Mandibulofacial dysostosis with alopecia results from ETAR gain-of-function mutations via allosteric effects on ligand binding.

Mutations of G protein-coupled receptors (GPCRs) cause various human diseases, but the mechanistic details are limited. Here, we establish p.E303K in the gene encoding the endothelin receptor type A (ETAR/EDNRA) as a recurrent mutation causing mandibulofacial dysostosis with alopecia (MFDA), with craniofacial changes similar to those caused by p.Y129F. Mouse models carrying either of these missense mutations exhibited a partial maxillary-to-mandibular transformation, which was rescued by deleting the ligand endothelin 3 (ET3/EDN3). Pharmacological experiments confirmed the causative ETAR mutations as gain of function, dependent on ET3. To elucidate how an amino acid substitution far from the ligand binding site can increase ligand affinity, we used molecular dynamics (MD) simulations. E303 is located at the intracellular end of transmembrane domain 6, and its replacement by a lysine increased flexibility of this portion of the helix, thus favoring G protein binding and leading to G protein-mediated enhancement of agonist affinity. The Y129F mutation located under the ligand binding pocket reduced the sodium-water network, thereby affecting the extracellular portion of helices in favor of ET3 binding. These findings provide insight into the pathogenesis of MFDA and into allosteric mechanisms regulating GPCR function, which may provide the basis for drug design targeting GPCRs.

Epigenetic and Transcriptional Regulation of Spontaneous and Sensory Activity Dependent Programs During Neuronal Circuit Development.

Spontaneous activity generated before the onset of sensory transduction has a key role in wiring developing sensory circuits. From axonal targeting, to synapse formation and elimination, to the balanced integration of neurons into developing circuits, this type of activity is implicated in a variety of cellular processes. However, little is known about its molecular mechanisms of action, especially at the level of genome regulation. Conversely, sensory experience-dependent activity implements well-characterized transcriptional and epigenetic chromatin programs that underlie heterogeneous but specific genomic responses that shape both postnatal circuit development and neuroplasticity in the adult. In this review, we focus on our knowledge of the developmental processes regulated by spontaneous activity and the underlying transcriptional mechanisms. We also review novel findings on how chromatin regulates the specificity and developmental induction of the experience-dependent program, and speculate their relevance for our understanding of how spontaneous activity may act at the genomic level to instruct circuit assembly and prepare developing neurons for sensory-dependent connectivity refinement and processing.

Different Ectopic Hoxa2 Expression Levels in Mouse Cranial Neural Crest Cells Result in Distinct Craniofacial Anomalies and Homeotic Phenotypes.

Providing appropriate positional identity and patterning information to distinct rostrocaudal subpopulations of cranial neural crest cells (CNCCs) is central to vertebrate craniofacial morphogenesis. Hox genes are not expressed in frontonasal and first pharyngeal arch (PA1) CNCCs, whereas a single Hox gene, Hoxa2, is necessary to provide patterning information to second pharyngeal arch (PA2) CNCCs. In frog, chick and mouse embryos, ectopic expression of Hoxa2 in Hox-negative CNCCs induced hypoplastic phenotypes of CNCC derivatives of variable severity, associated or not with homeotic transformation of a subset of PA1 structures into a PA2-like identity. Whether these different morphological outcomes are directly related to distinct Hoxa2 overexpression levels is unknown. To address this issue, we selectively induced Hoxa2 overexpression in mouse CNCCs, using a panel of mouse lines expressing different Hoxa2 ectopic expression levels, including a newly generated Hoxa2 knocked-in mouse line. While ectopic Hoxa2 expression at only 60% of its physiological levels was sufficient for pinna duplication, ectopic Hoxa2 expression at 100% of its normal level was required for complete homeotic repatterning of a subset of PA1 skeletal elements into a duplicated set of PA2-like elements. On the other hand, ectopic Hoxa2 overexpression at non-physiological levels (200% of normal levels) led to an almost complete loss of craniofacial skeletal structures. Moreover, ectopic Hoxa5 overexpression in CNCCs, while also resulting in severe craniofacial defects, did not induce homeotic changes of PA1-derived CNCCs, indicating Hoxa2 specificity in repatterning a subset of Hox-negative CNCCs. These results reconcile some discrepancies in previously published experiments and indicate that distinct subpopulations of CNCCs are differentially sensitive to ectopic levels of Hox expression.

A unique bipartite Polycomb signature regulates stimulus-response transcription during development.

Rapid cellular responses to environmental stimuli are fundamental for development and maturation. Immediate early genes can be transcriptionally induced within minutes in response to a variety of signals. How their induction levels are regulated and their untimely activation by spurious signals prevented during development is poorly understood. We found that in developing sensory neurons, before perinatal sensory-activity-dependent induction, immediate early genes are embedded into a unique bipartite Polycomb chromatin signature, carrying active H3K27ac on promoters but repressive Ezh2-dependent H3K27me3 on gene bodies. This bipartite signature is widely present in developing cell types, including embryonic stem cells. Polycomb marking of gene bodies inhibits mRNA elongation, dampening productive transcription, while still allowing for fast stimulus-dependent mark removal and bipartite gene induction. We reveal a developmental epigenetic mechanism regulating the rapidity and amplitude of the transcriptional response to relevant stimuli, while preventing inappropriate activation of stimulus-response genes.

Dlx5-augmentation in neural crest cells reveals early development and differentiation potential of mouse apical head mesenchyme.

Neural crest cells (NCCs) give rise to various tissues including neurons, pigment cells, bone and cartilage in the head. Distal-less homeobox 5 (Dlx5) is involved in both jaw patterning and differentiation of NCC-derivatives. In this study, we investigated the differentiation potential of head mesenchyme by forcing Dlx5 to be expressed in mouse NCC (NCCDlx5). In NCCDlx5 mice, differentiation of dermis and pigment cells were enhanced with ectopic cartilage (ec) and heterotopic bone (hb) in different layers at the cranial vertex. The ec and hb were derived from the early migrating mesenchyme (EMM), the non-skeletogenic cell population located above skeletogenic supraorbital mesenchyme (SOM). The ec developed within Foxc1+-dura mater with increased PDGFRα signalling, and the hb formed with upregulation of BMP and WNT/ß-catenin signallings in Dermo1+-dermal layer from E11.5. Since dermal cells express Runx2 and Msx2 in the control, osteogenic potential in dermal cells seemed to be inhibited by an anti-osteogenic function of Msx2 in normal context. We propose that, after the non-skeletogenic commitment, the EMM is divided into dermis and meninges by E11.5 in normal development. Two distinct responses of the EMM, chondrogenesis and osteogenesis, to Dlx5-augmentation in the NCCDlx5 strongly support this idea.

Lysine demethylase 7a regulates murine anterior-posterior development by modulating the transcription of Hox gene cluster.

Temporal and spatial colinear expression of the Hox genes determines the specification of positional identities during vertebrate development. Post-translational modifications of histones contribute to transcriptional regulation. Lysine demethylase 7A (Kdm7a) demethylates lysine 9 or 27 di-methylation of histone H3 (H3K9me2, H3K27me2) and participates in the transcriptional activation of developmental genes. However, the role of Kdm7a during mouse embryonic development remains to be elucidated. Herein, we show that Kdm7a-/- mouse exhibits an anterior homeotic transformation of the axial skeleton, including an increased number of presacral elements. Importantly, posterior Hox genes (caudally from Hox9) are specifically downregulated in the Kdm7a-/- embryo, which correlates with increased levels of H3K9me2, not H3K27me2. These observations suggest that Kdm7a controls the transcription of posterior Hox genes, likely via its demethylating activity, and thereby regulating the murine anterior-posterior development. Such epigenetic regulatory mechanisms may be harnessed for proper control of coordinate body patterning in vertebrates.

Probing the origin of matching functional jaws: roles of Dlx5/6 in cranial neural crest cells.

Gnathostome jaws derive from the first pharyngeal arch (PA1), a complex structure constituted by Neural Crest Cells (NCCs), mesodermal, ectodermal and endodermal cells. Here, to determine the regionalized morphogenetic impact of Dlx5/6 expression, we specifically target their inactivation or overexpression to NCCs. NCC-specific Dlx5/6 inactivation (NCC∆Dlx5/6) generates severely hypomorphic lower jaws that present typical maxillary traits. Therefore, differently from Dlx5/6 null-embryos, the upper and the lower jaws of NCC∆Dlx5/6 mice present a different size. Reciprocally, forced Dlx5 expression in maxillary NCCs provokes the appearance of distinct mandibular characters in the upper jaw. We conclude that: (1) Dlx5/6 activation in NCCs invariably determines lower jaw identity; (2) the morphogenetic processes that generate functional matching jaws depend on the harmonization of Dlx5/6 expression in NCCs and in distinct ectodermal territories. The co-evolution of synergistic opposing jaws requires the coordination of distinct regulatory pathways involving the same transcription factors in distant embryonic territories.

Barrelette map formation in the prenatal mouse brainstem.

The rodent whiskers are topographically mapped in brainstem sensory nuclei as neuronal modules known as barrelettes. Little is known about how the facial whisker pattern is copied into a brainstem barrelette topographic pattern, which serves as a template for the establishment of thalamic barreloid and, in turn, cortical barrel maps, and how precisely is the whisker pattern mapped in the brainstem during prenatal development. Here, we review recent insights advancing our understanding of the intrinsic and extrinsic patterning mechanisms contributing to establish topographical equivalence between the facial whisker pattern and the mouse brainstem during prenatal development and their relative importance.

Integrating into the Rhombomere Community across the Border.

During early hindbrain development, single neuroepithelial progenitors cross into neighboring rhombomere compartments and switch their molecular identity to match their new position. In this issue of Developmental Cell,Addison et al. (2018) show that this identity switch is mediated by non-cell-autonomous retinoid signaling that ensures a homogeneous segment identity.

Nuclear Pore Protein Meets Transcription Factor in Neural Fate.

How nuclear architecture contributes to transcriptional regulation in neural progenitor cells (NeuPCs) is poorly understood. A study by Toda et al. (2017) now shows that the nuclear pore protein Nup153 associates with the Sox2 transcription factor in the regulation of NeuPC maintenance and neural fate.

Differing contributions of the first and second pharyngeal arches to tympanic membrane formation in the mouse and chick.

We have proposed that independent origins of the tympanic membrane (TM), consisting of the external auditory meatus (EAM) and first pharyngeal pouch, are linked with distinctive middle ear structures in terms of dorsal-ventral patterning of the pharyngeal arches during amniote evolution. However, previous studies have suggested that the first pharyngeal arch (PA1) is crucial for TM formation in both mouse and chick. In this study, we compare TM formation along the anterior-posterior axis in these animals using Hoxa2 expression as a marker of the second pharyngeal arch (PA2). In chick, the EAM begins to invaginate at the surface ectoderm of PA2, not at the first pharyngeal cleft, and the entire TM forms in PA2. Chick-quail chimera that have lost PA2 and duplicated PA1 suggest that TM formation is achieved by developmental interaction between a portion of the EAM and the columella auris in PA2, and that PA1 also contributes to formation of the remaining part of the EAM. By contrast, in mouse, TM formation is highly associated with an interdependent relationship between the EAM and tympanic ring in PA1.

Gene bivalency at Polycomb domains regulates cranial neural crest positional identity.

The cranial neural crest cells are multipotent cells that provide head skeletogenic mesenchyme and are crucial for craniofacial patterning. We analyzed the chromatin landscapes of mouse cranial neural crest subpopulations in vivo. Early postmigratory subpopulations contributing to distinct mouse craniofacial structures displayed similar chromatin accessibility patterns yet differed transcriptionally. Accessible promoters and enhancers of differentially silenced genes carried H3K27me3/H3K4me2 bivalent chromatin marks embedded in large enhancer of zeste homolog 2-dependent Polycomb domains, indicating transcriptional poising. These postmigratory bivalent chromatin regions were already present in premigratory progenitors. At Polycomb domains, H3K27me3 antagonized H3K4me2 deposition, which was restricted to accessible sites. Thus, bivalent Polycomb domains provide a chromatin template for the regulation of cranial neural crest cell positional identity in vivo, contributing insights into the epigenetic regulation of face morphogenesis.

Hox2 Genes Are Required for Tonotopic Map Precision and Sound Discrimination in the Mouse Auditory Brainstem.

Tonotopy is a hallmark of auditory pathways and provides the basis for sound discrimination. Little is known about the involvement of transcription factors in brainstem cochlear neurons orchestrating the tonotopic precision of pre-synaptic input. We found that in the absence of Hoxa2 and Hoxb2 function in Atoh1-derived glutamatergic bushy cells of the anterior ventral cochlear nucleus, broad input topography and sound transmission were largely preserved. However, fine-scale synaptic refinement and sharpening of isofrequency bands of cochlear neuron activation upon pure tone stimulation were impaired in Hox2 mutants, resulting in defective sound-frequency discrimination in behavioral tests. These results establish a role for Hox factors in tonotopic refinement of connectivity and in ensuring the precision of sound transmission in the mammalian auditory circuit.

Developmental mechanisms of the tympanic membrane in mammals and non-mammalian amniotes.

The tympanic membrane is a thin layer that originates from the ectoderm, endoderm, and mesenchyme. Molecular-genetic investigations have revealed that interaction between epithelial and mesenchymal cells in the pharyngeal arches is essential for development of the tympanic membrane. We have recently reported that developmental mechanisms underlying the tympanic membrane seem to be different between mouse and chicken, suggesting that the tympanic membrane evolved independently in mammals and non-mammalian amniotes. In this review, we summarize previous studies of tympanic membrane formation in the mouse. We also discuss its formation in amniotes from an evolutionary point of view.

Developmental genetic bases behind the independent origin of the tympanic membrane in mammals and diapsids.

The amniote middle ear is a classical example of the evolutionary novelty. Although paleontological evidence supports the view that mammals and diapsids (modern reptiles and birds) independently acquired the middle ear after divergence from their common ancestor, the developmental bases of these transformations remain unknown. Here we show that lower-to-upper jaw transformation induced by inactivation of the Endothelin1-Dlx5/6 cascade involving Goosecoid results in loss of the tympanic membrane in mouse, but causes duplication of the tympanic membrane in chicken. Detailed anatomical analysis indicates that the relative positions of the primary jaw joint and first pharyngeal pouch led to the coupling of tympanic membrane formation with the lower jaw in mammals, but with the upper jaw in diapsids. We propose that differences in connection and release by various pharyngeal skeletal elements resulted in structural diversity, leading to the acquisition of the tympanic membrane in two distinct manners during amniote evolution.

Distinct effects of Hoxa2 overexpression in cranial neural crest populations reveal that the mammalian hyomandibular-ceratohyal boundary maps within the styloid process.

Most gnathostomata craniofacial structures derive from pharyngeal arches (PAs), which are colonized by cranial neural crest cells (CNCCs). The anteroposterior and dorsoventral identities of CNCCs are defined by the combinatorial expression of Hox and Dlx genes. The mechanisms associating characteristic Hox/Dlx expression patterns with the topology and morphology of PAs derivatives are only partially known; a better knowledge of these processes might lead to new concepts on the origin of taxon-specific craniofacial morphologies and of certain craniofacial malformations. Here we show that ectopic expression of Hoxa2 in Hox-negative CNCCs results in distinct phenotypes in different CNCC subpopulations. Namely, while ectopic Hoxa2 expression is sufficient for the morphological and molecular transformation of the first PA (PA1) CNCC derivatives into the second PA (PA2)-like structures, this same genetic alteration does not provoke the transformation of derivatives of other CNCC subpopulations, but severely impairs their development. Ectopic Hoxa2 expression results in the transformation of the proximal Meckel's cartilage and of the malleus, two ventral PA1 CNCCs derivatives, into a supernumerary styloid process (SP), a PA2-derived mammalian-specific skeletal structure. These results, together with experiments to inactivate and ectopically activate the Edn1-Dlx5/6 pathway, indicate a dorsoventral PA2 (hyomandibular/ceratohyal) boundary passing through the middle of the SP. The present findings suggest context-dependent function of Hoxa2 in CNCC regional specification and morphogenesis, and provide novel insights into the evolution of taxa-specific patterning of PA-derived structures.

Mouse Hoxa2 mutations provide a model for microtia and auricle duplication.

External ear abnormalities are frequent in newborns ranging from microtia to partial auricle duplication. Little is known about the molecular mechanisms orchestrating external ear morphogenesis. In humans, HOXA2 partial loss of function induces a bilateral microtia associated with an abnormal shape of the auricle. In mice, Hoxa2 inactivation at early gestational stages results in external auditory canal (EAC) duplication and absence of the auricle, whereas its late inactivation results in a hypomorphic auricle, mimicking the human HOXA2 mutant condition. By genetic fate mapping we found that the mouse auricle (or pinna) derives from the Hoxa2-expressing neural crest-derived mesenchyme of the second pharyngeal arch, and not from a composite of first and second arch mesenchyme as previously proposed based on morphological observation of human embryos. Moreover, the mouse EAC is entirely lined by Hoxa2-negative first arch mesenchyme and does not develop at the first pharyngeal cleft, as previously assumed. Conditional ectopic Hoxa2 expression in first arch neural crest is sufficient to induce a complete duplication of the pinna and a loss of the EAC, suggesting transformation of the first arch neural crest-derived mesenchyme lining the EAC into an ectopic pinna. Hoxa2 partly controls the morphogenesis of the pinna through the BMP signalling pathway and expression of Eya1, which in humans is involved in branchio-oto-renal syndrome. Thus, Hoxa2 loss- and gain-of-function approaches in mice provide a suitable model to investigate the molecular aetiology of microtia and auricle duplication.

Endothelin regulates neural crest deployment and fate to form great vessels through Dlx5/Dlx6-independent mechanisms.

Endothelin-1 (Edn1), originally identified as a vasoconstrictor peptide, is involved in the development of cranial/cardiac neural crest-derived tissues and organs. In craniofacial development, Edn1 binds to Endothelin type-A receptor (Ednra) to induce homeobox genes Dlx5/Dlx6 and determines the mandibular identity in the first pharyngeal arch. However, it remains unsolved whether this pathway is also critical for pharyngeal arch artery development to form thoracic arteries. Here, we show that the Edn1/Ednra signaling is involved in pharyngeal artery development by controlling the fate of neural crest cells through a Dlx5/Dlx6-independent mechanism. Edn1 and Ednra knock-out mice demonstrate abnormalities in pharyngeal arch artery patterning, which include persistent first and second pharyngeal arteries, resulting in additional branches from common carotid arteries. Neural crest cell labeling with Wnt1-Cre transgene and immunostaining for smooth muscle cell markers revealed that neural crest cells abnormally differentiate into smooth muscle cells at the first and second pharyngeal arteries of Ednra knock-out embryos. By contrast, Dlx5/Dlx6 knockout little affect the development of pharyngeal arch arteries and coronary arteries, the latter of which is also contributed by neural crest cells through an Edn-dependent mechanism. These findings indicate that the Edn1/Ednra signaling regulates neural crest differentiation to ensure the proper patterning of pharyngeal arch arteries, which is independent of the regional identification of the pharyngeal arches along the dorsoventral axis mediated by Dlx5/Dlx6.

Identification and developmental analysis of endothelin receptor type-A expressing cells in the mouse kidney.

The endothelin (Edn) system plays pleiotropic roles in renal function and various disease processes through two distinct G protein-coupled receptors, Edn receptors type-A (Ednra) and type-B (Ednrb). However, difficulties in the accurate identification of receptor-expressing cells in situ have made it difficult to dissect their diverse action in renal (patho)physiology. We have recently established mouse lines in which lacZ and EGFP are 'knocked-in' to the Ednra locus to faithfully mark Ednra-expressing cells. Here we analyzed these mice for their expression in the kidney to characterize Ednra-expressing cells. Ednra expression was first observed in undifferentiated mesenchymal cells around the ureteric bud at E12.5. Thereafter, Ednra expression was widely observed in vascular smooth muscle cells, JG cells and mesenchymal cells in the interstitium. After growth, the expression became confined to vascular smooth muscle cells, pericytes and renin-producing JG cells. By contrast, most cells in the nephron and vascular endothelial cells did not express Ednra. These results indicate that Ednra expression may be linked with non-epithelial fate determination and differentiation of metanephric mesenchyme. Ednra-lacZ/EGFP knock-in mice may serve as a useful tool in studies on renal function and pathophysiology of various renal diseases.