CHIP inhibits odontoblast differentiation through promoting DLX3 polyubiquitylation and degradation.

Dentin is the major hard tissue of teeth formed by differentiated odontoblasts. How odontoblast differentiation is regulated remains enigmatic. Here, we report that the E3 ubiquitin ligase CHIP is highly expressed in undifferentiated dental mesenchymal cells and downregulated after differentiation of odontoblasts. Ectopic expression of CHIP inhibits odontoblastic differentiation of mouse dental papilla cells, whereas knockdown of endogenous CHIP has opposite effects. Chip (Stub1) knockout mice display increased formation of dentin and enhanced expression of odontoblast differentiation markers. Mechanistically, CHIP interacts with and induces K63 polyubiquitylation of the transcription factor DLX3, leading to its proteasomal degradation. Knockdown of DLX3 reverses the enhanced odontoblastic differentiation caused by knockdown of CHIP. These results suggest that CHIP inhibits odontoblast differentiation by targeting its tooth-specific substrate DLX3. Furthermore, our results indicate that CHIP competes with another E3 ubiquitin ligase, MDM2, that promotes odontoblast differentiation by monoubiquitylating DLX3. Our findings suggest that the two E3 ubiquitin ligases CHIP and MDM2 reciprocally regulate DLX3 activity by catalyzing distinct types of ubiquitylation, and reveal an important mechanism by which differentiation of odontoblasts is delicately regulated by divergent post-translational modifications.

High-quality haploid genomes corroborate 29 chromosomes and highly conserved synteny of genes in Hyles hawkmoths (Lepidoptera: Sphingidae).

BACKGROUND: Morphological and traditional genetic studies of the young Pliocene genus Hyles have led to the understanding that despite its importance for taxonomy, phenotypic similarity of wing patterns does not correlate with phylogenetic relationship. To gain insights into various aspects of speciation in the Spurge Hawkmoth (Hyles euphorbiae), we assembled a chromosome-level genome and investigated some of its characteristics. RESULTS: The genome of a male H. euphorbiae was sequenced using PacBio and Hi-C data, yielding a 504 Mb assembly (scaffold N50 of 18.2 Mb) with 99.9% of data represented by the 29 largest scaffolds forming the haploid chromosome set. Consistent with this, FISH analysis of the karyotype revealed n = 29 chromosomes and a WZ/ZZ (female/male) sex chromosome system. Estimates of chromosome length based on the karyotype image provided an additional quality metric of assembled chromosome size. Rescaffolding the published male H. vespertilio genome resulted in a high-quality assembly (651 Mb, scaffold N50 of 22 Mb) with 98% of sequence data in the 29 chromosomes. The larger genome size of H. vespertilio (average 1C DNA value of 562 Mb) was accompanied by a proportional increase in repeats from 45% in H. euphorbiae (measured as 472 Mb) to almost 55% in H. vespertilio. Several wing pattern genes were found on the same chromosomes in the two species, with varying amounts and positions of repetitive elements and inversions possibly corrupting their function. CONCLUSIONS: Our two-fold comparative genomics approach revealed high gene synteny of the Hyles genomes to other Sphingidae and high correspondence to intact Merian elements, the ancestral linkage groups of Lepidoptera, with the exception of three simple fusion events. We propose a standardized approach for genome taxonomy using nucleotide homology via scaffold chaining as the primary tool combined with Oxford plots based on Merian elements to infer and visualize directionality of chromosomal rearrangements. The identification of wing pattern genes promises future understanding of the evolution of forewing patterns in the genus Hyles, although further sequencing data from more individuals are needed. The genomic data obtained provide additional reliable references for further comparative studies in hawkmoths (Sphingidae).

Activation of butterfly eyespots by Distal-less is consistent with a reaction-diffusion process.

Eyespots on the wings of nymphalid butterflies represent colorful examples of pattern formation, yet the developmental origins and mechanisms underlying eyespot center differentiation are still poorly understood. Using CRISPR-Cas9 we re-examine the function of Distal-less (Dll) as an activator or repressor of eyespots, a topic that remains controversial. We show that the phenotypic outcome of CRISPR mutations depends upon which specific exon is targeted. In Bicyclus anynana, exon 2 mutations are associated with both missing and ectopic eyespots, and also exon skipping. Exon 3 mutations, which do not lead to exon skipping, produce only null phenotypes, including missing eyespots, lighter wing coloration and loss of scales. Reaction-diffusion modeling of Dll function, using Wnt and Dpp as candidate morphogens, accurately replicates these complex crispant phenotypes. These results provide new insight into the function of Dll as a potential activator of eyespot development, scale growth and melanization, and suggest that the tuning of Dll expression levels can generate a diversity of eyespot phenotypes, including their appearance on the wing.This article has an associated 'The people behind the papers' interview.

The Hox gene Abdominal-B regulates the appendage development during the embryogenesis of scorpionflies.

The Homeotic Complex (Hox) genes encode conserved homeodomain transcription factors that specify segment identity and appendage morphology along the antero-posterior axis in bilaterian animals. The Hox gene Abdominal-B (Abd-B) is mainly expressed in the posterior segments of the abdomen and plays an important role in insect organogenesis. In Mecoptera, the potential function of this gene remains unclear yet. Here, we performed a de novo transcriptome assembly and identified an Abd-B ortholog in the scorpionfly Panorpa liui. Quantitative real-time reverse transcription PCR showed that Abd-B expression increased gradually in embryos 76 h post oviposition, and was mainly present in the more posterior abdominal segments. Embryonic RNA interference of Abd-B resulted in a set of abnormalities, including developmental arrest, malformed suckers and misspecification of posterior segment identity. These results suggest that Abd-B is required for the proper development of the posterior abdomen. Furthermore, in Abd-B RNAi embryos, the expression of the appendage marker Distal-less (Dll) was up-regulated and was additionally present on abdominal segments IX and X compared with wild embryos, suggesting that scorpionfly Abd-B may act to suppress proleg development and has gained the ability to repress Dll expression on the more posterior abdominal segments. This study provides additional information on both the functional and evolutionary roles of Abd-B across different insects.

DLX6 promotes cell proliferation and survival in oral squamous cell carcinoma.

OBJECTIVE: Distal-less homeobox 6 (DLX6) has been reported to play important roles in the development of craniofacial structures, inner ear, limb, and brain. We found in our previous investigation that DLX6 was significantly highly expressed in oral cancer tissues in The Cancer Genome Atlas database. This study aimed to explore its roles and regulation mechanism in oral squamous cell carcinoma. MATERIALS AND METHODS: We analyzed the expression of DLX6 and its association with overall survival in OSCC by real-time quantitative PCR. Besides, clone formation, proliferation, and apoptosis were detected after knocking down DLX6 and microarray analysis was performed to explore the possible regulatory mechanism. RESULTS: DLX6 was overexpressed in oral cancer tissues and was associated with advance tumor stage and poor prognosis. In vitro studies have shown that DLX6 promotes proliferation and inhibits cell apoptosis in oral cancer cells. Microarray analysis along with Western blotting results indicated that DLX6 significantly associated with malignant tumors and may regulate OSCC cells proliferation through EGFR-CCND1 axis. CONCLUSION: DLX6 promotes cell proliferation and suppresses cell apoptosis in oral cancer cells. EGFR-CCND1 pathway might be the potential mechanism participating in the regulating axis.

Dlx5 Represses the Transcriptional Activity of PPARγ.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master transcription factor in adipocyte differentiation, while distal-less homeobox 5 (Dlx5) is essential for initiating osteoblast differentiation by driving Runt-related transcription factor 2 expression. Considering that adipocytes and osteoblasts share common progenitors, there is a reciprocal correlation between bone and fat formation. However, the mechanism by which Dlx5 controls PPARγ remains unclear. We elucidated that Dlx5 physically binds to PPARγ during immunoprecipitation; in particular, the ligand-binding and DNA-binding domains of PPARγ were involved in the interaction. Transcriptional activity of PPARγ was significantly decreased by Dlx5 overexpression, whereas the opposite results were detected with Dlx5 knockdown. Rosiglitazone, a PPARγ agonist, further enhanced the PPARγ-induced transcriptional activity; however, Dlx5 overexpression effectively repressed the rosiglitazone-mediated increase in activity. Finally, DNA-binding affinity assay revealed that Dlx5 interrupts the interaction of PPARγ with the PPARγ response element promoter. In conclusion, our findings indicate that Dlx5 impedes PPARγ-induced activity, and it may be useful for managing diabetes drug-mediated obesity.

Metallothionein 3 Promotes Osteoblast Differentiation in C2C12 Cells via Reduction of Oxidative Stress.

Metallothioneins (MTs) are intracellular cysteine-rich proteins, and their expressions are enhanced under stress conditions. MTs are recognized as having the ability to regulate redox balance in living organisms; however, their role in regulating osteoblast differentiation is still unclear. In this research, we found that the expression of MT3, one member of the MT protein family, was specifically upregulated in the differentiation process of C2C12 myoblasts treated with bone morphogenetic protein 4 (BMP4). Transfection with MT3-overexpressing plasmids in C2C12 cells enhanced their differentiation to osteoblasts, together with upregulating the protein expression of bone specific transcription factors runt-related gene 2 (Runx2), Osterix, and distal-less homeobox 5 (Dlx5). Additionally, MT3 knockdown performed the opposite. Further studies revealed that overexpression of MT3 decreased reactive oxygen species (ROS) production in C2C12 cells treated with BMP4, and MT3 silencing enhanced ROS production. Treating C2C12 cells with antioxidant N-acetylcysteine also promoted osteoblast differentiation, and upregulated Runx2/Osterix/Dlx5, while ROS generator antimycin A treatment performed the opposite. Finally, antimycin A treatment inhibited osteoblast differentiation and Runx2/Osterix/Dlx5 expression in MT3-overexpressing C2C12 cells. These findings identify the role of MT3 in osteoblast differentiation and indicate that MT3 may have interesting potential in the field of osteogenesis research.

Distal-less homeobox 5 promotes the osteo-/dentinogenic differentiation potential of stem cells from apical papilla by activating histone demethylase KDM4B through a positive feedback mechanism.

Understanding the mechanism of osteo-/dentinogenic differentiation is beneficial for jaw bone and dental tissue regeneration. DLX5 is highly expressed in dental tissue-derived mesenchymal stem cells (MSCs) and is upregulated by lysine-specific demethylase 4B (KDM4B), enabling it to regulate osteo-/dentinogenic differentiation, while the function of DLX5 in osteo-/dentinogenesis has not been thoroughly elucidated to date. Therefore, we investigated DLX5 function using stem cells from apical papilla (SCAPs). SCAPs were obtained from the human wisdom tooth. Alkaline phosphatase (ALP) assay, Alizarin red staining (ARS), quantitative analysis of calcium, osteo-/dentinogenesis-related gene expression and in vivo transplantation were used to determine the osteo-/dentinogenic differentiation potential. Luciferase and ChIP assays were used to investigate the physical relationship between DLX5 and KDM4B. DLX5 and KDM4B were upregulated during osteogenic induction and were induced by BMP4 in SCAPs. Next, we found that DLX5 enhanced ALP activity, mineralization in vitro, and the expression of dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP1), osteopontin (OPN), and the key transcription factor osterix (OSX). Moreover, transplant experiments showed that DLX5 promoted osteo-/dentinogenesis in vivo. Interestingly, DLX5 enhanced KDM4B transcription by directly binding with its promoter. In addition, KDM4B upregulated DLX5 in SCAPs. These results indicate that DLX5 and KDM4B are positive effectors of BMP signaling and regulate each other via a positive feedback mechanism. DLX5 enhanced osteo-/dentinogenic differentiation via upregulated KDM4B in SCAPs, suggesting that activation of the DLX5/KDM4B signaling pathway might serve as an intrinsic mechanism that promotes tissue regeneration mediated by dental-derived MSCs.

Proximal fate marker homothorax marks the lateral extension of stalk-eyed fly Cyrtodopsis whitei.

The placement of eyes on insect head is an important evolutionary trait. The stalk-eyed fly, Cyrtodopsis whitei, exhibits a hypercephaly phenotype where compound eyes are located on lateral extension from the head while the antennal segments are placed inwardly on this stalk. This stalk-eyed phenotype is characteristic of the family Diopsidae in the Diptera order and dramatically deviates from other dipterans, such as Drosophila. Like other insects, the adult eye and antenna of stalk-eyed fly develop from a complex eye-antennal imaginal disc. We analyzed the markers involved in proximo-distal (PD) axis of the developing eye imaginal disc of the stalk-eyed flies. We used homothorax (hth) and distalless (dll), two highly conserved genes as the marker for proximal and distal fate, respectively. We found that lateral extensions between eye and antennal field of the stalk-eyed fly's eye-antennal imaginal disc exhibit robust Hth expression. Hth marks the head specific fate in the eye- and proximal fate in the antenna-disc. Thus, the proximal fate marker Hth expression evolves in the stalk-eyed flies to generate lateral extensions for the placement of the eye on the head. Moreover, during pupal eye metamorphosis, the lateral extension folds back on itself to place the antenna inside and the adult compound eye on the distal tip. Interestingly, the compound eye in other insects does not have a prominent PD axis as observed in the stalk-eyed fly.

MicroRNA-539 functions as a tumour suppressor in prostate cancer via the TGF-ß/Smad4 signalling pathway by down-regulating DLX1.

Prostate cancer (PCa) is the second leading cause of cancer-related death in males, primarily due to its metastatic potential. The present study aims to identify the expression of microRNA-539 (miR-539) in PCa and further investigate its functional relevance in PCa progression both in vitro and in vivo. Initially, microarray analysis was conducted to obtain the differentially expressed gene candidates and the regulatory miRNAs, after which the possible interaction between the two was determined. Next, ectopic expression and knock-down of the levels of miR-539 were performed in PCa cells to identify the functional role of miR-539 in PCa pathogenesis, followed by the measurement of E-cadherin, vimentin, Smad4, c-Myc, Snail1 and SLUG expression, as well as proliferation, migration and invasion of PCa cells. Finally, tumour growth was evaluated in nude mice through in vivo experiments. The results found that miR-539 was down-regulated and DLX1 was up-regulated in PCa tissues and cells. miR-539 was also found to target and negatively regulate DLX1 expression, which resulted in the inhibition of the TGF-ß/Smad4 signalling pathway. Moreover, the up-regulation of miR-539 or DLX1 gene silencing led to the inhibition of PCa cell proliferation, migration, invasion, EMT and tumour growth, accompanied by increased E-cadherin expression and decreased expression of vimentin, Smad4, c-Myc, Snail1 and SLUG. In conclusion, the overexpression of miR-539-mediated DLX1 inhibition could potentially impede EMT, proliferation, migration and invasion of PCa cells through the blockade of the TGF-ß/Smad4 signalling pathway, highlighting a potential miR-539/DLX1/TGF-ß/Smad4 regulatory axis in the treatment of PCa.

Distal-less homeobox 3, a negative regulator of myogenesis, is downregulated by microRNA-133.

Distal-less homeobox 3 (Dlx3), a member of the Dlx family of homeobox proteins, is a transcriptional activator of runt-related transcription factor 2 (Runx2) during osteogenic differentiation. It has been demonstrated that forced expression of Runx2 induces an osteogenic program and ectopic calcification in muscles. Therefore, it would be reasonable to predict that Dlx3 also affects myogenic differentiation. The relationship between Dlx3 and myogenesis, however, remains poorly understood. Therefore, in this study, the role and regulation of Dlx3 during myogenic differentiation were investigated. Expression level of Dlx3 was downregulated in human mesenchymal stem cells (MSCs), mouse MSCs, and C2C12 cells cultured in myogenic medium. Dlx3 level was inversely correlated with myogenic differentiation 1 and the muscle-specific microRNA, microRNA-133 (miR-133). The expression level of Runx2 was closely regulated by Dlx3 even under myogenic conditions. Overexpression of Dlx3 markedly downregulated expression levels of myogenic transcription factors and myotube formation in C2C12 cells, whereas Dlx3 knockdown enhanced myogenic differentiation. The Dlx3 3'-untranslated region (3'-UTR) has two potential binding sites for miR-133. Luciferase reporter assays demonstrated that Dlx3 is a direct target of miR-133a and miR-133b, and that the two target sites are redundantly active. Taken together, these results suggest that Dlx3 is a negative regulator of myogenic differentiation and that miR-133a and miR-133b enhance myogenic differentiation, partly through inhibition of Dlx3 expression via direct targeting of the Dlx3 3'-UTR.

Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex.

The microcrustacean Daphnia pulex is an important model for environmental, ecological, evolutionary and developmental genomics because its adaptive life history displays plasticity in response to environmental changes. Even though the whole-genome sequence is available and omics data have actively accumulated for this species, the available tools for analyzing gene function have thus far been limited to RNAi (RNA interference) and TALEN (the transcription activator-like effector nuclease) systems. The development of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is thus expected to further increase the genetic tractability of D. pulex and to advance the understanding of this species. In this study, we developed a genome editing system for D. pulex using CRISPR/Cas9 ribonucleoprotein complexes (Cas9 RNPs). We first assembled a CRISPR single-guide RNA (sgRNA) specific to the Distal-less gene (Dll), which encodes a homeodomain transcription factor essential for distal limb development in invertebrates and vertebrates. Then, we injected Cas9 RNPs into eggs and evaluated its activity in vivo by a T7 endonuclease I assay. Injected embryos showed defective formation of the second antenna and disordered development of appendages, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene.

UTR-specific knockdown of Distal-less and Sp8 leads to new phenotypic variants in the flour beetle Tribolium.

RNA interference (RNAi)-mediated knockdown serves as an effective technique for the functional analysis of developmental genes that is well established in many organisms. In the beetle Tribolium castaneum, double-stranded RNA is applied by simple injection and distributes systemically within the tissue. Thus, systematic testing for RNAi specificity and efficiency is easily possible in this organism. Generally, the use of non-overlapping dsRNA fragments yielding qualitatively identical phenotypes is the method of choice to verify target-specific knockdown effects. Here, we show that UTR-specific RNAi results in different effects regarding quality, severity and penetrance when compared to RNAi fragments directed at the coding region. Furthermore, when using 3'UTR-specific dsRNA, we first describe the Distal-lessRNAi antenna-to-leg transformation phenotype in the Tribolium larva, which has only been observed in the adult beetle and Drosophila so far. In addition, we unexpectedly observed sterility effects caused by 3'UTR-specific knockdown of the Tribolium-Sp8 orthologue that is not seen when dsRNA targeted a sequence within the coding-region or the 5'UTR that itself led to early embryonic lethality. We conclude that targeting UTR sequences by region-specific RNAi can reveal unexpected new aspects of gene function applicable in basic research and crop protection.

Dlx3 and GCM-1 functionally coordinate the regulation of placental growth factor in human trophoblast-derived cells.

Placental growth factor (PGF) is abundantly expressed by trophoblast cells within human placentae and is important for trophoblast development and placental vascularization. Circulating maternal serum levels of PGF are dynamically upregulated across gestation in normal pregnancies, whereas low circulating levels and placental production of PGF have been implicated in the pathogenesis of preeclampsia and other gestational diseases. However, the underlying molecular mechanism of regulating PGF expression in the human placenta remains poorly understood. In this study, we demonstrated that transcription factors Distal-less 3 (DLX3) and Glial cell missing-1 (GCM1) were both sufficient and required for PGF expression in human trophoblast-derived cells by overexpression and knockdown approaches. Surprisingly, while DLX3 and GCM1 were both positive regulators of PGF, co-overexpression of DLX3 and GCM1 led to an antagonist effect on PGF expression on the endogenous gene and a luciferase reporter. Further, deletion and site-directed mutagenesis studies identified a novel regulatory element on the PGF promoter mediating both DLX3- and GCM1-dependent PGF expression. This regulatory region was also found to be essential for the basal activity of the PGF promoter. Finally, Chromatin-immunoprecipitation (ChIP) assays revealed colocalization of DLX3 and GCM1 at the identified regulatory region on the PGF promoter. Taken together, our studies provide important insights into intrinsic regulation of human placental PGF expression through the functional coordination of DLX3 and GCM1, and are likely to further the understanding of pathogenesis of PGF dysregulation in preeclampsia and other disease conditions.

DLX3 promotes bone marrow mesenchymal stem cell proliferation through H19/miR-675 axis.

The underlying molecular mechanism of the increased bone mass phenotype in Tricho-dento-osseous (TDO) syndrome remains largely unknown. Our previous study has shown that the TDO point mutation c.533A>G, Q178R in DLX3 could increase bone density in a TDO patient and transgenic mice partially through delaying senescence in bone marrow mesenchymal stem cells (BMSCs). In the present study, we provided a new complementary explanation for TDO syndrome: the DLX3 (Q178R) mutation increased BMSCs proliferation through H19/miR-675 axis. We found that BMSCs derived from the TDO patient (TDO-BMSCs) had stronger proliferation ability than controls by clonogenic and CCK-8 assays. Next, experiments of overexpression and knockdown of wild-type DLX3 via lentiviruses in normal BMSCs confirmed the results by showing its negative role in cell proliferation. Through validated high-throughput data, we found that the DLX3 mutation reduced the expression of H19 and its coexpression product miR-675 in BMSCs. Function and rescue assays suggested that DLX3, long noncoding RNA H19, and miR-675 are negative factors in modulation of BMSCs proliferation as well as NOMO1 expression. The original higher proliferation rate and the expression of NOMO1 in TDO-BMSCs were suppressed after H19 restoration. Collectively, it indicates that DLX3 regulates BMSCs proliferation through H19/miR-675 axis. Moreover, the increased expression of NOMO1 and decreased H19/miR-675 expression in DLX3 (Q178R) transgenic mice, accompanying with accrual bone mass and density detected by micro-CT, further confirmed our hypothesis. In summary, we, for the first time, demonstrate that DLX3 mutation interferes with bone formation partially through H19/miR-675/NOMO1 axis in TDO syndrome.

Possible co-option of engrailed during brachiopod and mollusc shell development.

In molluscs, two homeobox genes, engrailed (en) and distal-less (dlx), are transcription factors that are expressed in correlation with shell development. They are expressed in the regions between shell-forming and non-shell-forming cells, likely defining the boundaries of shell-forming fields. Here we investigate the expression of two transcription factors in the brachiopod Lingula anatina We find that en is expressed in larval mantle lobes, whereas dlx is expressed in larval tentacles. We also demonstrate that the embryonic shell marker mantle peroxidase (mpox) is specifically expressed in mantle lobes. Our results suggest that en and mpox are possibly involved in brachiopod embryonic shell development. We discuss the evolutionary developmental origin of lophotrochozoan biomineralization through independent gene co-option.

DLX3 negatively regulates osteoclastic differentiation through microRNA-124.

Homeodomain gene Distal-less-3 (DLX3) plays an essential role in the development of bones. Mutations of DLX3 are closely associated with Tricho-Dento-Osseous (TDO) syndrome featured with increased bone formation. However, the mechanism regarding whether DLX3 regulates osteoclastogenesis remains largely unknown. In this study, we firstly examined the expression of DLX3 mounting during osteoclastic differentiation process, and then established stably expressing wild type DLX3 (WT-DLX3), a novel mutant DLX3 (Q178R) found in our laboratory recently (MT-DLX3) and Dlx3 knockdown cell lines (Dlx3-shRNA) in Raw 264.7 cells using corresponding lentiviruses. Next, we investigated the influence of DLX3 on these stable cells in the process of osteoclastogenesis. The results showed that the expression of osteoclastogenesis-related genes as well as tartrate-resistant acid phosphatase-positive multinucleated cells were lower in WT-DLX3 and MT-DLX3, but higher in Dlx3-shRNA compared with control cells. Besides, the microRNA-124 expression was higher in WT-DLX3 and MT-DLX3 but lower in Dlx3-shRNA. Moreover, the microRNA-124 expression level positively correlated with DLX3, negatively with osteoclastogenesis-related gene NFATc1. Our results indicate that DLX3 negatively regulates osteoclastic differentiation through microRNA-124, which is partially responsible for the increased bone density in TDO patient. DLX3 may be exploited for osteoclastogenesis regulator and potential therapeutic target of osteoporosis in future.

Regulation of neuronal migration, an emerging topic in autism spectrum disorders.

Autism spectrum disorders (ASD) encompass a group of neurodevelopmental diseases that demonstrate strong heritability, however, the inheritance is not simple and many genes have been associated with these disorders. ASD is regarded as a neurodevelopmental disorder, and abnormalities at different developmental stages are part of the disease etiology. This review provides a general background on neuronal migration during brain development and discusses recent advancements in the field connecting ASD and aberrant neuronal migration. We propose that neuronal migration impairment may be an important common pathophysiology in autism spectrum disorders (ASD). This review provides a general background on neuronal migration during brain development and discusses recent advancements in the field connecting ASD and aberrant neuronal migration.

Overexpression of DLX2 is associated with poor prognosis and sorafenib resistance in hepatocellular carcinoma.

The mechanism underlying poor prognosis and sorafenib resistance in patients with hepatocellular carcinoma (HCC) is unknown and, to date, no useful predictive biomarkers of sorafenib resistance have been identified. Distal-less homeobox 2 (DLX2) is a transcription factor involved in cell cycle regulation that is closely correlated with cancer prognosis. In this study, we showed that DLX2 is overexpressed in HCC tissues and cell lines and that the level of DLX2 overexpression is positively correlated with histological grade, metastasis and Ki67 expression, which are indicators of poor prognosis. We also found that DLX2 accumulates in proliferating HCC cells, where it is associated with the expression of proliferating cell nuclear antigen (PCNA), Cyclin D1 and Cyclin A. Flow cytometry and cell counting kit-8 (CCK-8) assays indicated that DLX2 depletion causes cell cycle arrest at the G1 phase and hinders cell proliferation. Moreover, the sensitivity of HCC cells to sorafenib is restored when the DLX2 gene is knocked down using a short interfering RNA. We demonstrated that DLX2 facilitates sorafenib resistance by promoting the expression of markers of epithelial-mesenchymal transition and by activating the extracellular signal-regulated protein kinase pathway. Our findings reveal that DLX2 plays a regulatory role in HCC cell proliferation and suggests that targeting DLX2 represents a novel strategy to increase sorafenib efficacy in the management of HCC. In conclusion, DLX2 is a novel marker of poor prognosis and sorafenib resistance in patients with HCC.

A Single-Wing Removal Method to Assess Correspondence Between Gene Expression and Phenotype in Butterflies: The Case of Distal-less.

It is often desirable but difficult to retrieve information on the mature phenotype of an immature tissue sample that has been subjected to gene expression analysis. This problem cannot be ignored when individual variation within a species is large. To circumvent this problem in the butterfly wing system, we developed a new surgical method for removing a single forewing from a pupa using Junonia orithya; the operated pupa was left to develop to an adult without eclosion. The removed right forewing was subjected to gene expression analysis, whereas the non-removed left forewing was examined for color patterns. As a test case, we focused on Distal-less (Dll), which likely plays an active role in inducing elemental patterns, including eyespots. The Dll expression level in forewings was paired with eyespot size data from the same individual. One third of the operated pupae survived and developed wing color patterns. Dll expression levels were significantly higher in males than in females, although male eyespots were smaller in size than female eyespots. Eyespot size data showed weak but significant correlations with the Dll expression level in females. These results demonstrate that a single-wing removal method was successfully applied to the butterfly wing system and suggest the weak and non-exclusive contribution of Dll to eyespot size determination in this butterfly. Our novel methodology for establishing correspondence between gene expression and phenotype can be applied to other candidate genes for color pattern development in butterflies. Conceptually similar methods may also be applicable in other developmental systems.