Andre L.P. Tavares

TGFβ‐mediated RhoA expression is necessary for epithelial‐mesenchymal transition in the embryonic chick heart

Endothelia in the atrioventricular canal (AVC) of the embryonic heart undergo an epithelial‐mesenchymal transition (EMT) and migrate into the underlying extracellular matrix. We explore here whether RhoA mediates this EMT. RhoA was detected in all cells of the chick heart during the stages studied. Expression was elevated when EMT was actively occurring. Explants treated with C3 exoenzyme in collagen gel cultures showed a significant decrease in mesenchymal cell numbers. siRNA was used to inhibit RhoA mRNA, and both activated endothelial and mesenchymal cells decreased significantly with treatment. Loss of RhoA produced a reduction of RhoB, cyclin‐b2, and β‐catenin messages showing that these genes are regulated downstream of RhoA. In contrast, runx‐2 was not reduced. Inhibition of TGFβ3 or TGFβ2 activity caused a large reduction of RhoA message. These data place RhoA in TGFβ regulated pathways for both endothelial activation and mesenchymal invasion and demonstrate a functional requirement during EMT. Developmental Dynamics 235:1589–1598, 2006.

Ectodermal-derived Endothelin1 is required for patterning the distal and intermediate domains of the mouse mandibular arch

Morphogenesis of the vertebrate head relies on proper dorsal-ventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches. Endothelin-1 (Edn1)-induced signaling through the endothelin-A receptor (Ednra) is crucial for cranial NCC patterning within the mandibular portion of the first pharyngeal arch, from which the lower jaw arises. Deletion of Edn1, Ednra or endothelin-converting enzyme in mice causes perinatal lethality due to severe craniofacial birth defects. These include homeotic transformation of mandibular arch-derived structures into more maxillary-like structures, indicating a loss of NCC identity. All cranial NCCs express Ednra whereas Edn1 expression is limited to the overlying ectoderm, core paraxial mesoderm and pharyngeal pouch endoderm of the mandibular arch as well as more caudal arches. To define the developmental significance of Edn1 from each of these layers, we used Cre/loxP technology to inactivate Edn1 in a tissue-specific manner. We show that deletion of Edn1 in either the mesoderm or endoderm alone does not result in cellular or molecular changes in craniofacial development. However, ectodermal deletion of Edn1 results in craniofacial defects with concomitant changes in the expression of early mandibular arch patterning genes. Importantly, our results also both define for the first time in mice an intermediate mandibular arch domain similar to the one defined in zebrafish and show that this region is most sensitive to loss of Edn1. Together, our results illustrate an integral role for ectoderm-derived Edn1 in early arch morphogenesis, particularly in the intermediate domain.

Negative regulation of endothelin signaling by SIX1 is required for proper maxillary development

Jaw morphogenesis is a complex event mediated by inductive signals that establish and maintain the distinct developmental domains required for formation of hinged jaws, the defining feature of gnathostomes. The mandibular portion of pharyngeal arch 1 is patterned dorsally by Jagged-Notch signaling and ventrally by endothelin receptor A (EDNRA) signaling. Loss of EDNRA signaling disrupts normal ventral gene expression, the result of which is homeotic transformation of the mandible into a maxilla-like structure. However, loss of Jagged-Notch signaling does not result in significant changes in maxillary development. Here we show in mouse that the transcription factor SIX1 regulates dorsal arch development not only by inducing dorsal Jag1 expression but also by inhibiting endothelin 1 (Edn1) expression in the pharyngeal endoderm of the dorsal arch, thus preventing dorsal EDNRA signaling. In the absence of SIX1, but not JAG1, aberrant EDNRA signaling in the dorsal domain results in partial duplication of the mandible. Together, our results illustrate that SIX1 is the central mediator of dorsal mandibular arch identity, thus ensuring separation of bone development between the upper and lower jaws. Summary: In mouse, SIX1 represses endothelin 1 and induces Jag1 expression in the dorsal mandibular arch. Loss of this signaling leads to partial duplication of the mandible in place of the posterior maxilla.

Endosomal regulation of contact inhibition through the AMOT:YAP pathway

It was shown previously that endotubin, an integral membrane protein of endosomes, regulates the trafficking of tight junction proteins between endosomes and the tight junctions. Here it is shown that endotubin regulates YAP localization on endosomes through its interaction with AMOT and thus may play a role in contact inhibition.

Cre recombinase-regulated Endothelin1 transgenic mouse lines: novel tools for analysis of embryonic and adult disorders

Endothelin-1 (EDN1) influences both craniofacial and cardiovascular development and a number of adult physiological conditions by binding to one or both of the known endothelin receptors, thus initiating multiple signaling cascades. Animal models containing both conventional and conditional loss of the Edn1 gene have been used to dissect EDN1 function in both embryos and adults. However, while transgenic Edn1 over-expression or targeted genomic insertion of Edn1 has been performed to understand how elevated levels of Edn1 result in or exacerbate disease states, an animal model in which Edn1 over-expression can be achieved in a spatiotemporal-specific manner has not been reported. Here we describe the creation of Edn1 conditional over-expression transgenic mouse lines in which the chicken β-actin promoter and an Edn1 cDNA are separated by a strong stop sequence flanked by loxP sites. In the presence of Cre, the stop cassette is removed, leading to Edn1 expression. Using the Wnt1-Cre strain, in which Cre expression is targeted to the Wnt1-expressing domain of the central nervous system (CNS) from which neural crest cells (NCCs) arise, we show that stable chicken β-actin-Edn1 (CBA-Edn1) transgenic lines with varying EDN1 protein levels develop defects in NCC-derived tissues of the face, though the severity differs between lines. We also show that Edn1 expression can be achieved in other embryonic tissues utilizing other Cre strains, with this expression also resulting in developmental defects. CBA-Edn1 transgenic mice will be useful in investigating diverse aspects of EDN1-mediated-development and disease, including understanding how NCCs achieve and maintain a positional and functional identity and how aberrant EDN1 levels can lead to multiple physiological changes and diseases.

Runx2‐I is an Early Regulator of Epithelial–Mesenchymal Cell Transition in the Chick Embryo

Background: Although normally linked to bone and cartilage development, the Runt‐related transcription factor, RUNX2, was reported in the mouse heart during development of the valves. We examined RUNX2 expression and function in the developing avian heart as it related to the epithelial–mesenchymal transition (EMT) in the atrioventricular canal. EMT can be separated into an activation stage involving hypertrophy and cell separation and an invasion stage where cells invade the extracellular matrix. The localization and activity of RUNX2 was explored in relation to these steps in the heart. As RUNX2 was also reported in cancer tissues, we examined its expression in the progression of esophageal cancer in staged tissues. Results: A specific isoform, RUNX2‐I, is present and required for EMT by endothelia of the atrioventricular canal. Knockdown of RUNX2‐I inhibits the cell–cell separation that is characteristic of initial activation of EMT. Loss of RUNX2‐I altered expression of EMT markers to a greater extent during activation than during subsequent cell invasion. Transforming growth factor beta 2 (TGFβ2) mediates activation during cardiac endothelial EMT. Consistent with a role in activation, RUNX2‐I is regulated by TGFβ2 and its activity is independent of similarly expressed Snai2 in regulation of EMT. Examination of RUNX2 expression in esophageal cancer showed its upregulation concomitant with the development of dysplasia and continued expression in adenocarcinoma. Conclusions: These data introduce the RUNX2‐I isoform as a critical early transcription factor mediating EMT in the developing heart after induction by TGFβ2. Its expression in tumor tissue suggests a similar role for RUNX2 in the EMT of metastasis. Developmental Dynamics 247:542–554, 2018.

Nkx2.5 regulates endothelin converting enzyme-1 during pharyngeal arch patterning

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 mediate 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.