Craniosynostosis is a common birth defect affecting 1 of the 2200 live births causing severe skull and cognitive defects, due to premature cranial suture fusion. The current surgical treatments require invasive calvaria vault remodeling and cranial bone resection in the baby. We demonstrate that inhibition of miR-200a in PMIS-miR-200a mice results in coronal suture fusion (craniosynostosis). Therefore, we use overexpression of miR-200a to prevent suture fusion in Twist1 mutant mice, a well-known model for craniosynostosis. We developed a PEGylated-peptide nanoparticle system to deliver plasmid DNA expressing miR-200a directly to the sutures of postnatal day 4 (P4) Twist1 mutant mice before suture fusion. Injection of the miR-200a nanoparticles under the scalp before suture fusion at P7 to P10 inhibited suture fusion. Treatments increased Gli1- and Six2-positive suture stem cells and the thickness of the periosteum layer. The treated Twist1+/- mice increased body weight and were alert and active. We demonstrate an effective noninvasive gene therapy treatment for craniosynostosis.

Ischemic injury and adverse post-infarction myocardial remodeling are major causes of heart failure worldwide. Strategies designed to offset cell death with cardiomyocyte regeneration have, to date, not translated to routine clinical practice. Mir-200c modulates the transcripts of cardiogenic transcription factors (TFs) Tbx5 , Gata4 , Mef2c to promote myocyte maturation. We hypothesized that inhibition of mir-200c activity would result in postnatal myocyte de-differentiation and replication sufficient to ameliorate post-infarction decompensation after ischemic injury. To test this hypothesis, we preformed left coronary ligation on Wild-Type (WT) and transgenic mice expressing an RNA inhibitor against miR-200c ( PMIS-C ). Echocardiographic left ventricular (LV) ejection fraction (EF) fell from 88.41 ± 1.08% (WT) and 90% ± 1.42% ( PMIS-C ) (p=NS) to 28% ± 11.55% (WT) and 36% ± 9.35% at 1 day post injury (DPI) (p =NS). At 21DPI, LVEF was 27% ± 4.31% (WT) and 64% ± 4.25% ( PMIS-C ) [p ≤ 0.0001]. Post-infarction LV chamber dilation was reversed in PMIS-C mice (1.136 ± 0.19 v 0.67 ± 0.06 p ≤ 0.004 LV Vol/mass). By 9 WPI, PMIS-C heart function within non-significantly levels of sham and function prior to injury. These results indicated that inhibiting miR-200c following ischemic injury can recover cardiac function and prevent LV dilation. Moreover, trichrome stain showed a decrease in fibrosis 3 WPI (WT: 57% ± 17.46 v PMIS-C : 12% ± 3.01 p ≤ 0.001) and 9 WPI. To understand the mechanism causing this phenomenon, we analyzed expression of cardiac progenitor markers. Fold change in expression of TFs Tbx5, Gata4, Mef2c, and Isl1 were increased at 1 and 3 DPI in PMIS-C . At 1 WPI, PMIS-C border zone CMs express markers of a “progenitor-like” cell state seen during cardiogenesis. Conclusions: Inhibiting miR-200c abrogates post-infarction LV remodeling and significantly restores LV systolic function. Ongoing studies are investigating the therapeutic use of PMIS-C through delivery via AAV and nanoparticles.

Transcription factors such as Tbx5, Gata4, Mef2c and Pitx2 are required during cardiac development, and in adult cardiac homeostasis. We demonstrate that the gene dosage and modulation of these factors are mediated in vivoby the miR-200 family. These microRNAs (miRs) are expressed during the earlystages of heart development, and they regulate a highly complex gene regulatory network (GRN). To study the in vivo and in vitro function of specific miR-200 family members, we inhibited individual members of the miR-200 family during embryonic development. Inhibition of a single miR-200 family member within the cluster caused defects in the left ventricle and cardiomyocyte maturation during development. Inhibition of the entire miR-200 family resulted in a ventricular septal defect and embryonic lethality by embryonic day (E)16.5. In cardiomyocytes, the miR-200 family targets the transcripts of Tbx5, Gata4, Mef2c and Pitx2. Inhibition of this family increased expression of Tbx5, Gata4, Mef2c and Pitx2 in the left ventricle and atria across multiple stages of cardiac development. Embryos with reduced levels of a single miR-200 family member were non-lethal, and pups survived into adulthood. On postnatal day (P)1, these pups had a reduced heart rate and increased ventricular wall thickness. Each miR-200 family has distinct heart phenotypes in cell specific differentiation and maturation. snRNA-sequencing revealed a new cardiomyocyte cell state, suggesting these cells were less differentiated due to inhibition of the miR-200 family. We have identified several new transcription factors regulated by miR-200during heart development. The miR-200 family members are critical regulators of early cardiac development through maintaining gene dosage of Tbx5, Gata4, Mef2c and Pitx2, which affects cardiomyocyte differentiation and maturation. The high throughput multimodal analyses of early heart development after miR-200 inhibition revealed a new cardiomyocyte population, with characteristics of an immature cell state and chromatin modifications near Tbx5 transcriptional targets.

Congenital heart defects (CHDs) are be caused by mutations in genes that drive cardiac development, such as Tbx5 , Gata4 , and Mef2c . Cardiac development, and its transcriptional regulators are also regulated by microRNAs (miRs). These small RNAs target the transcripts of genes in numerous cardiac cell types during embryonic development. We and others have shown that the miR-200 family modulates the transcripts of cardiogenic transcription factors (TFs) Tbx5 , Gata4 , and Mef3c. However, the relationship between these miRs and cardiogenic TFs during in vivo cardiac development is poorly understood. During cardiogenesis, miR-200 family members are highly expressed (E14.5) but reduced in adults (3mo) (Ct Value: miR-200a : 24.3 ± 0.59 v 38.3 ± 0.21; miR-200c : 25.0 ± 0.64 v 32.5 ± 0.16). Using our miR-200 family inhibitor mice models (PMIS), we have found these miRs are required for cardiac development. PMIS-miR-200 embryos are found with a ventral septal defect and poor ventricle wall development, which is lethal by e16.5. At e14.5, PMIS-miR-200 hearts have a significant increase in expression of Tbx5, Gata4, and Mef2c compared to Wild-Type. This induced expression of these TFs is seen within CMs of the ventricle at E14.5. snMulti-Omics of WT and PMIS-miR-200 hearts found a population of CMs enriched in the PMIS-miR-200 hearts. These CMs are marked by expression of Tbx5 , Nppa , and Sox5 . RNA velocity analysis found these CMs to be “progenitor-like” and associated with an early pseudotime. Expression of Tbx5 , Nppa , and Sox5 correlated along the pseudotime with the “progenitor-like” CMs. ATAC-seq showed enrichment of Tbx5 and Mef2c motifs within this new CM cell state. Conclusions: The miR-200 family is a modulator of cardiogenic TFs expression and activity during development. Inhibition of miR-200 induces a CM cell state with “progenitor-like” qualities. Future directions will determine the role of miR- 200 in adult cardiac disease, such as ischemic injury. Our work provides new insights into gene dosage, modulated by miRs, that is required and necessary during cardiac development.

Wolf-Hirschhorn syndrome (WHS) is a developmental disorder attributed to a partial deletion on the short arm of chromosome 4. WHS patients suffer from oral manifestations including cleft lip and palate, hypodontia, and taurodontism. WHS candidate 1 (WHSC1) gene is a H3K36-specific methyltransferase that is deleted in every reported case of WHS. Mutation in this gene also results in tooth anomalies in patients. However, the correlation between genetic abnormalities and the tooth anomalies has remained controversial. In our study, we aimed to clarify the role of WHSC1 in tooth development. We profiled the Whsc1 expression pattern during mouse incisor and molar development by immunofluorescence staining and found Whsc1 expression is reduced as tooth development proceeds. Using real-time quantitative reverse transcription PCR, Western blot, chromatin immunoprecipitation, and luciferase assays, we determined that Whsc1 and Pitx2, the initial transcription factor involved in tooth development, positively and reciprocally regulate each other through their gene promoters. miRNAs are known to regulate gene expression posttranscriptionally during development. We previously reported miR-23a/b and miR-24-1/2 were highly expressed in the mature tooth germ. Interestingly, we demonstrate here that these two miRs directly target Whsc1 and repress its expression. Additionally, this miR cluster is also negatively regulated by Pitx2. We show the expression of these two miRs and Whsc1 are inversely correlated during mouse mandibular development. Taken together, our results provide new insights into the potential role of Whsc1 in regulating tooth development and a possible molecular mechanism underlying the dental defects in WHS.

OBJECTIVE:The objective of the study was to investigate differential gene expression between murine right and left maxilla-mandibular (MxMn) complexes. SETTING AND SAMPLE POPULATION:Wild-type (WT) C57BL/6 embryonic (E) day 14.5 (n = 3) and 18.5 (n = 3) murine embryos. METHODS:The E14.5 and 18.5 embryos were harvested and hemi-sectioned the MxMn complexes into right and left halves in the mid-sagittal plane. We isolated total RNA using Trizol reagent and further purified using the RNA-easy kit (QIAGEN). We confirmed equal expression of house-keeping genes in right and left halves using RT-PCR and then performed paired-end whole mRNA sequencing in LC Sciences (Houston, TX) followed by differential transcript analyses (>1 or <-1 log fold change; p < .05; q < .05; and FPKM >0.5 in 2/3 samples). The Mouse Genome Informatics and Online Mendelian Inheritance in Man databases as well as gnomAD constraint scores were used to prioritize differentially expressed transcripts. RESULTS:There were 19 upregulated and 19 downregulated transcripts at E14.5 and 8 upregulated and 17 downregulated transcripts at E18.5 time-points. These differentially expressed transcripts were statistically significant and shown to be associated with craniofacial phenotypes in mouse models. These transcripts also have significant gnomAD constraint scores and are enriched in biological processes critical for embryogenesis. CONCLUSIONS:We identified significant differential expression of transcripts between E14.5 and 18.5 murine right and left MxMn complexes. These findings when extrapolated to humans, they may provide a biological basis for facial asymmetry. Further experiments are required to validate these findings in murine models with craniofacial asymmetry.

Stem cells are potent to self‐renew and differentiate which contribute to the regenerative process of various mature tissue types. It is important to understand how stem cell behaviors are regulated to give further insights to the application of stem cell therapies in tissue regeneration and repair. We use the developing murine lower incisor as a model to study stem cell maintenance and differentiation, which are housed in a niche and are fated to only a handful of differentiated cell types. Tooth development is a complex process involving rigid temporal and reciprocal regulation of transcription factors including Sox2 and Lef‐1, and signaling pathways including Wnt. We are particularly interested in the regulation between Lef‐1 and miR‐26b due to their inverse expression profiles during tooth development. Lef1 harbors a miR‐26b binding site in its 3’‐UTR and is negatively regulated by miR‐26b. In this study, we aim to understand the role of Lef‐1 and miR‐26b in dental epithelial stem cell (DESC) differentiation, determine the molecular mechanism of miR‐26b regulated Lef‐1 expression and identify genetic pathways for tooth development and regeneration.

Wolf‐Hirschhorn syndrome (WHS) is a developmental disorder attributed to a partial deletion on the short arm of chromosome 4. WHS patients suffer from oral manifestations including cleft lip and palate (CLP), hypodontia and taurodontism. However, the causative factors and underlying mechanisms of these oral anomalies are relatively unknown. Wolf‐Hirschhorn syndrome candidate 1 (WHSC1) is a H3K36‐specific methyltransferase that is frequently deleted in WHS. This gene has been associated with craniofacial defects including CLP and defects in occipital ossification. In our study, we aim to understand the role of WHSC1 in tooth development.

The developmental role ofLef-1in ectodermal organs has been characterized usingLef-1murine knockout models. We generated aLef-1conditional over-expression (COEL) mouse to determine the role ofLef-1expression in epithelial structures at later stages of development after endogenous expression switches to the mesenchyme.Lef-1over expression (OE) in the oral epithelium creates a new dental epithelial stem cell niche that significantly increases incisor growth. These data indicate thatLef-1expression is switched off in the dental epithelial at early stages to maintain the stem cell niche and regulate incisor growth. Bioinformatics analyses indicated thatmiR-26bexpression increased coinciding with decreasedLef-1expression in the dental epithelium. We generated a murine model over-expressingmiR-26bthat targets endogenousLef-1expression andLef-1-related developmental mechanisms.miR-26bOE mice have ectodermal organ defects including a lack of incisors, molars, and hair similar to theLef-1null mice.miR-26bOE rescues theLef-1OE phenotype demonstrating a critical genetic and developmental role formiR-26bin the temporal and spatial expression ofLef-1in epithelial tissues.Lef-1expression regulates Wnt signaling and Wnt target genes as well as cell proliferation mechanisms, whilemiR-26bOE reduced the levels of Wnt target gene expression. The extra stem cell compartment in theCOELmice expressedLef-1suggesting thatLef-1is a stem cell factor, which was absent in themiR-26b OE/COELrescue mice. This is the first demonstration of a microRNA OE mouse model that has ectodermal organ defects. These findings demonstrate that the levels ofLef-1are critical for development and establish a role formiR-26bin the regulation of ectodermal organ development through the control ofLef-1expression and an endogenous stem cell niche.

The developmental role ofLef-1in ectodermal organs has been characterized usingLef-1murine knockout models. We generated aLef-1conditional over-expression (COEL) mouse to determine the role ofLef-1expression in epithelial structures at later stages of development after endogenous expression switches to the mesenchyme.Lef-1over expression (OE) in the oral epithelium creates a new dental epithelial stem cell niche that significantly increases incisor growth. These data indicate thatLef-1expression is switched off in the dental epithelial at early stages to maintain the stem cell niche and regulate incisor growth. Bioinformatics analyses indicated thatmiR-26bexpression increased coinciding with decreasedLef-1expression in the dental epithelium. We generated a murine model over-expressingmiR-26bthat targets endogenousLef-1expression andLef-1-related developmental mechanisms.miR-26bOE mice have ectodermal organ defects including a lack of incisors, molars, and hair similar to theLef-1null mice.miR-26bOE rescues theLef-1OE phenotype demonstrating a critical genetic and developmental role formiR-26bin the temporal and spatial expression ofLef-1in epithelial tissues.Lef-1expression regulates Wnt signaling and Wnt target genes as well as cell proliferation mechanisms, whilemiR-26bOE reduced the levels of Wnt target gene expression. The extra stem cell compartment in theCOELmice expressedLef-1suggesting thatLef-1is a stem cell factor, which was absent in themiR-26b OE/COELrescue mice. This is the first demonstration of a microRNA OE mouse model that has ectodermal organ defects. These findings demonstrate that the levels ofLef-1are critical for development and establish a role formiR-26bin the regulation of ectodermal organ development through the control ofLef-1expression and an endogenous stem cell niche.

Orofacial clefts are common developmental disorders that pose significant clinical, economical and psychological problems. We conducted genome-wide association analyses for cleft palate only (CPO) and cleft lip with or without palate (CL/P) with ~17 million markers in sub-Saharan Africans. After replication and combined analyses, we identified novel loci for CPO at or near genome-wide significance on chromosomes 2 (near CTNNA2) and 19 (near SULT2A1). In situ hybridization of Sult2a1 in mice showed expression of SULT2A1 in mesenchymal cells in palate, palatal rugae and palatal epithelium in the fused palate. The previously reported 8q24 was the most significant locus for CL/P in our study, and we replicated several previously reported loci including PAX7 and VAX1.

Tooth development proceeds through a series of steps wherein epithelial and mesenchymal tissue layers cooperate to form a tooth bud, and cells in the tooth bud are organized and differentiated into a mature tooth. Throughout this process, the changes in gene expression that are required for tooth formation are prompted by transcription factors. Lef‐1 is a transcription factor and a major effector of WNT signaling, and the loss of Lef‐1 expression or an ectopic increase in Lef‐1 results in dramatic dental anomalies in mice. The transcriptional mechanisms responsible for maintaining appropriate levels of Lef‐1 expression and downstream targets are not well understood. We show that Sox2, a major regulator of dental stem cells inhibits Lef‐1 transcriptional activation. Endogenous Sox2 directly binds to Lef‐1 and we have identified specific motifs in the Sox2 and Lef‐1 proteins regulating this interaction. By immobilizing different GST‐labeled Sox2 domains we show that the HMG domain of Sox2 is required for the Sox2‐Lef1 protein interaction. Functionally, Sox2 is capable of disrupting the association of Lef‐1 protein with its consensus DNA binding sequence. Taken together, these data suggest a novel mechanism wherein the Sox2 protein functions to negatively regulate the expression of Lef‐1 and Lef‐1 target genes during odontogenisis. These new mechanisms may also regulate other epithelial organs during development. Support or Funding Information Iowa Institute for Oral Health Research, College of Dentistry, The University of Iowa, Iowa City, IA.

Iroquois 1 (Irx1) is a homobox transcription factor that was identified in craniofacial tissues through RNA‐sequencing and bioinformatics analyses. Irx1 is expressed in specific dental tissues during development and adult stages. Irx1 is present in the outer enamel epithelium and the stratum intermedium at prenatal stages. The expression of Irx1 is maintained until tooth eruption and at this stage it appears to play a role in root formation. Heterozygous Irx1 knockout mice show Irx1 expression in the Hertwig Epithelial Root Sheath and in the outer enamel epithelium at post‐natal stage p12 in molars. Epithelial rests of Malassez and isolated cells in the inter‐radicular cementum are positive for Irx1 expression. The presence of Irx1 is nonexistent in late stages on the root surface. Differences in Irx1 levels can be found in cultured cementoblast and periodontal ligament cells. Cementoblasts have a higher level of Irx1 than periodontal ligament cells. These findings were confirmed by real‐time PCR and protein levels. Under mineralization conditions cementoblasts show a reduction in the level of Irx1 expression. The presence of Irx1 during tooth development is indicative of the implication of this novel transcription factor in tooth formation. Moreover, the positive expression of Irx1 in cementoblasts under non‐mineralization conditions and its reduction of expression under‐mineralization conditions may suggest it regulates genes involved in root formation and possibly the origin progenitor cells of the cementum. Support or Funding Information Miguel Romero‐Bustillos would like to acknowledge the support received from the NIH under the R90 DE024296‐03 grant.

MicroRNAs (miRs) are small non‐coding RNAs that bind to target mRNAs and negatively regulate their expression. The expression of miRs are altered in many cancers and cancer cell types. We previously generated a miR‐17‐92 knockdown mouse model using the Plasmid based MicroRNA Inhibitor System (PMIS). With this new PMIS system we found that thyroid development was abnormal or absence in these mice. Thus, thyroid development requires miR‐17 for normal development. We investigated the role of miR‐17 in SW579 thyroid cancer cells, which are derived from human thyroid tissue. PMIS‐miR‐17, specifically targeting miR‐17‐5p completely inhibited the activity of miR‐17‐5p in SW579 cells. However, inhibiting miR‐17‐5p activity in SW579 thyroid cancer cells also promotes tumor growth in a nude mouse model for thyroid cancer. The tumor formation was due to increase cell proliferation and aberrant gene expression. RNA‐seq data shows knocking down miR‐17 in thyroid cancer cells induces large‐scale changes in miR‐17‐5p target gene expression, including PDGFR and MMP2, which may play oncogenic roles in tumorigenesis. Bioinformatics analyses has identified several new gene expression networks regulated by miR‐17 in thyroid cancer. We have identified several new gene expression mechanisms for miR‐17 and more importantly regulation of miR‐17‐92 expression and processing of the miR‐17‐92 cluster during thyroid development and thyroid cancer.Support or Funding InformationIowa Institute for Oral Health Research, College of Dentistry, The University of Iowa, Iowa City, IA

The t(15;17) translocation, found in 95% of acute promyelocytic leukemia, encodes a promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) fusion protein. Complete remission of acute promyelocytic leukemia can be obtained by treating patients with all-trans retinoic acid, and PML-RARalpha plays a major role in mediating retinoic acid effects in leukemia cells. A main model proposed for acute promyelocytic leukemia is that PML-RARalpha exerts its oncogenic effects by repressing the expression of retinoic acid-inducible genes critical to myeloid differentiation. By applying subtraction cloning to acute promyelocytic leukemia cells, we identified a retinoic acid-induced gene, PRAM-1 (PML-RARalpha target gene encoding an Adaptor Molecule-1), which encodes a novel adaptor protein sharing structural homologies with the SLAP-130/fyb adaptor. PRAM-1 is expressed and regulated during normal human myelopoiesis. In U937 myeloid precursor cells, PRAM-1 expression is inhibited by expression of PML-RARalpha in the absence of ligand and de novo superinduced by retinoic acid. PRAM-1 associates with other adaptors, SLP-76 and SKAP-55HOM, in myeloid cell lines and with protein tyrosine kinase lyn. By providing the first evidence that PML-RARalpha dysregulates expression of an adaptor protein, our data open new insights into signaling events that are disrupted during transformation by PML-RARalpha and induced by retinoic acid during de novo differentiation of acute promyelocytic leukemia cells.

The interaction between CD95 (Fas) and CD95L (Fas ligand) initiates apoptosis in a variety of cell types. Although the regulation of CD95L expression on activated T cells is an area of intense study, knowledge related to the induction of CD95L promoter activity in primary T cells is lacking. In this report we describe the generation of a novel transgenic mouse strain, CD95LP-Luc, in which murine CD95L promoter sequence controls the expression of a luciferase reporter gene. We use these mice to illustrate several important findings related to transcriptional regulation of CD95L in primary T cells. We demonstrate that maximal CD95L promoter activity occurs only after prolonged T cell stimulation and requires costimulation through CD28. We provide evidence that thymocytes express CD95L/luciferase after strong TCR ligation and that inducible CD95L promoter activation is present, but unequal, in both Th1 and Th2 effector cells. We also illustrate that while agonist peptide presentation by APCs generates robust proliferation during a primary T cell response, the same stimulus induces only modest CD95L promoter activity. These results suggest alternate explanations for the well-characterized delay in CD95-mediated activation-induced cell death following initial ligation of the TCR.

Significant fibrosis and acinar atrophy are characteristics of chronic pancreatitis; however, because of the lack of a reproducible model, early phases of these changes are poorly understood. We have developed a model of severe hyperstimulation and obstruction pancreatitis (SHOP) to better define the mechanisms of early pancreatic fibrogenesis. Sprague-Dawley rats were used and SHOP was induced by complete pancreatic duct obstruction and daily cerulein hyperstimulation (50 (μg/kg intraperitoneally). Animals were killed at 24, 48, 72, and 96 hours. Control animals underwent sham operation and received no cerulein. Pancreata were prepared for hematoxylin and eosin and sirius red (collagen-specific) staining and for hydroxyproline assay (measure of total collagen content). We found moderate amounts of edema and inflammation but minimal parenchymal necrosis. Significant loss of acinar cell mass was noted by 48 hours, and normal acinar cells were essentially absent by 96 hours. Tissue collagen content increased with time and large amounts of interstitial collagen were detected by 72 hours. In conclusion, SHOP is a novel model of early pancreatic fibrosis associated with minimal necrosis and a significant decrease in acinar cell mass, making it an ideal model to study the early cellular mechanisms of pancreatic fibrogenesis.