Expression of the homeobox proteins Dlx-5 and TLX1 during the development of cat testis / Expresión de las proteínas homeobox Dlx-5 y TLX1 durante el desarrollo del testículo de gato

SUMMARY: Recent studies have shown that homeobox proteins play an important role in the formation and development of tissues and organs in the embryonic period. In our study, the distribution of Dlx-5 and TLX proteins, which are members of the homeobox family, in the testis, epididymis and ductus deferens ducts of some cat breeds were investigated. For this purpose, in the study, 18 testes younger than six months (immature) and older than one year (mature) were examined under a light microscope using an immunohistochemical method (indirect streptavidin-biotin complex). While it was determined that Dlx-5 and TLX1 proteins were expressed at varying levels in cells in immature and mature cat testicles, epithelial cells of ductus epididymis and ductus deferens, and smooth muscle cells of ductus deferens, no differences were observed between cat breeds. Dlx-5 immunoreactivity was more intense in the testes, epididymis and deferens ducts of immature and mature compared to TLX1. These results suggested that both proteins play important roles in the development of male feline genital organs and in the secretion and differentiation of cells, and also further observation of Dlx-5 expression suggested that this protein may be more effective than TLX1 in testicular development and physiological processes.

RESUMEN: Estudios recientes han demostrado que las proteínas homeobox juegan un papel importante en la formación y desarrollo de tejidos y órganos en el período embrionario. En nuestro estudio, se investigó la distribución de las proteínas Dlx-5 y TLX, que son miembros de la familia homeobox, en los testículos, en el epidídimo y en los conductos deferentes de algunas razas de gatos. En el estudio fueron examinados, 18 testículos de animales menores de seis meses (inmaduros) y mayores de un año (maduros) bajo un microscopio óptico utilizando un método inmunohistoquímico (complejo indirecto de estreptavidina-biotina). Si bien se determinó que las proteínas Dlx-5 y TLX1 se expresaron en niveles variables en las células de los testículos de gatos inmaduros y maduros, las células epiteliales del epidídimo y del conducto deferente y las células del músculo liso del conducto deferente, no se observaron diferencias entre las razas de gatos. La inmunorreactividad de Dlx-5 fue más intensa en los testículos, epidídimo y conductos deferentes de gatos inmaduros y maduros en comparación con TLX1. Estos resultados sugieren que ambas proteínas tienen un rol importante en el desarrollo de los órganos genitales felinos masculinos y en la secreción y diferenciación de células, y también la observación de la expresión de Dlx-5 sugirió que esta proteína puede ser más efectiva que TLX1 en el desarrollo testicular y en los procesos fisiológicos.

Sp7/osterix positively regulates dlx2b and bglap to affect tooth development and bone mineralization in zebrafish larvae

Osterix (or Sp7) is an important transcription factor that promotes osteoblast differentiation by modulating the expression ofa range of target genes. Although many studies have focused on Osterix/Sp7 regulatory mechanisms, the detailed functionshave not been fully elucidated. Toward this end, in this study, we used CRISPR/Cas9 technology to knock out the zebrafishsp7 gene, and then analyzed its phenotype and biological function. Two knockout sp7 mutant lines were successfullyobtained. The bone mineralization level was significantly reduced in the zebrafish sp7-/- homozygote, resulting inabnormal tooth development in the larvae. Quantitative real-time polymerase chain reaction showed that loss of sp7 led todown-regulated expression of the dlx2b and bglap genes related to tooth development and bone mineralization, respectively. Moreover, cell transfection experiments demonstrated that Sp7 directly regulates the expression of dlx2b and bglapthrough Sp7-binding sites on the promoter regions of these two genes. Overall, this study provides new insight into the roleof Sp7 in bone mineralization and tooth development.

Expression of nestin, transcription factor SOX3 and homeobox protein DLX-4 in the facial tissue of children with cleft lip and palate / Expresión de nestina, factor de transcripción SOX3 y proteína homeobox DLX-4 en el tejido facial de niños con fisura labiopalatina

The failure of fusion of nasal and maxillary processes results in cleft lip and palate (CLP), which is one of the most common birth defects. The morphopathogenesis of this pathology is multifactorial and still largely unclear. The aim of this study was to evaluate the presence of nestin, transcriptor factor SOX3 (Sox3) and homeobox protein DLX-4 (Dlx-4) in complete unilateral (CU) and complete bilateral (CB) CLP affected facial tissue. Oral mucosa tissue samples were obtained from 17 CUCLP and 13 CBCLP patients during surgical cleft correction and 6 unaffected control subjects. Obtained tissue sections were stained with hematoxylin and eosin and by immunohistochemistry for nestin, Sox3 and Dlx-4. The intensity of staining was graded semiquantitatively. Nestin-positive structures were detected in all CUCLP and CBCLP patients' tissue samples, varying from moderate number of nestin-positive structures to numerous. Sox3 immunoreactivity was more prominent in epithelial cells in both patient groups with frequently patchy distribution. Mainly moderate number of Dlx-4-positive cells was observed in most of tissue samples. Statistically significant moderate positive correlation was found between nestin and Sox3 factors in CUCLP patient group (Spearman's rank correlation coefficient = .517, P = .034). Increase of nestinpositive structures suggests its role in the regulation of the remodeling of tissue in both CUCLP and CBCLP affected tissue. Dominance of Sox3 positivity in cleft affected epithelium indicates its possible role in (compensatory) formation of defective oral epithelium of CUCLP and CBCLP patients. The reduced expression of Dlx-4 implicates its limited regulatory role on the craniofacial development in CUCLP and CBCLP affected tissue.

La falla en la fusión de los procesos nasal y maxilar son causante de la fisura labiopalatina (FLP), que es uno de los defectos congénitos más comunes. La morfopatogenia de esta patología es multifactorial y aún poco clara. El objetivo de este estudio fue evaluar la presencia de nestina, el factor transcriptor SOX3 (Sox3) y la proteína homeobox DLX-4 (Dlx-4) en todo el tejido facial afectado por FLP bilateral unilateral (FU) y bilateral completa (FB). Se obtuvieron muestras de tejido de mucosa oral de 17 pacientes FUFLP y 13 FBFLP durante la corrección quirúrgica de la fisura y de 6 sujetos de control no afectados. Las secciones de tejido obtenidas se tiñeron con hematoxilina y eosina y mediante inmunohistoquímica para nestina, Sox3 y Dlx-4. La intensidad de la tinción fue graduada semicuantitativamente. Se detectaron estructuras positivas para nestina en todas las muestras de tejido de pacientes FUFLP y FBFLP, variando desde un número moderado a numerosas estructuras positivas para nestina. La inmunorreactividad de Sox3 fue más prominente en las células epiteliales en ambos grupos de pacientes con distribución frecuentemente irregular. Se observó un número principalmente moderado de células Dlx-4-positivas en la mayoría de las muestras de tejido. Se encontró una correlación positiva moderada estadísticamente significativa entre los factores de nestina y Sox3 en el grupo de pacientes FUFLP (coeficiente de correlación de rangos de Spearman = 0,517, P = 0,034). El aumento de estructuras positivas para nestina sugiere su papel en la regulación de la remodelación de tejido, tanto en tejido afectado por FUFLP como FBFLP. La dominancia de la positividad de Sox3 en el epitelio afectado de la fisura, indica su posible papel en la formación (compensatoria) del epitelio oral defectuoso de pacientes FUFLP y FBFLP. La expresión reducida de Dlx-4 implica su función reguladora limitada en el desarrollo craneofacial en tejido afectado por FUFLP y FBFLP.

Changes of distal-less homeobox genes 5 and Msh homeobox 1 in a rat model of bisphosphonate related osteonecrosis of the jaw / 中华口腔医学杂志

Objective@#To further study the effects of distal-less homeobox gene 5 (Dlx-5) and Msh homeobox 1 (Msx-1) in the pathogenic mechanism of bisphosphonate related osteonecrosis of the jaw (BRONJ) .@*Methods@#Twenty-four SD rats were divided into two groups, the experimental group was injected intraperitoneally with zoledronic acid for 12 weeks (0.2 mg/kg, three times a week), and the control group was injected with saline solution for 12 weeks. The first mandibular molars were extracted after 12 weeks. All of the animals were sacrificed eight weeks after teeth extraction. The BRONJ was diagnosed by gross observation, X-ray examination and histopathlolgical examination. Through real-time PCR, the expression level of Dlx-5 and Msx-1 were detected in the mandible of BRONJ samples and normal samples.@*Results@#X-ray examination and histopathlolgical analysis showed the presence of BRONJ. The results of real-time PCR showed that the expression levels of Dlx-5 were increased (P=0.001) and the expression level of Msx-1 was decreased (P=0.001) in the experimental group compared with the control group.@*Conclusions@#Dlx-5 and Msx-1 genes play roles in the pathogenic mechanism of BRONJ.

Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System

Assessing the cell-type expression pattern of a certain gene can be achieved by using cell-type-specific gene manipulation. Recently, cre-recombinase-dependent gene-silencing tool, pSico has become popular in neuroscientific research. However, pSico has a critical limitation that gene-silenced cell cannot be identified by fluorescence, due to an excision of the reporter gene for green fluorescence protein (GFP). To overcome this limitation, we newly developed pSico-Red, with mCherry gene as a reporter outside two loxP sites, so that red mCherry signal is detected in all transfected cells. When a cell expresses cre, GFP is excised and shRNA is enabled, resulting in disappearance of GFP. This feature of pSico-Red provides not only cell-type-specific gene-silencing but also identification of cre expressing cells. Using this system, we demonstrated for the first time the neuronal expression of the Bestrophin-1 (Best1) in thalamic reticular nucleus (TRN) and TRN-neuron-specific gene-silencing of Best1. We combined adeno-associated virus (AAV) carrying Best1-shRNA in pSico-Red vector and transgenic mouse expressing cre under the promoter of distal-less homeobox 5/6 (DLX5/6), a marker for inhibitory neurons. Firstly, we found that almost all of inhibitory neurons in TRN express Best1 by immunohistochemistry. Using pSico-Red virus, we found that 80% of infected TRN neurons were DLX5/6-cre positive but parvalbumin negative. Finally, we found that Best1 in DLX5/6-cre positive neurons were significantly reduced by Best1-shRNA. Our study demonstrates that TRN neurons strongly express Best1 and that pSico-Red is a valuable tool for cell-type-specific gene manipulation and identification of specific cell population.

Role of Genes in Odontogenesis.

With the discovery of the homeobox genes in craniofacial biology researchers across the globe have studied in depth the genetic patterning of the craniofacial region. With respect to craniofacial development –, Barx, Dlx, Gsc, Lim, Msx, Otx, Prx; part of the Hox cluster are important. Barx gene are strongly expressed only in the mesenchyme of the developing molars. Dlx gene expression is noted in the mandibular and maxillary arch ectomesenchyme. Msx genes are expressed in the area of epithelial mesenchymal interactions in the brachial arches in the area of future dentition and also expressed in the formation of skull, facial primordial and sense organs. Msx-1 is seen to be expressed in various stages of tooth formation i.e bud and cap stage of organogenesis. Lim genes which control morphogenesis of the first brachial arch, are expressed in the maxillo-mandibular ectomesenchyme. Prx gene expression is seen in the proximal portion of the mandibular arch. The role of hox genes in the morphogenesis of the jaws and the dentition is immense. Thus it has been proved beyond doubt that the genes have a major role in organogenesis than what human beings have ever envisaged. This review will give the scientific community an overview of all the genes affecting odontogenesis.

Prospecting molecular markers to distinguish Cichla kelberi, C. monoculus and C. piquiti / Prospecção de marcadores moleculares para distinção de Cichla kelberi, Cichla monoculus e Cichla piquiti

Peacock bass, a fish of the genus Cichla, is an exotic species from the upper river Paraná floodplain in which the species Cichla kelberi and C. piquiti have been confirmed, coupled to the specie C. monoculus upstream in the Capivara and Taquaruçu dams. The introduction of this genus has caused negative impacts on the diversity of native species. Current research prospects DNA sequences capable of distinguishing the three species and provide molecular data for the taxonomic characterization of the species in the upper Paraná River basin. Sequencing of nuclear (tmo4c4, dlx2 and bmp4) and mitochondrial (cox1, cytb) loci were done from fish of the three species of the genus Cichla reported in the literature of the upper Paraná River basin. Sequence analysis provided molecular differentiation for the species through the usage of loci cytb, dlx2 and cox1. Since the latter only distinguished C. piquiti from the other Cichla species, the loci bmp4 and tmo4c4 were not adequate to accomplish our aim.

O tucunaré é um peixe pertencente ao gênero Cichla, sendo exótico na planície de inundação do alto rio Paraná, onde foi confirmada a presença das espécies Cichla kelberi e C. piquiti e, logo a montante, C. monoculus, nas represas de Capivara e Taquaruçu. A introdução deste gênero tem ocasionado impacto negativo à diversidade de espécies nativas. Visando providenciar dados moleculares para auxiliar na caracterização taxonômica das espécies deste gênero na bacia do alto rio Paraná, os objetivos deste trabalho foram prospectar sequências de DNA capazes de distinguir as três espécies. Procedeu-se o sequenciamento de loci nucleares (tmo4c4, dlx2 e bmp4) e mitocondriais (cox1, cytb) de peixes das três espécies do gênero Cichla relatadas na literatura da bacia do alto rio Paraná. As análises das sequências possibilitaram a diferenciação molecular para estas espécies pela utilização dos loci cytb, dlx2 e cox1; este último, somente para distinguir C. piquiti das demais espécies de Cichla. A utilização dos loci bmp4 e tmo4c4 não foi adequada para este propósito.

Differential Expression of STATs and Its Function in Osteogenesis of Mesenchymal Stem Cells / 대한정형외과연구학회지

PURPOSE: To investigate the role of STAT5 during osteogenesis differentiation of human mesenchymal stem cells(hMSC). MATERIALS AND METHODS: To assess the expression pattern of STATs during osteogenic differentiation, we performed western blot analysis and RT-PCR. By RNA interference, we have performed effect of STAT5A and STAT5B in osteogenesis. As a result of Luciferase assay, the promoter activity of DLX5 was decreased by STAT5A. RESULTS: To assess the expression pattern of STATs during osteogenic differentiation, we have performed western blot and RT-PCR after 14 day differentiation period. STAT1, 2, 3 and 4 showed no change in expression level during differentiation, while STAT5A and STAT5B displayed steady increase compared to the control. When STAT5A was knock down, the level of osteogenesis was increased. The transcriptional activity of DLX5 was decreased about 70% by STAT5A. CONCLUSION: The expression of STAT5A and STAT5B increased during osteogenesis of hMSC. Also, we shown that STAT5A regulated the transcriptional activity of DLX5. These results indicate that STAT5A acts as a pivotal transcription factor in osteogenesis of hMSC.

Does Dlx abnormal expression regulate the migration of cranial neural crest cells and development of the first brachial arch? / 中国组织工程研究

BACKGROUND: Dlx gene family is highly expressed in the cranial neural crest cells, and regulates the cranial neural crest cel migration and differentiation. OBJECTIVE: To review the mechanism that the highly-expressed Dlx genes mediate the cranial neural crest cel migration and differentiation. METHODS: An online search of CNKI and Medline databases was performed for articles published before 2013 using keywords of “cranial neural crest cells, migration of cranial neural crest cells, Dlx, Dlx overexpression, Fgf, chodrogenesis, osteogenesis” in Chinese and English, respectively. Relevant articles were summarized from three aspects: the migration of cranial neural crest cells, Dlx over-expression’s impact on the migration of cranial neural crest cells, interaction between the environment and Dlx genes. A total of 63 articles were included. According to inclusion criteria, 43 articles were retained at last. RESULTS AND CONCLUSION: Dlx abnormal-expression wil lead to cel -cel adhesion. Dlx over-expression wil induce most of the cranial neural crest cells aggregate and migrate to a wrong place, and result in skeletal dysmorphology. Dlx over-expression wil also lead to ectopic chondrogenesis, and the interaction between cel factors can be the possible reason for this.

Dlx3 and Dlx5 Inhibit Adipogenic Differentiation of Human Dental Pulp Stem Cells

Dlx3 and Dlx5 are homeobox domain proteins and are well-known regulators of osteoblastic differentiation. Since possible reciprocal relationships between osteogenic and adipogenic differentiation in mesenchymal stem cells exist, we examined the regulatory role of Dlx3 and Dlx5 on adipogenic differentiation using human dental pulp stem cells. Over-expression of Dlx3 and Dlx5 stimulated osteogenic differentiation but inhibited adipogenic differentiation of human dental pulp stem cells. Dlx3 and Dlx5 suppressed the expression of adipogenic marker genes such as C/EBPalpha, PPARgamma, aP2 and lipoprotein lipase. Adipogenic stimuli suppressed the mRNA levels of Dlx3 and Dlx5, whereas osteogenic stimuli enhanced the expression of Dlx3 and Dlx5 in 3T3-L1 preadipocytes. These results suggest that Dlx3 and Dlx5 exert a stimulatory effect on osteogenic differentiation of stem cells through the inhibition of adipogenic differentiation as well as direct stimulation.

Rat Dlx5 is expressed in the subventricular zone and promotes neuronal differentiation

The molecular mechanisms and potential clinical applications of neural precursor cells have recently been the subject of intensive study. Dlx5, a homeobox transcription factor related to the distal-less gene in Drosophila, was shown to play an important role during forebrain development. The subventricular zone (SVZ) in the adult brain harbors the largest abundance of neural precursors. The anterior SVZ (SVZa) contains the most representative neural precursors in the SVZ. Further research is necessary to elucidate how Dlx5-related genes regulate the differentiation of SVZa neural precursors. Here, we employed immunohistochemistry and molecular biology techniques to study the expression of Dlx5 and related homeobox genes Er81 and Islet1 in neonatal rat brain and in in vitro cultured SVZa neural precursors. Our results show that Dlx5 and Er81 are also highly expressed in the SVZa, rostral migratory stream, and olfactory bulb. Islet1 is only expressed in the striatum. In cultured SVZa neural precursors, Dlx5 mRNA expression gradually decreased with subsequent cell passages and was completely lost by passage four. We also transfected a Dlx5 recombinant plasmid and found that Dlx5 overexpression promoted neuronal differentiation of in vitro cultured SVZa neural precursors. Taken together, our data suggest that Dlx5 plays an important role during neuronal differentiation.

No Association Between Single Nucleotide Polymorphisms in Distal-Less Homeobox-6 (DLX6) and Autism Spectrum Disorders (ASD) from the Korean Male Population

OBJECTIVES: Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is characterized by abnormalities of social functioning, communication and behavior. The association of the 7q21-34 region with ASD has been reported. The DLX6 gene, which is located at the 7q22 region, is one of the positional and functional candidate genes for ASD. We found that there is no association between DLX6 polymorphisms and ASD in the Korean male population. METHODS: We selected three single nucleotide polymorphisms (SNPs) that might be implicated in the change of the DLX6 gene expression. The genomic DNA was collected from the venous blood of 147 male controls and 179 male patients with ASD. The genotypes of the selected SNPs were determined using the Illumina GoldenGate assay, and the statistical analyses were performed using HapAnalyzer software and SAS Enterprise. RESULTS: We found no association of the three SNPs in the DLX6 gene with ASD in the Korean male population. CONCLUSION: Our study suggests that the three SNPs in the DLX6 gene are not associated with ASD, and we need to analyze the previously reported regions for their associations with ASD.

Expression of Msx1 and Dlx1 during Dumbo rat head development: correlation with morphological features

The Dumbo rat possesses some characteristics that evoke several human syndromes, such as Treacher-Collins: shortness of the maxillary, zygomatic and mandibular bones, and low position of the ears. Knowing that many homeobox genes are candidates in craniofacial development, we investigated the involvement of the Msx1 and Dlx1 genes in the Dumbo phenotype with the aim of understanding their possible role in abnormal craniofacial morphogenesis and examining the possibility of using Dumbo rat as an experimental model for understanding abnormal craniofacial development. We studied the expression of these genes during craniofacial morphogenesis by RT-PCR method. We used Dumbo embryos at E12 and E14 and included the Wistar strain as a control. Semi-quantitative PCR analysis demonstrated that Msx1 and Dlx1 are expressed differently between Dumbo and Wistar rats, indicating that their low expression may underly the Dumbo phenotype.

The effect of DLX4 on the apoptosis of choriocarcinoma cells and its related mechanism / 肿瘤

Objective: To explore the effect of homeodomain protein DLX4 on apoptosis of choriocarcinoma cells and its related mechanism. Methods: RNA interference (RNAi) was used to knockdown DLX4 expression in JEG-3 cells. Then the inhibitory effect of DLX4 on apoptosis was determined by flow cytometry and the expression of apoptosis-related protein cleaved caspase-3 and caspase-8 and bax was detected by western blotting. Results: RNA interference targeted for DLX4 effectively and specifically inhibited DLX4 mRNA expression and decreased DLX4 protein level in JEG-3 cells. The apoptotic rate of JEG-3 cells was increased, the expression levels of cleaved both caspase-3 and caspase-8 were elevated but the bax protein expression did not change after knocking down DLX4. Conclusion: RNA interference silences DLX4 expression and induces apoptosis of JEG-3 cells. The mechanism for apoptosis was related with transcriptional regulation on caspase-3 and caspase-8.

The inhibitory effect of DLX4 RNAi on adhesion and invasion ability of choriocarcinoma cells / 中国癌症杂志

Background and purpose:Choriocarcinoma is a highly malignant tumor,which metastasis could occur at early stage,the molecular mechanism is still under investigation.DLX4 is a member of homeodomain transcriptional factors,and its abnormal expression has a close correlation with mammary cancer,colon caner and so on.Based on our previous study that DLX4 was highly expressed in choriocarcinoma JEG-3 cell lines,we used RNAi to knock down the expression of DLX4,and aimed to explore the effect of DLX4 on the adhesion,movement and invasion ability of choriocarcinoma cells.Methods:DLX4 siRNA and control siRNA plasmid were constructed and transfected into choriocarcinoma cell line JEG-3 cells with LipofectamineTM2000.JEG-3 cells that expressed high level of DLX4 siRNA and control siRNA plasmid were selected and maintained with G418.The effect of RNAi was detected by western blot,and then the adhesion,movement and invasion ability of JEG-3 cells were investigated by cell-ECM adhesion assay,Transwell chamber and Transwell-Matrigel model,respectively.Results:DLX4 RNAi inhibited DLX4 protein expression specifically and effectively.The adhesion rate of the cells in JEG-3 siDLX4 group was(30.6?4.2)%,which was significantly less than(57.2?4.0)% in JEG-3 without transfenction and(51.6?3.9)% in JEG-3 scDLX4 groups(P0.05).Conclusions:The knock-down of DLX4 by RNAi could prevent the adhesion and invasion ability of choriocarcinoma cells,which provides a new thought for further research in invasion and metastasis of choriocarcinoma.

The Roles of S1P on Placental Development / 체질인류학회지

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, has various functions to affect many signalling pathways leading to cellular proliferation and differentiation and to regulate of cell migration, invasion, and angiogenesis. However, there are little reports about the relation between trophoblast stem cells and S1P. Thus, the physiologic effects of S1P on trophoblast stem (TS) cells were investigated in this study. S1P was involved in early stage development of trophoblast via upregulation of Eomesodermine mRNA expression and suppressed differentiation of TS cells through activation of extracellular-signal regulated kinase (ERK) activation. Other actions of S1P were the activation of p38 and the induction of Dlx-3 mRNA expression for angiogenesis in TS cells. Interestingly, TS cells cultured with S1P for 4 days in thrombin-fibrinogen gel culture system, specific culture system for endothelial cells, showed good healthy appearance, but TS cells cultured without S1P got severe damages. Taken together, we suggest that S1P has very important roles on placenta such as development of early stage trophoblast, suppression of differentiation, and angiogenesis on placenta.