Taro Kitazawa

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.

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.

Gene bivalency at Polycomb domains regulates cranial neural crest positional identity

Epigenetic regulation of craniofacial development in mice is discussed. The epigenetics of face-making How is it that our earlobes are attached to our ears and not our chins? Diverse bits of facial structure are derived from migrating neural crest cells. The cells start out similar but end up building very different facial structures. Neural crest cells destined for one structure can be rerouted to develop others, however. Minoux et al. found that neural crest cells share prepatterned poised chromatin states that are established before the cells migrate and retained during migration. Different developmental programs are unlocked when the migrating cells near their final location and interact with local patterning signals. Science, this issue p. eaal2913 INTRODUCTION Craniofacial morphogenesis involves the cranial neural crest (NC) cells, a vertebrate-specific multipotent cell population that provides most of the head skeletogenic mesenchyme. Cranial NC cells delaminate from different points along the developing neural tube and migrate into distinct facial and pharyngeal arch processes, where they give rise to distinctly shaped cartilage and bone elements, which in turn assemble into a harmonious face. How do distinct cranial NC cell subpopulations acquire their regional identity, allowing them to generate the specific subsets of craniofacial elements appropriate to their position? Premigratory NC cells that contribute to frontonasal, maxillary, or mandibular processes share similar patterning potential, because they can replace each other in building a whole craniofacial skeleton. Such plasticity is maintained during and after migration until subpopulation-specific transcriptional identity and positional patterning programs are established as a result of interactions with their local surrounding environment. We asked how chromatin regulation may allow cranial NC cells to maintain broad patterning competence through migration while being poised to respond to local cues and induce position-specific transcriptional subprograms. RATIONALE We used genome-wide RNA sequencing (RNA-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq), and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and integrated the information to propose a model to explain how cranial NC subpopulations maintain broad patterning competence through chromatin epigenetic regulation and how transcription factor–dependent responses to local cues can modify the chromatin pattern to establish unique subpopulation-specific transcriptional subprograms. To this aim, we microdissected the Hox-free frontonasal, maxillary, mandibular, and the Hox-expressing second pharyngeal arch processes of E10.5 mouse embryos. We isolated the NC cell subpopulations from each of these prominences by cell sorting and analyzed their transcriptional state, as well as the H3K27me3, H3K4me2, and H3K27Ac histone modification and chromatin accessibility profiles at promoters and enhancers. We then compared these data sets with the transcriptional, histone mark, and chromatin accessibility profiles of the Hox-free NC premigratory progenitors and of E10.5 frontonasal, maxillary, mandibular, and second pharyngeal arch NC cell subpopulations in which we conditionally inactivated the Polycomb H3K27 methyltransferase gene enhancer of zeste homolog 2 (Ezh2cKO mutants). RESULTS Early postmigratory NC subpopulations contributing to distinct mouse craniofacial structures displayed similar chromatin accessibility patterns yet differed transcriptionally. The differentially expressed genes (positional genes) displayed accessible and H3K27me3+/H3K4me2+ bivalent enhancers and promoters, and were embedded in large Ezh2-dependent Polycomb domains, in the NC cell subpopulations in which they were silenced, indicating transcriptional poising. These postmigratory chromatin domains of poised gene regulation were inherited from NC premigratory progenitors. At Polycomb domains, H3K27me3 antagonized H3K4me2 deposition, which was restricted to accessible promoter and enhancer elements, preventing ectopic activation at inappropriate positions. DISCUSSION Our findings explain how cranial NC cell plasticity is maintained through migration until postmigratory stages. We propose that an Ezh2-dependent poised chromatin organization underlies the positional plasticity of cranial premigratory NC cell progenitors. This chromatin prepattern is maintained through migration. In response to position-specific environmental signals encountered by the NC cells during or after their migration, the regulatory elements and promoters of positional genes switch from a poised to an active chromatin state, contributing to establish NC subpopulation–specific transcriptional identities. This work contributes novel insights into the epigenetic regulation of face morphogenesis. Epigenetic regulation of cranial NC cell identity. A Polycomb-dependent poised chromatin organization underlies the positional plasticity of cranial premigratory NC cell progenitors. This chromatin prepattern is maintained through migration. In response to local cues encountered by the NC cells during or after their migration, the regulatory elements (E) and promoters (P) of differentially expressed genes switch from a poised to an active chromatin state, establishing transcriptional identities specific to subpopulations of NC cells. 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.

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 Meckels 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.

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.

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. Summary: Comparison of tissue interactions during tympanic membrane development in mouse and chick indicates similarities and differences in the developmental mechanisms and evolution of middle ear morphology in mammals and diapsids.

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.

Craniofacial traits determined by neural crest cells-restricted expression of Dlx5/6: probing the origin of matching functional jaws

During gnathostome development, lower and upper jaws derive from the first pharyngeal arch (PA1), a complex structure constituted by Neural Crest Cells (NCCs), mesodermal, ectodermal and endodermal cell populations. Lower jaw (mandibular) identity depends on endothelin-1 (Edn1)-mediated activation of Dlx5/6 in PA1 NCCs. Transient expression of Dlx5/6 in ectodermal cells is also necessary for correct jaw morphogenesis. Here we inactivate or overexpress Dlx5/6 specifically in NCCs to determine the morphogenetic impact of their expression in these cells. Invalidation of Dlx5/6 in NCCs (NCCΔDlx5/6) generates severely hypomorphic lower jaws that present typical maxillary traits. Reciprocally, forced expression of Dlx5 in maxillary NCCs (NCCDlx5), provokes the transformation of the upper jaw into a structure that presents distinct mandibular characters. Therefore, similarly to Edn1-signalling mutants, the NCCΔDlx5/6 jaw transformation engenders an asymmetric mouth that is strikingly different from the symmetric jaws obtained after constitutive Dlx5/6 inactivation. Our data demonstrate that: 1) Dlx5/6 expression in NCCs is necessary and sufficient to specify mandibular identity; 2) these same genes must also be regulated in other cell types to generate functional matching jaws capable to support mastication. These finding are critical to understand the developmental and evolutionary origin of distinct and synergic anatomical structures.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 (NCCΔDlx5/6) generates severely hypomorphic lower jaws that present typical maxillary traits. Therefore, differently from the symmetric jaws obtained after constitutive Dlx5/6 inactivation, NCCΔDlx5/6 embryos present a strikingly asymmetric mouth. 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.

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.