Shaping faces: genetic and epigenetic control of craniofacial morphogenesis.

Major differences in facial morphology distinguish vertebrate species. Variation of facial traits underlies the uniqueness of human individuals, and abnormal craniofacial morphogenesis during development leads to birth defects that significantly affect quality of life. Studies during the past 40 years have advanced our understanding of the molecular mechanisms that establish facial form during development, highlighting the crucial roles in this process of a multipotent cell type known as the cranial neural crest cell. In this Review, we discuss recent advances in multi-omics and single-cell technologies that enable genes, transcriptional regulatory networks and epigenetic landscapes to be closely linked to the establishment of facial patterning and its variation, with an emphasis on normal and abnormal craniofacial morphogenesis. Advancing our knowledge of these processes will support important developments in tissue engineering, as well as the repair and reconstruction of the abnormal craniofacial complex.

monaLisa: an R/Bioconductor package for identifying regulatory motifs.

SUMMARY: Proteins binding to specific nucleotide sequences, such as transcription factors, play key roles in the regulation of gene expression. Their binding can be indirectly observed via associated changes in transcription, chromatin accessibility, DNA methylation and histone modifications. Identifying candidate factors that are responsible for these observed experimental changes is critical to understand the underlying biological processes. Here, we present monaLisa, an R/Bioconductor package that implements approaches to identify relevant transcription factors from experimental data. The package can be easily integrated with other Bioconductor packages and enables seamless motif analyses without any software dependencies outside of R. AVAILABILITY AND IMPLEMENTATION: monaLisa is implemented in R and available on Bioconductor at https://bioconductor.org/packages/monaLisa with the development version hosted on GitHub at https://github.com/fmicompbio/monaLisa. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

DPP9 enzyme activity controls survival of mouse migratory tongue muscle progenitors and its absence leads to neonatal lethality due to suckling defect.

Dipeptidyl peptidase 9 (DPP9) is an intracellular N-terminal post-proline-cleaving enzyme whose physiological function remains largely unknown. We investigated the role of DPP9 enzyme in vivo by characterizing knock-in mice expressing a catalytically inactive mutant form of DPP9 (S729A; DPP9ki/ki mice). We show that DPP9ki/ki mice die within 12-18h after birth. The neonatal lethality can be rescued by manual feeding, indicating that a suckling defect is the primary cause of neonatal lethality. The suckling defect results from microglossia, and is characterized by abnormal formation of intrinsic muscles at the distal tongue. In DPP9ki/ki mice, the number of occipital somite-derived migratory muscle progenitors, forming distal tongue intrinsic muscles, is reduced due to increased apoptosis. In contrast, intrinsic muscles of the proximal tongue and extrinsic tongue muscles, which derive from head mesoderm, develop normally in DPP9ki/ki mice. Thus, lack of DPP9 activity in mice leads to impaired tongue development, suckling defect and subsequent neonatal lethality due to impaired survival of a specific subset of migratory tongue muscle progenitors.

Facial whisker pattern is not sufficient to instruct a whisker-related topographic map in the mouse somatosensory brainstem.

Facial somatosensory input is relayed by trigeminal ganglion (TG) neurons and serially wired to brainstem, thalamus and cortex. Spatially ordered sets of target neurons generate central topographic maps reproducing the spatial arrangement of peripheral facial receptors. Facial pattern provides a necessary template for map formation, but may be insufficient to impose a brain somatotopic pattern. In mice, lower jaw sensory information is relayed by the trigeminal nerve mandibular branch, whose axons target the brainstem dorsal principal sensory trigeminal nucleus (dPrV). Input from mystacial whiskers is relayed by the maxillary branch and forms a topographic representation of rows and whiskers in the ventral PrV (vPrV). To investigate peripheral organisation in imposing a brain topographic pattern, we analysed Edn1(-/-) mice, which present ectopic whisker rows on the lower jaw. We found that these whiskers were innervated by mandibular TG neurons which initially targeted dPrV. Unlike maxillary TG neurons, the ectopic whisker-innervating mandibular neuron cell bodies and pre-target central axons did not segregate into a row-specific pattern nor target the dPrV with a topographic pattern. Following periphery-driven molecular repatterning to a maxillary-like identity, mandibular neurons partially redirected their central projections from dPrV to vPrV. Thus, while able to induce maxillary-like molecular features resulting in vPrV final targeting, a spatially ordered lower jaw ectopic whisker pattern is insufficient to impose row-specific pre-target organisation of the central mandibular tract or a whisker-related matching pattern of afferents in dPrV. These results provide novel insights into periphery-dependent versus periphery-independent mechanisms of trigeminal ganglion and brainstem patterning in matching whisker topography.

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.

MicroRNA-196b is transcribed from an autonomous promoter and is directly regulated by Cdx2 and by posterior Hox proteins during embryogenesis.

The miR-196 miRNA gene family located within the Hox gene clusters has been shown to function during embryogenesis and to be aberrantly expressed in various malignancies, including leukaemia, melanoma, and colorectal cancer. Despite its involvement in numerous biological processes, the control of miR-196 expression is still poorly defined. We identified the miR-196b promoter and found that the mature miR-196b originates from a large, non-coding primary transcript, which starts within an autonomous TATA box promoter and is not in physical continuity with either the Hoxa10 or Hoxa9 main primary transcripts. A ~680bp genomic fragment, spanning the pri-miR-196b transcription start site, is sufficient to recapitulate the neural tube expression pattern of miR-196 during embryogenesis. This region contains potential binding sites for Cdx and 5'Hox transcription factors. Two of these sites revealed to be necessary for neural tube expression and were bound in vivo by Cdx2 and Hoxd13. We show that Cdx2 is required for miR-196 expression and that both Cdx2 and 5'Hox, but not 3'Hox, are able to activate the miR-196b promoter. The possible role of Cdx2- and 5'Hox-mediated regulation of miR-196 expression in vertebrate anterior-posterior (AP) axis formation during embryogenesis is discussed.

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.

Assembly of the auditory circuitry by a Hox genetic network in the mouse brainstem.

Rhombomeres (r) contribute to brainstem auditory nuclei during development. Hox genes are determinants of rhombomere-derived fate and neuronal connectivity. Little is known about the contribution of individual rhombomeres and their associated Hox codes to auditory sensorimotor circuitry. Here, we show that r4 contributes to functionally linked sensory and motor components, including the ventral nucleus of lateral lemniscus, posterior ventral cochlear nuclei (VCN), and motor olivocochlear neurons. Assembly of the r4-derived auditory components is involved in sound perception and depends on regulatory interactions between Hoxb1 and Hoxb2. Indeed, in Hoxb1 and Hoxb2 mutant mice the transmission of low-level auditory stimuli is lost, resulting in hearing impairments. On the other hand, Hoxa2 regulates the Rig1 axon guidance receptor and controls contralateral projections from the anterior VCN to the medial nucleus of the trapezoid body, a circuit involved in sound localization. Thus, individual rhombomeres and their associated Hox codes control the assembly of distinct functionally segregated sub-circuits in the developing auditory brainstem.

Parallel pathways from motor and somatosensory cortex for controlling whisker movements in mice.

Mice can gather tactile sensory information by actively moving their whiskers to palpate objects in their immediate surroundings. Whisker sensory perception therefore requires integration of sensory and motor information, which occurs prominently in the neocortex. The signalling pathways from the neocortex for controlling whisker movements are currently poorly understood in mice. Here, we delineate two pathways, one originating from primary whisker somatosensory cortex (wS1) and the other from whisker motor cortex (wM1), that control qualitatively distinct movements of contralateral whiskers. Optogenetic stimulation of wS1 drove retraction of contralateral whiskers while stimulation of wM1 drove rhythmic whisker protraction. To map brainstem pathways connecting these cortical areas to whisker motor neurons, we used a combination of anterograde tracing using adenoassociated virus injected into neocortex and retrograde tracing using monosynaptic rabies virus injected into whisker muscles. Our data are consistent with wS1 driving whisker retraction by exciting glutamatergic premotor neurons in the rostral spinal trigeminal interpolaris nucleus, which in turn activate the motor neurons innervating the extrinsic retractor muscle nasolabialis. The rhythmic whisker protraction evoked by wM1 stimulation might be driven by excitation of excitatory and inhibitory premotor neurons in the brainstem reticular formation innervating both intrinsic and extrinsic muscles. Our data therefore begin to unravel the neuronal circuits linking the neocortex to whisker motor neurons.

Mapping the face in the somatosensory brainstem.

The facial somatosensory map in the cortex is derived from facial representations that are first established at the brainstem level and then serially 'copied' at each stage of the somatosensory pathway. Recent studies have provided insights into the molecular mechanisms involved in the development of somatotopic maps of the face and whiskers in the trigeminal nuclei of the mouse brainstem. This work has revealed that early molecular regionalization and positional patterning of trigeminal ganglion and brainstem target neurons are established by homeodomain transcription factors, the expression of which is induced and maintained by signals from the brain and face. Such position-dependent information is fundamental in transforming the early spatial layout of sensory receptors into a topographic connectivity map that is conferred to higher brain levels.

Perinatal induction of Cre recombination with tamoxifen.

Temporal control of site-specific recombination is commonly achieved by using a tamoxifen-inducible form of Cre or Flp recombinases. Although powerful protocols of induction have been developed for gene inactivation at adult stages or during embryonic development, induction of recombination at late gestational or early postnatal stages is still difficult to achieve. In this context, using the ubiquitous CMV-CreER(T2) transgenic mice, we have tested and validated two procedures to achieve recombination just before and just after birth. The efficiency of recombination was evaluated in the brain, which is known to be more problematic to target. For the late gestation treatment with tamoxifen, different protocols of complementary administration of progesterone and estrogen were tested. However, delayed delivery and/or mortality of pups due to difficult delivery were always observed. To circumvent this problem, pups were collected from tamoxifen-treated pregnant dams by caesarian section at E18.5 and given to foster mothers. For postnatal treatment, different dosages of tamoxifen were administered by intragastric injection to the pups during 3 or 4 days after birth. The efficiency of these treatments was analyzed at P7 using a transgenic reporter line. They were also validated with the Hoxa5 conditional allele. In conclusion, we have developed efficient procedures that allow achieving efficient recombination of floxed alleles at perinatal stages. These protocols will allow investigating the late/adult functions of many developmental genes, whose characterization has been so far restricted to embryonic development.

Pattern and polarity in the development and evolution of the gnathostome jaw: both conservation and heterotopy in the branchial arches of the shark, Scyliorhinus canicula.

The acquisition of jaws constitutes a landmark event in vertebrate evolution, one that in large part potentiated their success and diversification. Jaw development and patterning involves an intricate spatiotemporal series of reciprocal inductive and responsive interactions between the cephalic epithelia and the cranial neural crest (CNC) and cephalic mesodermal mesenchyme. The coordinated regulation of these interactions is critical for both the ontogenetic registration of the jaws and the evolutionary elaboration of variable jaw morphologies and designs. Current models of jaw development and evolution have been built on molecular and cellular evidence gathered mostly in amniotes such as mice, chicks and humans, and augmented by a much smaller body of work on the zebrafish. These have been partnered by essential work attempting to understand the origins of jaws that has focused on the jawless lamprey. Chondrichthyans (cartilaginous fish) are the most distant group to amniotes within extant gnathostomes, and comprise the crucial clade uniting amniotes and agnathans; yet despite their critical phylogenetic position, evidence of the molecular and cellular underpinnings of jaw development in chondrichthyans is still lacking. Recent advances in genome and molecular developmental biology of the lesser spotted dogfish shark, Scyliorhinus canicula, make it ideal for the molecular study of chondrichthyan jaw development. Here, following the 'Hinge and Caps' model of jaw development, we have investigated evidence of heterotopic (relative changes in position) and heterochronic (relative changes in timing) shifts in gene expression, relative to amniotes, in the jaw primordia of S. canicula embryos. We demonstrate the presence of clear proximo-distal polarity in gene expression patterns in the shark embryo, thus establishing a baseline molecular baüplan for branchial arch-derived jaw development and further validating the utility of the 'Hinge and Caps' model in comparative studies of jaw development and evolution. Moreover, we correlate gene expression patterns with the absence of a lambdoidal junction (formed where the maxillary first arch meets the frontonasal processes) in chondrichthyans, further highlighting the importance of this region for the development and evolution of jaw structure in advanced gnathostomes.

Cdx mediates neural tube closure through transcriptional regulation of the planar cell polarity gene Ptk7.

The vertebrate Cdx genes (Cdx1, Cdx2 and Cdx4) encode homeodomain transcription factors with well-established roles in anteroposterior patterning. To circumvent the peri-implantation lethality inherent to Cdx2 loss of function, we previously used the Cre-loxP system to ablate Cdx2 at post-implantation stages and confirmed a crucial role for Cdx2 function in events related to axial extension. As considerable data suggest that the Cdx family members functionally overlap, we extended this analysis to assess the consequence of concomitant loss of both Cdx1 and Cdx2. Here, we report that Cdx1-Cdx2 double mutants exhibit a severely truncated anteroposterior axis. In addition, these double mutants exhibit fused somites, a widened mediolateral axis and craniorachischisis, a severe form of neural tube defect in which early neurulation fails and the neural tube remains open. These defects are typically associated with deficits in planar cell polarity (PCP) signaling in vertebrates. Consistent with this, we found that expression of Ptk7, which encodes a gene involved in PCP, is markedly reduced in Cdx1-Cdx2 double mutants, and is a candidate Cdx target. Genetic interaction between Cdx mutants and a mutant allele of Scrib, a gene involved in PCP signaling, is suggestive of a role for Cdx signaling in the PCP pathway. These findings illustrate a novel and pivotal role for Cdx function upstream of Ptk7 and neural tube closure in vertebrates.

Genetic identification of medullary neurons underlying congenital hypoventilation.

Mutations in the transcription factors encoded by PHOX2B or LBX1 correlate with congenital central hypoventilation disorders. These conditions are typically characterized by pronounced hypoventilation, central apnea, and diminished chemoreflexes, particularly to abnormally high levels of arterial PCO2. The dysfunctional neurons causing these respiratory disorders are largely unknown. Here, we show that distinct, and previously undescribed, sets of medullary neurons coexpressing both transcription factors (dB2 neurons) account for specific respiratory functions and phenotypes seen in congenital hypoventilation. By combining intersectional chemogenetics, intersectional labeling, lineage tracing, and conditional mutagenesis, we uncovered subgroups of dB2 neurons with key functions in (i) respiratory tidal volumes, (ii) the hypercarbic reflex, (iii) neonatal respiratory stability, and (iv) neonatal survival. These data provide functional evidence for the critical role of distinct medullary dB2 neurons in neonatal respiratory physiology. In summary, our work identifies distinct subgroups of dB2 neurons regulating breathing homeostasis, dysfunction of which causes respiratory phenotypes associated with congenital hypoventilation.

Opposing roles for Hoxa2 and Hoxb2 in hindbrain oligodendrocyte patterning.

Oligodendrocytes are the myelin-forming cells of the vertebrate CNS. Little is known about the molecular control of region-specific oligodendrocyte development. Here, we show that oligodendrogenesis in the mouse rostral hindbrain, which is organized in a metameric series of rhombomere-derived (rd) territories, follows a rhombomere-specific pattern, with extensive production of oligodendrocytes in the pontine territory (r4d) and delayed and reduced oligodendrocyte production in the prepontine region (r2d, r3d). We demonstrate that segmental organization of oligodendrocytes is controlled by Hox genes, namely Hoxa2 and Hoxb2. Specifically, Hoxa2 loss of function induced a dorsoventral enlargement of the Olig2/Nkx2.2-expressing oligodendrocyte progenitor domain, whereas conditional Hoxa2 overexpression in the Olig2(+) domain inhibited oligodendrogenesis throughout the brain. In contrast, Hoxb2 deletion resulted in a reduction of the pontine oligodendrogenic domain. Compound Hoxa2(-/-)/Hoxb2(-/-) mutant mice displayed the phenotype of Hoxb2(-/-) mutants in territories coexpressing Hoxa2 and Hoxb2 (rd3, rd4), indicating that Hoxb2 antagonizes Hoxa2 during rostral hindbrain oligodendrogenesis. This study provides the first in vivo evidence that Hox genes determine oligodendrocyte regional identity in the mammalian brain.

Cdx regulates Dll1 in multiple lineages.

Vertebrate Cdx genes encode homeodomain transcription factors related to caudal in Drosophila. The murine Cdx homologues Cdx1, Cdx2 and Cdx4 play important roles in anterior-posterior patterning of the embryonic axis and the intestine, as well as axial elongation. While our understanding of the ontogenic programs requiring Cdx function has advanced considerably, the molecular bases underlying these functions are less well understood. In this regard, Cdx1-Cdx2 conditional mutants exhibit abnormal somite formation, while loss of Cdx1-Cdx2 in the intestinal epithelium results in a shift in differentiation toward the Goblet cell lineage. The aim of the present study was to identify the Cdx-dependent mechanisms impacting on these events. Consistent with prior work implicating Notch signaling in these pathways, we found that expression of the Notch ligand Dll1 was reduced in Cdx mutants in both the intestinal epithelium and paraxial mesoderm. Cdx members occupied the Dll1 promoter both in vivo and in vitro, while genetic analysis indicated interaction between Cdx and Dll1 pathways in both somitogenesis and Goblet cell differentiation. These findings suggest that Cdx members operate upstream of Dll1 to convey different functions in two distinct lineages.

Molecular mechanisms of cranial neural crest cell migration and patterning in craniofacial development.

During vertebrate craniofacial development, neural crest cells (NCCs) contribute much of the cartilage, bone and connective tissue that make up the developing head. Although the initial patterns of NCC segmentation and migration are conserved between species, the variety of vertebrate facial morphologies that exist indicates that a complex interplay occurs between intrinsic genetic NCC programs and extrinsic environmental signals during morphogenesis. Here, we review recent work that has begun to shed light on the molecular mechanisms that govern the spatiotemporal patterning of NCC-derived skeletal structures - advances that are central to understanding craniofacial development and its evolution.

Ongoing roles of Phox2 homeodomain transcription factors during neuronal differentiation.

Transcriptional determinants of neuronal identity often stay expressed after their downstream genetic program is launched. Whether this maintenance of expression plays a role is for the most part unknown. Here, we address this question for the paralogous paired-like homeobox genes Phox2a and Phox2b, which specify several classes of visceral neurons at the progenitor stage in the central and peripheral nervous systems. By temporally controlled inactivation of Phox2b, we find that the gene, which is required in ventral neural progenitors of the hindbrain for the production of branchio-visceral motoneuronal precursors, is also required in these post-mitotic precursors to maintain their molecular signature - including downstream transcription factors - and allow their tangential migration and the histogenesis of the corresponding nuclei. Similarly, maintenance of noradrenergic differentiation during embryogenesis requires ongoing expression of Phox2b in sympathetic ganglia, and of Phox2a in the main noradrenergic center, the locus coeruleus. These data illustrate cases where the neuronal differentiation program does not unfold as a transcriptional `cascade' whereby downstream events are irreversibly triggered by an upstream regulator, but instead require continuous transcriptional input from it.

Comparative analysis of Hmx expression and the distribution of neuronal somata in the trigeminal ganglion in lamprey and shark: insights into the homology of the trigeminal nerve branches and the evolutionary origin of the vertebrate jaw.

The evolutionary origin of the jaw remains one of the most enigmatic events in vertebrate evolution. The trigeminal nerve is a key component for understanding jaw evolution, as it plays a crucial role as a sensorimotor interface for the effective manipulation of the jaw. This nerve is also found in the lamprey, an extant jawless vertebrate. The trigeminal nerve has three major branches in both the lamprey and jawed vertebrates. Although each of these branches was classically thought to be homologous between these two taxa, this homology is now in doubt. In the present study, we compared expression patterns of Hmx, a candidate genetic marker of the mandibular nerve (rV3, the third branch of the trigeminal nerve in jawed vertebrates), and the distribution of neuronal somata of trigeminal nerve branches in the trigeminal ganglion in lamprey and shark. We first confirmed the conserved expression pattern of Hmx1 in the shark rV3 neuronal somata, which are distributed in the caudal part of the trigeminal ganglion. By contrast, lamprey Hmx genes showed peculiar expression patterns, with expression in the ventrocaudal part of the trigeminal ganglion similar to Hmx1 expression in jawed vertebrates, which labeled the neuronal somata of the second branch. Based on these results, we propose two alternative hypotheses regarding the homology of the trigeminal nerve branches, providing new insights into the evolutionary origin of the vertebrate jaw.