Browsing by Author "Vincentz, Joshua W."

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    Exclusion of Dlx5/6 expression from the distal-most mandibular arches enables BMP-mediated specification of the distal cap
    (Proceedings of the National Academy of Sciences, 2016-07-05) Vincentz, Joshua W.; Casasnovas, Jose J.; Barnes, Ralston M.; Que, Jianwen; Clouthier, David E.; Wang, Jun; Firulli, Anthony B.; Department of Pediatrics, IU School of Medicine
    Cranial neural crest cells (crNCCs) migrate from the neural tube to the pharyngeal arches (PAs) of the developing embryo and, subsequently, differentiate into bone and connective tissue to form the mandible. Within the PAs, crNCCs respond to local signaling cues to partition into the proximo-distally oriented subdomains that convey positional information to these developing tissues. Here, we show that the distal-most of these subdomains, the distal cap, is marked by expression of the transcription factor Hand1 (H1) and gives rise to the ectomesenchymal derivatives of the lower incisors. We uncover a H1 enhancer sufficient to drive reporter gene expression within the crNCCs of the distal cap. We show that bone morphogenic protein (BMP) signaling and the transcription factor HAND2 (H2) synergistically regulate H1 distal cap expression. Furthermore, the homeodomain proteins distal-less homeobox 5 (DLX5) and DLX6 reciprocally inhibit BMP/H2-mediated H1 enhancer regulation. These findings provide insights into how multiple signaling pathways direct transcriptional outcomes that pattern the developing jaw.
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    Hand factor ablation causes defective left ventricular chamber development and compromised adult cardiac function
    (PLOS, 2017-07-21) Vincentz, Joshua W.; Toolan, Kevin P.; Zhang, Wenjun; Firulli, Anthony B.; Pediatrics, School of Medicine
    Coordinated cardiomyocyte growth, differentiation, and morphogenesis are essential for heart formation. We demonstrate that the bHLH transcription factors Hand1 and Hand2 play critical regulatory roles for left ventricle (LV) cardiomyocyte proliferation and morphogenesis. Using an LV-specific Cre allele (Hand1LV-Cre), we ablate Hand1-lineage cardiomyocytes, revealing that DTA-mediated cardiomyocyte death results in a hypoplastic LV by E10.5. Once Hand1-linage cells are removed from the LV, and Hand1 expression is switched off, embryonic hearts recover by E16.5. In contrast, conditional LV loss-of-function of both Hand1 and Hand2 results in aberrant trabeculation and thickened compact zone myocardium resulting from enhanced proliferation and a breakdown of compact zone/trabecular/ventricular septal identity. Surviving Hand1;Hand2 mutants display diminished cardiac function that is rescued by concurrent ablation of Hand-null cardiomyocytes. Collectively, we conclude that, within a mixed cardiomyocyte population, removal of defective myocardium and replacement with healthy endogenous cardiomyocytes may provide an effective strategy for cardiac repair., The left ventricle of the heart drives blood flow throughout the body. Impaired left ventricle function, associated either with heart failure or with certain, severe cardiac birth defects, constitutes a significant cause of mortality. Understanding how heart muscle grows is vital to developing improved treatments for these diseases. Unfortunately, genetic tools necessary to study the left ventricle have been lacking. Here we engineer the first mouse line to enable specific genetic study of the left ventricle. We show that, unlike in the adult heart, the embryonic left ventricle is remarkably tolerant of cell death, as remaining cells have the capacity to proliferate and to restore heart function. Conversely, disruption of two related genes, Hand1 and Hand2, within the left ventricle causes cells to assume the wrong identity, and to consequently overgrow and impair cardiac function. Ablation of these mutant cells rescues heart function. We conclude that selective removal of defective heart muscle and replacement with healthy cells may provide an effective therapy to treat heart failure.
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    HAND transcription factors cooperatively specify the aorta and pulmonary trunk
    (Elsevier, 2021) Vincentz, Joshua W.; Firulli, Beth A.; Toolan, Kevin P.; Osterwalder, Marco; Pennacchio, Len A.; Firulli, Anthony B.; Pediatrics, School of Medicine
    Congenital heart defects (CHDs) affecting the cardiac outflow tract (OFT) constitute a significant cause of morbidity and mortality. The OFT develops from migratory cell populations which include the cardiac neural crest cells (cNCCs) and secondary heart field (SHF) derived myocardium and endocardium. The related transcription factors HAND1 and HAND2 have been implicated in human CHDs involving the OFT. Although Hand1 is expressed within the OFT, Hand1 NCC-specific conditional knockout mice (H1CKOs) are viable. Here we show that these H1CKOs present a low penetrance of OFT phenotypes, whereas SHF-specific Hand1 ablation does not reveal any cardiac phenotypes. Further, HAND1 and HAND2 appear functionally redundant within the cNCCs, as a reduction/ablation of Hand2 on an NCC-specific H1CKO background causes pronounced OFT defects. Double conditional Hand1 and Hand2 NCC knockouts exhibit persistent truncus arteriosus (PTA) with 100% penetrance. NCC lineage-tracing and Sema3c in situ mRNA expression reveal that Sema3c-expressing cells are mis-localized, resulting in a malformed septal bridge within the OFTs of H1CKO;H2CKO embryos. Interestingly, Hand1 and Hand2 also genetically interact within the SHF, as SHF H1CKOs on a heterozygous Hand2 background exhibit Ventricular Septal Defects (VSDs) with incomplete penetrance. Previously, we identified a BMP, HAND2, and GATA-dependent Hand1 OFT enhancer sufficient to drive reporter gene expression within the nascent OFT and aorta. Using these transcription inputs as a probe, we identify a novel Hand2 OFT enhancer, suggesting that a conserved BMP-GATA dependent mechanism transcriptionally regulates both HAND factors. These findings support the hypothesis that HAND factors interpret BMP signaling within the cNCCs to cooperatively coordinate OFT morphogenesis.
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    Hand1 phosphoregulation within the distal arch neural crest is essential for craniofacial morphogenesis
    (The Company of Biologists, 2014-08) Firulli, Beth A.; Fuchs, Robyn K.; Vincentz, Joshua W.; Clouthier, David E.; Firulli, Anthony B.; Department of Pediatrics, IU School of Medicine
    In this study we examine the consequences of altering Hand1 phosphoregulation in the developing neural crest cells (NCCs) of mice. Whereas Hand1 deletion in NCCs reveals a nonessential role for Hand1 in craniofacial development and embryonic survival, altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, in NCCs results in severe mid-facial clefting and neonatal death. Hand1 phosphorylation mutants exhibit a non-cell-autonomous increase in pharyngeal arch cell death accompanied by alterations in Fgf8 and Shh pathway expression. Together, our data indicate that the extreme distal pharyngeal arch expression domain of Hand1 defines a novel bHLH-dependent activity, and that disruption of established Hand1 dimer phosphoregulation within this domain disrupts normal craniofacial patterning.
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    Hand2 is an essential regulator for two Notch-dependent functions within the embryonic endocardium
    (Elsevier, 2014-12-24) VanDusen, Nathan J.; Casanovas, Jose; Vincentz, Joshua W.; Firulli, Beth A.; Osterwalder, Marco; Lopez-Rios, Javier; Zeller, Rolf; Zhou, Bin; Grego-Bessa, Joaquim; De La Pompa, José Luis; Shou, Weinian; Firulli, Anthony B.; Department of Pediatrics, IU School of Medicine
    The basic-helix-loop-helix (bHLH) transcription factor Hand2 plays critical roles during cardiac morphogenesis via expression and function within myocardial, neural crest, and epicardial cell populations. Here, we show that Hand2 plays two essential Notch-dependent roles within the endocardium. Endocardial ablation of Hand2 results in failure to develop a patent tricuspid valve, intraventricular septum defects, and hypotrabeculated ventricles, which collectively resemble the human congenital defect tricuspid atresia. We show endocardial Hand2 to be an integral downstream component of a Notch endocardium-to-myocardium signaling pathway and a direct transcriptional regulator of Neuregulin1. Additionally, Hand2 participates in endocardium-to-endocardium-based cell signaling, with Hand2 mutant hearts displaying an increased density of coronary lumens. Molecular analyses further reveal dysregulation of several crucial components of Vegf signaling, including VegfA, VegfR2, Nrp1, and VegfR3. Thus, Hand2 functions as a crucial downstream transcriptional effector of endocardial Notch signaling during both cardiogenesis and coronary vasculogenesis.
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    Loss of Hand2 in a population of Periostin lineage cells results in pronounced bradycardia and neonatal death
    (Elsevier, 2014-04-15) VanDusen, Nathan J.; Vincentz, Joshua W.; Firulli, Beth A.; Howard, Marthe J.; Rubart, Michael; Firulli, Anthony B.; Department of Pediatrics, IU School of Medicine
    The Periostin Cre (Postn-Cre) lineage includes endocardial and neural crest derived mesenchymal cells of the cardiac cushions, neural crest-derived components of the sympathetic and enteric nervous systems, and cardiac fibroblasts. In this study, we use the Postn-Cre transgenic allele to conditionally ablate Hand2 (H2CKO). We find that Postn-Cre H2CKOs die shortly after birth despite a lack of obvious cardiac structural defects. To ascertain the cause of death, we performed a detailed comparison of the Postn-Cre lineage and Hand2 expression at mid and late stages of embryonic development. Gene expression analyses demonstrate that Postn-Cre ablates Hand2 from the adrenal medulla as well as the sphenopalatine ganglia of the head. In both cases, Hand2 loss-of-function dramatically reduces expression of Dopamine Beta Hydroxylase (Dbh), a gene encoding a crucial catecholaminergic biosynthetic enzyme. Expression of the genes Tyrosine Hydroxylase (Th) and Phenylethanolamine N-methyltransferase (Pnmt), which also encode essential catecholaminergic enzymes, were severely reduced in postnatal adrenal glands. Electrocardiograms demonstrate that 3-day postnatal Postn-Cre H2CKO pups exhibit sinus bradycardia. In conjunction with the aforementioned gene expression analyses, these results strongly suggest that the observed postnatal lethality occurs due to a catecholamine deficiency and subsequent heart failure.
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    Mis-Expression of a Cranial Neural Crest Cell-Specific Gene Program in Cardiac Neural Crest Cells Modulates HAND Factor Expression, Causing Cardiac Outflow Tract Phenotypes
    (MDPI, 2020-04-20) Vincentz, Joshua W.; Clouthier, David E.; Firulli, Anthony B.; Pediatrics, School of Medicine
    Congenital heart defects (CHDs) occur with such a frequency that they constitute a significant cause of morbidity and mortality in both children and adults. A significant portion of CHDs can be attributed to aberrant development of the cardiac outflow tract (OFT), and of one of its cellular progenitors known as the cardiac neural crest cells (NCCs). The gene regulatory networks that identify cardiac NCCs as a distinct NCC population are not completely understood. Heart and neural crest derivatives (HAND) bHLH transcription factors play essential roles in NCC morphogenesis. The Hand1PA/OFT enhancer is dependent upon bone morphogenic protein (BMP) signaling in both cranial and cardiac NCCs. The Hand1PA/OFT enhancer is directly repressed by the endothelin-induced transcription factors DLX5 and DLX6 in cranial but not cardiac NCCs. This transcriptional distinction offers the unique opportunity to interrogate NCC specification, and to understand why, despite similarities, cranial NCC fate determination is so diverse. We generated a conditionally active transgene that can ectopically express DLX5 within the developing mouse embryo in a Cre-recombinase-dependent manner. Ectopic DLX5 expression represses cranial NCC Hand1PA/OFT-lacZ reporter expression more effectively than cardiac NCC reporter expression. Ectopic DLX5 expression induces broad domains of NCC cell death within the cranial pharyngeal arches, but minimal cell death in cardiac NCC populations. This study shows that transcription control of NCC gene regulatory programs is influenced by their initial specification at the dorsal neural tube.
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    Variation in a Left Ventricle–Specific Hand1 Enhancer Impairs GATA Transcription Factor Binding and Disrupts Conduction System Development and Function
    (American Heart Association, 2019-08-01) Vincentz, Joshua W.; Firulli, Beth A.; Toolan, Kevin P.; Arking, Dan E.; Sotoodehnia, Nona; Wan, Juyi; Chen, Peng-Sheng; de Gier-de Vries, Corrie; Christoffels, Vincent M.; Rubart-von der Lohe, Michael; Firulli, Anthony B.; Pediatrics, School of Medicine
    Rationale The ventricular conduction system (VCS) rapidly propagates electrical impulses through the working myocardium of the ventricles to coordinate chamber contraction. Genome-wide association studies (GWAS) have associated nucleotide polymorphisms, most are located within regulatory intergenic or intronic sequences, with variation in VCS function. Two highly correlated polymorphisms (r2>0.99) associated with VCS functional variation (rs13165478 and rs13185595) occur 5’ to the gene encoding the bHLH transcription factor HAND1. Objective Here, we test the hypothesis that these polymorphisms influence HAND1 transcription thereby influencing VCS development and function. Methods and Results We employed transgenic mouse models to identify an enhancer that is sufficient for left ventricle (LV) cis-regulatory activity. Two evolutionarily conserved GATA transcription factor cis-binding elements within this enhancer are bound by GATA4 and are necessary for cis-regulatory activity, as shown by in vitro DNA binding assays. CRISPR/Cas9-mediated deletion of this enhancer dramatically reduces Hand1 expression solely within the LV but does not phenocopy previously published mouse models of cardiac Hand1 loss-of-function. Electrophysiological and morphological analyses reveals that mice homozygous for this deleted enhancer display a morphologically abnormal VCS, and a conduction system phenotype consistent with right bundle branch block. Using 1000 Genomes Project data, we identify three additional SNPs, located within the Hand1 LV enhancer, that compose a haplotype with rs13165478 and rs13185595. One of these SNPs, rs10054375, overlaps with a critical GATA cis-regulatory element within the Hand1 LV enhancer. This SNP, when tested in electrophoretic mobility shift assays (EMSA), disrupts GATA4 DNA-binding. Modeling two of these SNPs in mice causes diminished Hand1 expression and mice present with abnormal VCS function. Conclusions Together, these findings reveal that SNP rs10054375, which is located within a necessary and sufficient LV-specific Hand1 enhancer, exhibits reduces GATA DNA-binding in EMSA and this enhancer in total, is required for VCS development and function in mice and perhaps humans.