation of p110α promoted branching morphogenesis and blocked club cell differentiation in both proximal and distal airways. Mechanistically, the LIM homeodomain gene Islet-1 (Isl1), fibroblast growth factor 10 (Fgf10), and SRY (sex-determining region Y)-box9 (Sox9) were found to be downstream targets of p110α. The significantly increased expressions of Isl1, Fgf10, and Sox9 resulted in the stimulation of branching in mutant lungs. Activation of p110α-mediated signaling also increased the expression of phosphatase and tensin homolog deleted on chromosome 10 (Pten) and hairy/enhancer of split 1 (Hes1), which in turn blocked club cell differentiation. Thus, the signaling pathway by which PI3K/p110α-regulated epithelial–mesenchymal interactions may entail Isl1–Fgf10–Sox9 and Pten–Hes1 networks, which consequently regulate branching morphogenesis and club cell differentiation, respectively.

Regulation of LIM homeodomain 2 (Lhx2) in the distal limb bud during patterning and development

During limb morphogenesis, fibroblast growth factors (Fgfs) secreted from the apical ectodermal ridge (AER) direct proximodistal outgrowth of the limb, while sonic hedgehog (Shh) released from the zone of polarizing activity modulate anteroposterior expansion of the limb. Fgfs and Shh maintain each other’s expression through an autoregulatory loop. The transcription factor LIM homeodomain 2 (Lhx2) is expressed in the distal sub‐AER mesoderm and is important for Shh expression. We have identified a chicken enhancer upstream of the LHX2 promoter that is active in the distal sub‐AER LHX2 expression domain that contains putative transcription factor binding sites for known downstream targets of FGF signaling that are critical for activity. Therefore, we hypothesized that LASARM1 interacts with the LHX2 promoter and that FGF works through LASARM1 to regulate LHX2 expression. To determine this, we performed chromosome conformation capture (3C) on cells isolated from distal chicken limb buds and compared them to non‐LHX2 expressing tail cells. Promoter and enhancer specific primers were used to isolate ligated promoter‐enhancer fragments by end‐point PCR. Amplified sequences were then sequence validated. A 3C ligation product library was used as a control. We also evaluated the role of FGF to regulate LASARM1 activity in subAER mesoderm by analyzing LASARM1 activity following inhibition of FGF signaling by an FGF‐receptor inhibitor. End‐point PCR revealed promoter‐enhancer ligated amplicons in distal limb bud cells, but not in tail cells. DNA sequencing of enhancer‐ligated fragments confirmed LHX2‐LASARM1 fusion fragments for distal limb bud cells. Interestingly, disruption of FGF signaling via a specific FGF receptor inhibitor did not alter LASARM1 activity, although LHX2 expression was markedly reduced. These findings suggest that LASARM1 interacts with the LHX2 promoter during limb patterning, but additional enhancers are required to mediate the FGF‐mediated up‐regulation of LHX2. Evaluation of additional LHX2 enhancers is underway. Investigations regarding the relationship between FGF and LHX2 will provide important insights into the mechanisms underlying LHX2 regulation and the FGF‐to‐SHH reciprocal loop.

LHX2 Regulates Shh Expression in the Limb Independent of the ZPA Regulatory Sequence

During limb development, proximal‐distal patterning is controlled by fibroblast growth factors (FGFs) secreted from the apical ectodermal ridge (AER), while anterior‐posterior expansion is coordinated by sonic hedgehog (SHH) released from the zone of polarizing activity (ZPA). Both FGFs and SHH regulate each other through an autoregulatory loop. The transcription factor LIM homeodomain 2 (LHX2) is an intermediate of the Fgf‐to‐Shh portion of the loop. In the absence of Lhx2 (and its redundant family member, Lhx9), Shh expression is markedly reduced. However, the mechanism by which LHX2 mediates FGF‐regulation of Shh is unclear. Shh is regulated by a limb‐specific enhancer called the ZPA Regulatory Sequence (ZRS). Using in silicoanalysis, our lab has identified two putative LHX2 binding sites within the ZRS. We hypothesized that LHX2 regulates Shh expression directly via the ZRS. To determine if LHX2 binding sites were necessary for ZRS activity, we mutated both LHX2 binding sites within a control tk‐ZRS‐eGFP reporter construct using site‐directed mutagenesis. We electroporated these constructs into the presumptive limb buds of chicken embryos (Hamburger‐Hamilton stage 14) and assessed for changes in ZRS activity in the limb buds 48 hours post transfection via fluorescence microscopy. We found that the mutation of both LHX2 binding sites within the ZRS resulted in activity similar to our control. These results suggest that LHX2 may not bind directly to the ZRS in its regulation of Shh. Instead, LHX2 may use another pathway to regulate Shhexpression. Further experiments are needed to confirm this connection. Investigating the mechanism by which LHX2 regulates Shh will provide insight into how LHX2 contributes to coordinated patterned‐limb development.

Alternative LIM homeodomain splice variants are dynamically regulated at key developmental steps in vertebrates.

BackgroundAlternative splicing provides a broad strategy to amplify the genome. Yet how alternative splicing influences neurodevelopment or indeed which variants are translated at developmental choice points remains poorly explored. Here we focused on a gene important for neurodevelopment, the Lim homeodomain transcription factor, Lhx9. Lhx9 has two noncanonical splice variants, Lhx9a and Lhx9b which compared with the canonical variant Lhx9c have a truncated homeodomain and an alternative C‐terminal sequence, suggesting that, if translated, these variants could differently impact on cellular function.ResultsWe created a unique antibody tool designed to selectively detect noncanonical Lhx9 variants (Lhx9ab) and used this to examine the protein expression dynamics in embryos. Lhx9ab variants were translated and dynamically expressed similarly between mouse and chicken at key developmental choice points in the spinal cord, limbs and urogenital ridge. Within the spinal cord, enrichment of Lhx9c vs Lhx9ab expression was observed during key migration and axonal projection choice points.ConclusionsThese data support the notion that the expression dynamics between canonical and noncanonical Lhx9 variants could play an important role in spinal neuron maturation. More broadly, determining the temporal dynamics of alternative protein variants is a key entry point to understand how splicing influences developmental processes.

Synergistic effects of ISL1 and KDM6B on non-alcoholic fatty liver disease through the regulation of SNAI1

BackgroundThe increasing incidence of non-alcoholic fatty liver disease (NAFLD) has been reported worldwide, which urges understanding of its pathogenesis and development of more effective therapeutical methods for this chronic disease. In this study, we aimed to investigate the effects of a LIM homeodomain transcription factor, islet1 (ISL1) on NAFLD.MethodsMale C57BL/6J mice were fed with a diet high in fat content to produce NAFLD models. These models were then treated with overexpressed ISL1 (oe-ISL1), oe-Lysine-specific demethylase 6B (KDM6B), oe-SNAI1, or short hairpin RNA against SNAI1. We assessed triglyceride and cholesterol contents in the plasma and liver tissues and determined the expressions of ISL1, KDM6B and SNAI1 in liver tissues. Moreover, the in vitro model of lipid accumulation was constructed using fatty acids to explore the in vitro effect of ISL1/KDM6B/SNAI1 in NAFLD.ResultsThe results showed that the expressions of ISL1, KDM6B, and SNAI1 where decreased, but contents of triglyceride and cholesterol increased in mice exposed to high-fat diet. ISL1 inhibited lipogenesis and promoted lipolysis and exhibited a synergizing effect with KDM6B to upregulate the expression of SNAI1. Moreover, both KDM6B and SNAI1 could inhibit lipogenesis and induce lipolysis. Importantly, the therapeutic effects of ISL1 on in vitro model of lipid accumulations was also confirmed through the modulation of KDM6B and SNAI1.ConclusionsTaken together, these findings highlighted that ISL1 effectively ameliorated NAFLD by inducing the expressions of KDM6B and SNAI1, which might be a promising drug for the treatment of NAFLD.