IU Indianapolis ScholarWorks :: Browsing by Author "Firulli, Beth A."

Browsing by Author "Firulli, Beth A."

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Defective Hand1 phosphoregulation uncovers essential roles for Hand1 in limb morphogenesis
(The Company of Biologists Ltd, 2017-07-01) Firulli, Beth A.; Milliar, Hannah; Toolan, Kevin P.; Harkin, Jade; Fuchs, Robyn K.; Robling, Alex G.; Firulli, Anthony B.; Anatomy and Cell Biology, School of Medicine
The morphogenesis of the vertebrate limbs is a complex process in which cell signaling and transcriptional regulation coordinate diverse structural adaptations in diverse species. In this study, we examine the consequences of altering Hand1 dimer choice regulation within developing vertebrate limbs. Although Hand1 deletion via the limb-specific Prrx1-Cre reveals a non-essential role for Hand1 in mouse limb morphogenesis, altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, results in a severe truncation of proximal-anterior limb elements. Molecular analysis reveals a non-cell-autonomous mechanism that causes widespread cell death within the embryonic limb bud. In addition, we observe changes in proximal-anterior gene regulation, including a reduction in the expression of Irx3, Irx5, Gli3 and Alx4, all of which are upregulated in Hand2 limb conditional knockouts. A reduction of Hand2 and Shh gene dosage improves the integrity of anterior limb structures, validating the importance of the Twist-family bHLH dimer pool in limb morphogenesis., Summary: Altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, results in a severe truncation of anterior-proximal limb elements in mice.
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.
The HAND1 frameshift A126FS mutation does not cause hypoplastic left heart syndrome in mice
(Oxford University Press, 2017-12-01) Firulli, Beth A.; Toolan, Kevin P.; Harkin, Jade; Millar, Hannah; Pineda, Santiago; Firulli, Anthony B.; Pediatrics, School of Medicine
Aims: To test if a human Hand1 frame shift mutation identified in human samples is causative of hypoplastic left heart syndrome (HLHS). Methods and results: HLHS is a poorly understood single ventricle congenital heart defect that affects two to three infants in every 10 000 live births. The aetiologies of HLHS are largely unknown. The basic helix-loop-helix transcription factor HAND1 is required for normal heart development. Interrogation of HAND1 sequence from fixed HLHS tissues identified a somatic frame-shift mutation at Alanine 126 (NP_004812.1 p.Ala126Profs13X defined as Hand1A126fs). Hand1A126fs creates a truncated HAND1 protein that predictively functions as dominant negative. To determine if this mutation is causative of HLHS, we engineered a conditional Hand1A126fs mouse allele. Activation of this allele with Nkx2.5Cre results in E14.5 lethality accompanied by cardiac outflow tract and intraventricular septum abnormalities. Using αMHC-Cre or Mef2CAHF-Cre to activate Hand1A126fs results in reduced phenotype and limited viability. Left ventricles of Hand1A126FS mutant mice are not hypoplastic. Conclusions: Somatically acquired Hand1A126FS mutation is not causative of HLHS. Hand1A126FS mutation does exhibit embryonic lethal cardiac defects that reflect a dominant negative function supporting the critical role of Hand1 in cardiogenesis.
HAND1 loss-of-function within the embryonic myocardium reveals survivable congenital cardiac defects and adult heart failure
(Oxford University Press, 2020-03) Firulli, Beth A.; George, Rajani M.; Harkin, Jade; Toolan, Kevin P.; Gao, Hongyu; Liu, Yunlong; Zhang, Wenjun; Field, Loren J.; Liu, Ying; Shou, Weinian; Payne, Ronald Mark; Rubart-von der Lohe, Michael; Firulli, Anthony B.; Pediatrics, School of Medicine
Aims: To examine the role of the basic Helix-loop-Helix (bHLH) transcription factor HAND1 in embryonic and adult myocardium. Methods and results: Hand1 is expressed within the cardiomyocytes of the left ventricle (LV) and myocardial cuff between embryonic days (E) 9.5-13.5. Hand gene dosage plays an important role in ventricular morphology and the contribution of Hand1 to congenital heart defects requires further interrogation. Conditional ablation of Hand1 was carried out using either Nkx2.5 knockin Cre (Nkx2.5Cre) or α-myosin heavy chain Cre (αMhc-Cre) driver. Interrogation of transcriptome data via ingenuity pathway analysis reveals several gene regulatory pathways disrupted including translation and cardiac hypertrophy-related pathways. Embryo and adult hearts were subjected to histological, functional, and molecular analyses. Myocardial deletion of Hand1 results in morphological defects that include cardiac conduction system defects, survivable interventricular septal defects, and abnormal LV papillary muscles (PMs). Resulting Hand1 conditional mutants are born at Mendelian frequencies; but the morphological alterations acquired during cardiac development result in, the mice developing diastolic heart failure. Conclusion: Collectively, these data reveal that HAND1 contributes to the morphogenic patterning and maturation of cardiomyocytes during embryogenesis and although survivable, indicates a role for Hand1 within the developing conduction system and PM development.
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.
Hand2 elevates cardiomyocyte production during zebrafish heart development and regeneration
(The Company of Biologists, 2014-08) Schindler, Yocheved L.; Garske, Kristina M.; Wang, Jinhu; Firulli, Beth A.; Firulli, Anthony B.; Poss, Kenneth D.; Yelon, Deborah; Department of Pediatrics, IU School of Medicine
Embryonic heart formation requires the production of an appropriate number of cardiomyocytes; likewise, cardiac regeneration following injury relies upon the recovery of lost cardiomyocytes. The basic helix-loop-helix (bHLH) transcription factor Hand2 has been implicated in promoting cardiomyocyte formation. It is unclear, however, whether Hand2 plays an instructive or permissive role during this process. Here, we find that overexpression of hand2 in the early zebrafish embryo is able to enhance cardiomyocyte production, resulting in an enlarged heart with a striking increase in the size of the outflow tract. Our evidence indicates that these increases are dependent on the interactions of Hand2 in multimeric complexes and are independent of direct DNA binding by Hand2. Proliferation assays reveal that hand2 can impact cardiomyocyte production by promoting division of late-differentiating cardiac progenitors within the second heart field. Additionally, our data suggest that hand2 can influence cardiomyocyte production by altering the patterning of the anterior lateral plate mesoderm, potentially favoring formation of the first heart field at the expense of hematopoietic and vascular lineages. The potency of hand2 during embryonic cardiogenesis suggested that hand2 could also impact cardiac regeneration in adult zebrafish; indeed, we find that overexpression of hand2 can augment the regenerative proliferation of cardiomyocytes in response to injury. Together, our studies demonstrate that hand2 can drive cardiomyocyte production in multiple contexts and through multiple mechanisms. These results contribute to our understanding of the potential origins of congenital heart disease and inform future strategies in regenerative medicine.
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.
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.
Neonatal Deletion of Hand1 and Hand2 within Murine Cardiac Conduction System Reveals a Novel Role for HAND2 in Rhythm Homeostasis
(MDPI, 2022-07-04) George, Rajani M.; Guo, Shuai; Firulli, Beth A.; Rubart, Michael; Firulli, Anthony B.; Pediatrics, School of Medicine
The cardiac conduction system, a network of specialized cells, is required for the functioning of the heart. The basic helix loop helix factors Hand1 and Hand2 are required for cardiac morphogenesis and have been implicated in cardiac conduction system development and maintenance. Here we use embryonic and post-natal specific Cre lines to interrogate the role of Hand1 and Hand2 in the function of the murine cardiac conduction system. Results demonstrate that loss of HAND1 in the post-natal conduction system does not result in any change in electrocardiogram parameters or within the ventricular conduction system as determined by optical voltage mapping. Deletion of Hand2 within the post-natal conduction system results in sex-dependent reduction in PR interval duration in these mice, suggesting a novel role for HAND2 in regulating the atrioventricular conduction. Surprisingly, results show that loss of both HAND factors within the post-natal conduction system does not cause any consistent changes in cardiac conduction system function. Deletion of Hand2 in the embryonic left ventricle results in inconsistent prolongation of PR interval and susceptibility to atrial arrhythmias. Thus, these results suggest a novel role for HAND2 in homeostasis of the murine cardiac conduction system and that HAND1 loss potentially rescues the shortened HAND2 PR phenotype.
Nkx2.5 regulates Endothelin Converting Enzyme-1 during pharyngeal arch patterning
(Wiley, 2017-03) Iklé, Jennifer M.; Tavares, Andre L. P.; King, Marisol; Ding, Ding; Colombo, Sophie; Firulli, Beth A.; Firulli, Firulli; Targoff, Kimara L.; Yelon, Deborah; Clouthier, David E.; Anatomy and Cell Biology, School of Medicine
In gnathostomes, dorsoventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches is crucial for the development of hinged jaws. One of the key signals that mediates this process is Endothelin-1 (EDN1). Loss of EDN1 binding to the Endothelin-A receptor (EDNRA) results in loss of EDNRA signaling and subsequent facial birth defects in humans, mice and zebrafish. A rate-limiting step in this crucial signaling pathway is the conversion of immature EDN1 into a mature active form by Endothelin converting enzyme-1 (ECE1). However, surprisingly little is known about how Ece1 transcription is induced or regulated. We show here that Nkx2.5 is required for proper craniofacial development in zebrafish and acts in part by upregulating ece1 expression. Disruption of nkx2.5 in zebrafish embryos results in defects in both ventral and dorsal pharyngeal arch-derived elements, with changes in ventral arch gene expression consistent with a disruption in Ednra signaling. ece1 mRNA rescues the nkx2.5 morphant phenotype, indicating that Nkx2.5 functions through modulating Ece1 expression or function. These studies illustrate a new function for Nkx2.5 in embryonic development and provide new avenues with which to pursue potential mechanisms underlying human facial disorders.

Browsing by Author "Firulli, Beth A."

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