GATA3 is essential for separating patterning domains during facial morphogenesis.

Neural crest cells (NCCs) within the mandibular and maxillary prominences of the first pharyngeal arch are initially competent to respond to signals from either region. However, mechanisms that are only partially understood establish developmental tissue boundaries to ensure spatially correct patterning. In the 'hinge and caps' model of facial development, signals from both ventral prominences (the caps) pattern the adjacent tissues whereas the intervening region, referred to as the maxillomandibular junction (the hinge), maintains separation of the mandibular and maxillary domains. One cap signal is GATA3, a member of the GATA family of zinc-finger transcription factors with a distinct expression pattern in the ventral-most part of the mandibular and maxillary portions of the first arch. Here, we show that disruption of Gata3 in mouse embryos leads to craniofacial microsomia and syngnathia (bony fusion of the upper and lower jaws) that results from changes in BMP4 and FGF8 gene regulatory networks within NCCs near the maxillomandibular junction. GATA3 is thus a crucial component in establishing the network of factors that functionally separate the upper and lower jaws during development.

Pbx4 limits heart size and fosters arch artery formation by partitioning second heart field progenitors and restricting proliferation.

Vertebrate heart development requires the integration of temporally distinct differentiating progenitors. However, few signals are understood that restrict the size of the later-differentiating outflow tract (OFT). We show that improper specification and proliferation of second heart field (SHF) progenitors in zebrafish lazarus (lzr) mutants, which lack the transcription factor Pbx4, produces enlarged hearts owing to an increase in ventricular and smooth muscle cells. Specifically, Pbx4 initially promotes the partitioning of the SHF into anterior progenitors, which contribute to the OFT, and adjacent endothelial cell progenitors, which contribute to posterior pharyngeal arches. Subsequently, Pbx4 limits SHF progenitor (SHFP) proliferation. Single cell RNA sequencing of nkx2.5+ cells revealed previously unappreciated distinct differentiation states and progenitor subpopulations that normally reside within the SHF and arterial pole of the heart. Specifically, the transcriptional profiles of Pbx4-deficient nkx2.5+ SHFPs are less distinct and display characteristics of normally discrete proliferative progenitor and anterior, differentiated cardiomyocyte populations. Therefore, our data indicate that the generation of proper OFT size and arch arteries requires Pbx-dependent stratification of unique differentiation states to facilitate both homeotic-like transformations and limit progenitor production within the SHF.

Neural crest cells bulldoze through the microenvironment using Aquaporin 1 to stabilize filopodia.

Neural crest migration requires cells to move through an environment filled with dense extracellular matrix and mesoderm to reach targets throughout the vertebrate embryo. Here, we use high-resolution microscopy, computational modeling, and in vitro and in vivo cell invasion assays to investigate the function of Aquaporin 1 (AQP-1) signaling. We find that migrating lead cranial neural crest cells express AQP-1 mRNA and protein, implicating a biological role for water channel protein function during invasion. Differential AQP-1 levels affect neural crest cell speed and direction, as well as the length and stability of cell filopodia. Furthermore, AQP-1 enhances matrix metalloprotease activity and colocalizes with phosphorylated focal adhesion kinases. Colocalization of AQP-1 with EphB guidance receptors in the same migrating neural crest cells has novel implications for the concept of guided bulldozing by lead cells during migration.

Physiological electric fields induce directional migration of mammalian cranial neural crest cells.

During neurulation, cranial neural crest cells (CNCCs) migrate long distances from the neural tube to their terminal site of differentiation. The pathway traveled by the CNCCs defines the blueprint for craniofacial construction, abnormalities of which contribute to three-quarters of human birth defects. Biophysical cues like naturally occurring electric fields (EFs) have been proposed to be one of the guiding mechanisms for CNCC migration from the neural tube to identified position in the branchial arches. Such endogenous EFs can be mimicked by applied EFs of physiological strength that has been reported to guide the migration of amphibian and avian neural crest cells (NCCs), namely galvanotaxis or electrotaxis. However, the behavior of mammalian NCCs in external EFs has not been reported. We show here that mammalian CNCCs migrate towards the anode in direct current (dc) EFs. Reversal of the field polarity reverses the directedness. The response threshold was below 30 â€‹mV/mm and the migration directedness and displacement speed increased with increase in field strength. Both CNCC line (O9-1) and primary mouse CNCCs show similar galvanotaxis behavior. Our results demonstrate for the first time that the mammalian CNCCs respond to physiological EFs by robust directional migration towards the anode in a voltage-dependent manner.

AP-2α and AP-2ß cooperatively orchestrate homeobox gene expression during branchial arch patterning.

The evolution of a hinged moveable jaw with variable morphology is considered a major factor behind the successful expansion of the vertebrates. DLX homeobox transcription factors are crucial for establishing the positional code that patterns the mandible, maxilla and intervening hinge domain, but how the genes encoding these proteins are regulated remains unclear. Herein, we demonstrate that the concerted action of the AP-2α and AP-2ß transcription factors within the mouse neural crest is essential for jaw patterning. In the absence of these two proteins, the hinge domain is lost and there are alterations in the size and patterning of the jaws correlating with dysregulation of homeobox gene expression, with reduced levels of Emx, Msx and Dlx paralogs accompanied by an expansion of Six1 expression. Moreover, detailed analysis of morphological features and gene expression changes indicate significant overlap with various compound Dlx gene mutants. Together, these findings reveal that the AP-2 genes have a major function in mammalian neural crest development, influencing patterning of the craniofacial skeleton via the DLX code, an effect that has implications for vertebrate facial evolution, as well as for human craniofacial disorders.

A new heart for a new head in vertebrate cardiopharyngeal evolution.

It has been more than 30 years since the publication of the new head hypothesis, which proposed that the vertebrate head is an evolutionary novelty resulting from the emergence of neural crest and cranial placodes. Neural crest generates the skull and associated connective tissues, whereas placodes produce sensory organs. However, neither crest nor placodes produce head muscles, which are a crucial component of the complex vertebrate head. We discuss emerging evidence for a surprising link between the evolution of head muscles and chambered hearts - both systems arise from a common pool of mesoderm progenitor cells within the cardiopharyngeal field of vertebrate embryos. We consider the origin of this field in non-vertebrate chordates and its evolution in vertebrates.

Ontogeny of the digestive tract of Brycon amazonicus (Teleostei, Bryconidae) under culture conditions: from hatching to juvenile stage.

In the present study, the morphological development of the Brycon amazonicus digestive tract is described to provide basic knowledge for nutritional studies and, therefore, increase the survival of this species during larviculture. Samples were collected from hatching up to 25 days of age, measured, processed and observed under a stereomicroscope and light microscopy. Newly hatched larvae presented their digestive tract as a straight tube, dorsal to the yolk sac, lined with a single layer of undifferentiated cells. At 24 h post-hatching (hPH), the buccopharyngeal cavity was open, but the posterior region of the digestive tube remained closed. At 25 hPH, the digestive tube was completely open and could be divided into buccopharyngeal cavity, oesophagus and intestine. At 35 hPH, the intestine presented a dilatation in the proximal region, which had the function of storing food. Differentiation of the stomach started at 83 hPH, and mucous cells were observed in the epithelium. These cells are important in the production of mucus, whose function is to protect the organ against acidity, although the gastric glands began developing only from 171 hPH, when three stomach regions were observed: cardiac, fundic and pyloric. The gastric glands were observed in the cardiac region, indicating that this organ already had digestive functionality. From 243 hPH, the absorption and assimilation of nutrients were already possible but, only from 412 hPH, the digestive tract was completely developed and functional.

Endothelium in the pharyngeal arches 3, 4 and 6 is derived from the second heart field.

Oxygenated blood from the heart is directed into the systemic circulation through the aortic arch arteries (AAAs). The AAAs arise by remodeling of three symmetrical pairs of pharyngeal arch arteries (PAAs), which connect the heart with the paired dorsal aortae at mid-gestation. Aberrant PAA formation results in defects frequently observed in patients with lethal congenital heart disease. How the PAAs form in mammals is not understood. The work presented in this manuscript shows that the second heart field (SHF) is the major source of progenitors giving rise to the endothelium of the pharyngeal arches 3 - 6, while the endothelium in the pharyngeal arches 1 and 2 is derived from a different source. During the formation of the PAAs 3 - 6, endothelial progenitors in the SHF extend cellular processes toward the pharyngeal endoderm, migrate from the SHF and assemble into a uniform vascular plexus. This plexus then undergoes remodeling, whereby plexus endothelial cells coalesce into a large PAA in each pharyngeal arch. Taken together, our studies establish a platform for investigating cellular and molecular mechanisms regulating PAA formation and alterations that lead to disease.

Bapx1 is required for jaw joint development in amphibians.

The acquisition of a movable jaw and a jaw joint are key events in gnathostome evolution. Jaws are derived from the neural crest derived pharyngeal skeleton and the transition from jawless to jawed vertebrates consists of major morphological changes, which must have a genetic foundation. Recent studies on the effects of bapx1 knockdown in fish and chicken indicate that bapx1 has acquired such a role in primary jaw joint development during vertebrate evolution, but evidence from amphibians is missing so far. In the present study, we use Ambystoma mexicanum, Bombina orientalis, and Xenopus laevis to investigate the effects of bapx1 knockdown on the development of these three different amphibians. Using morpholinos we downregulated the expression of bapx1 and obtain morphants with altered mandibular arch morphology. In the absence of bapx1 Meckels cartilage and the palatoquadrate jaw joint initially develop separately but during further development the joint cavity between both fills with chondrocytes. This results in the fusion of both cartilages and the loss of the jaw joint. Despite this the jaw itself remains usable for feeding and breathing. We show that bapx1 plays a role in jaw joint maintenance during development and that the morphants morphology possibly mirrors the morphology of the jawless ancestors of the gnathostomes.

Pharyngeal arch deficiencies affect taste bud development in the circumvallate papilla with aberrant glossopharyngeal nerve formation.

BACKGROUND: The pharyngeal arches (PAs) generate cranial organs including the tongue. The taste placodes, formed in particular locations on the embryonic tongue surface, differentiate into taste buds harbored in distinct gustatory papillae. The developing tongue also has a complex supply of cranial nerves through each PA. However, the relationship between the PAs and taste bud development is not fully understood. RESULTS: Ripply3 homozygous mutant mice, which have impaired third/fourth PAs, display a hypoplastic circumvallate papilla and lack taste buds, although the taste placode is normally formed. Formation of the glossopharyngeal ganglia is defective and innervation toward the posterior tongue is completely missing in Ripply3 mutant embryos at E12.5. Moreover, the distribution of neuroblasts derived from the epibranchial placode is severely, but not completely, atenuated, and the neural crest cells are diminished in the third PA region of Ripply3 mutant embryos at E9.5-E10.5. In Tbx1 homozygous mutant embryos, which exhibit another type of deficiency in PA development, the hypoplastic circumvallate papilla is observed along with abnormal formation of the glossopharyngeal ganglia and severely impaired innervation. CONCLUSIONS: PA deficiencies affect multiple aspects of taste bud development, including formation of the cranial ganglia and innervation to the posterior tongue.

Rh protein expression in branchial neuroepithelial cells, and the role of ammonia in ventilatory control in fish.

Bill Milsom has made seminal contributions to our understanding of ventilatory control in a wide range of vertebrates. Teleosts are particularly interesting, because they produce a 3rd, potentially toxic respiratory gas (ammonia) in large amounts. Fish are well known to hyperventilate under high environmental ammonia (HEA), but only recently has the potential role of ammonia in normal ventilatory control been investigated. It is now clear that ammonia can act directly as a ventilatory stimulant in trout, independent of its effects on acid-base balance. Even in ureotelic dogfish sharks, acute elevations in ammonia cause increases in ventilation. Peripherally, the detection of elevated ammonia resides in gill arches I and II in trout, and in vitro, neuroepithelial cells (NECs) from these arches are sensitive to ammonia, responding with elevations in intracellular Ca(2+) ([Ca(2+)]i). Centrally, hyperventilatory responses to ammonia correlate more closely with concentrations of ammonia in the brain than in plasma or CSF. After chronic HEA exposure, ventilatory responsiveness to ammonia is lost, associated with both an attenuation of the [Ca(2+)]i response in NECs, and the absence of elevation in brain ammonia concentration. Chronic exposure to HEA also causes increases in the mRNA expression of several Rh proteins (ammonia-conductive channels) in both brain and gills. "Single cell" PCR techniques have been used to isolate the individual responses of NECs versus other gill cell types. We suggest several circumstances (post-feeding, post-exercise) where the role of ammonia as a ventilatory stimulant may have adaptive benefits for O2 uptake in fish.

Sonic Hedgehog promotes the survival of neural crest cells by limiting apoptosis induced by the dependence receptor CDON during branchial arch development.

Cell-adhesion molecule-related/Downregulated by Oncogenes (CDO or CDON) was identified as a receptor for the classic morphogen Sonic Hedgehog (SHH). It has been shown that, in cell culture, CDO also behaves as a SHH dependence receptor: CDO actively triggers apoptosis in absence of SHH via a proteolytic cleavage in CDO intracellular domain. We present evidence that CDO is also pro-apoptotic in the developing neural tube where SHH is known to act as a survival factor. SHH, produced by the ventral foregut endoderm, was shown to promote survival of facial neural crest cells (NCCs) that colonize the first branchial arch (BA1). We show here that the survival activity of SHH on neural crest cells is due to SHH-mediated inhibition of CDO pro-apoptotic activity. Silencing of CDO rescued NCCs from apoptosis observed upon SHH inhibition in the ventral foregut endoderm. Thus, the pair SHH/dependence receptor CDO may play an important role in neural crest cell survival during the formation of the first branchial arch.

Osmoregulation and branchial plasticity after acute freshwater transfer in red drum, Sciaenops ocellatus.

Red drum, Sciaenops ocellatus, is an estuarine-dependent fish species commonly found in the Gulf of Mexico and along the coast of the southeastern United States. This economically important species has demonstrated freshwater tolerance; however, the physiological mechanisms and costs related to freshwater exposure remain poorly understood. The current study therefore investigated the physiological response of red drum using an acute freshwater transfer protocol. Plasma osmolality, Cl⁻, Mg²âº and Ca²âº were all significantly reduced by 24h post-transfer; Cl⁻ and Mg²âº recovered to control levels by 7days post-transfer. No effect of transfer was observed on muscle water content; however, muscle Cl⁻ was significantly reduced. Interestingly, plasma and muscle Na⁺ content was unaffected by freshwater transfer. Intestinal fluid was absent by 24h post-transfer indicating cessation of drinking. Branchial gene expression analysis showed that both CFTR and NKCC1 exhibited significant down-regulation at 8 and 24h post-transfer, respectively, although transfer had no impact on NHE2, NHE3 or Na⁺, K⁺ ATPase (NKA) activity. These general findings are supported by immunohistochemical analysis, which revealed no apparent NKCC containing cells in the gills at 7days post transfer while NKA cells localization was unaffected. The results of the current study suggest that red drum can effectively regulate Na⁺ balance upon freshwater exposure using already present Na⁺ uptake pathways while also down-regulating ion excretion mechanisms.

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.

Hh signaling regulates patterning and morphogenesis of the pharyngeal arch-derived skeleton.

The proper function of the craniofacial skeleton requires the proper shaping of many individual skeletal elements. Neural crest cells generate much of the craniofacial skeleton and morphogenesis of skeletal elements occurs in transient, reiterated structures termed pharyngeal arches. The shape of individual elements depends upon intrinsic patterning within the neural crest as well as extrinsic signals to the neural crest from adjacent tissues within the arches. Hedgehog (Hh) signaling is known to play roles in craniofacial development, yet its involvement in intrinsic and extrinsic patterning of the craniofacial skeleton is still not well understood. Here, we show that morphogenetic movements of the pharyngeal arches and patterning of the neural crest require Hh signaling. Loss of Hh signaling, in smoothened (smo) mutants, disrupts the expression of some Dlx genes as well as other markers of dorsal/ventral patterning of the neural crest. Transplantation of wild-type neural crest cells into smo mutants rescues this defect, demonstrating that the neural crest requires reception of Hh signals for proper patterning. Despite the rescue, morphogenesis of the facial skeleton is not fully recovered. Through transplant analyses, we find two additional requirements for Hh signaling. The endoderm requires the reception of Hh signals for proper morphogenetic movements of the pharyngeal arches and the neural crest require the reception of Hh signaling for the activity of a reverse signal that maintains sonic hedgehog expression in the endoderm. Collectively, these results demonstrate that Hh signaling is essential to establish intrinsic and extrinsic patterning information for the craniofacial skeleton.

Hes1 is required for the development of pharyngeal organs and survival of neural crest-derived mesenchymal cells in pharyngeal arches.

Hes genes are required to maintain diverse progenitor cell populations during embryonic development. Loss of Hes1 results in a spectrum of malformations of pharyngeal endoderm-derived organs, including the ultimobranchial body (progenitor of C cells), parathyroid, thymus and thyroid glands, together with highly penetrant C-cell aplasia (81%) and parathyroid aplasia (28%). The hypoplastic parathyroid and thymus are mostly located around the pharyngeal cavity, even at embryonic day (E) 15.5 to E18.5, indicating the failure of migration of the organs. To clarify the relationship between these phenotypes and neural crest cells, we examine fate mapping of neural crest cells colonized in pharyngeal arches in Hes1 null mutants by using the Wnt1-Cre/R26R reporter system. In null mutants, the number of neural crest cells labeled by X-gal staining is markedly decreased in the pharyngeal mesenchyme at E12.5 when the primordia of the thymus, parathyroid and ultimobranchial body migrate toward their destinations. Furthermore, phospho-Histone-H3-positive proliferating cells are reduced in number in the pharyngeal mesenchyme at this stage. Our data indicate that the development of pharyngeal organs and survival of neural-crest-derived mesenchyme in pharyngeal arches are critically dependent on Hes1. We propose that the defective survival of neural-crest-derived mesenchymal cells in pharyngeal arches directly or indirectly leads to deficiencies of pharyngeal organs.

The SH2 tyrosine phosphatase shp2 is required for mammalian limb development.

The tyrosine phosphatase Shp2 is recruited into tyrosine-kinase signalling pathways through binding of its two amino-terminal SH2 domains to specific phosphotyrosine motifs, concurrent with its re-localization and stimulation of phosphatase activity. Shp2 can potentiate signalling through the MAP-kinase pathway and is required during early mouse development for gastrulation. Chimaeric analysis can identify, by study of phenotypically normal embryos, tissues that tolerate mutant cells (and therefore do not require the mutated gene) or lack mutant cells (and presumably require the mutated gene during their developmental history). We therefore generated chimaeric mouse embryos to explore the cellular requirements for Shp2. This analysis revealed an obligatory role for Shp2 during outgrowth of the limb. Shp2 is specifically required in mesenchyme cells of the progress zone (PZ), directly beneath the distal ectoderm of the limb bud. Comparison of Ptpn11 (encoding Shp2)-mutant and Fgfr1 (encoding fibroblast growth factor receptor-1)-mutant chimaeric limbs indicated that in both cases mutant cells fail to contribute to the PZ of phenotypically normal chimaeras, leading to the hypothesis that a signal transduction pathway, initiated by Fgfr1 and acting through Shp2, is essential within PZ cells. Rather than integrating proliferative signals, Shp2 probably exerts its effects on limb development by influencing cell shape, movement or adhesion. Furthermore, the branchial arches, which also use Fgfs during bud outgrowth, similarly require Shp2. Thus, Shp2 regulates phosphotyrosine-signalling events during the complex ectodermal-mesenchymal interactions that regulate mammalian budding morphogenesis.

Alcama mediates Edn1 signaling during zebrafish cartilage morphogenesis.

The zebrafish pharyngeal cartilage is derived from the pharyngeal apparatus, a vertebrate-specific structure derived from all three germ layers. Developmental aberrations of the pharyngeal apparatus lead to birth defects such as Treacher-Collins and DiGeorge syndromes. While interactions between endoderm and neural crest (NC) are known to be important for cartilage formation, the full complement of molecular players involved and their roles remain to be elucidated. Activated leukocyte cell adhesion molecule a (alcama), a member of the immunoglobulin (Ig) superfamily, is among the prominent markers of pharyngeal pouch endoderm, but to date no role has been assigned to this adhesion molecule in the development of the pharyngeal apparatus. Here we show that alcama plays a crucial, non-autonomous role in pharyngeal endoderm during zebrafish cartilage morphogenesis. alcama knockdown leads to defects in NC differentiation, without affecting NC specification or migration. These defects are reminiscent of the phenotypes observed when Endothelin 1 (Edn1) signaling, a key regulator of cartilage development is disrupted. Using gene expression analysis and rescue experiments we show that Alcama functions downstream of Edn1 signaling to regulate NC differentiation and cartilage morphogenesis. In addition, we also identify a role for neural adhesion molecule 1.1 (nadl1.1), a known interacting partner of Alcama expressed in neural crest, in NC differentiation. Our data shows that nadl1.1 is required for alcama rescue of NC differentiation in edn1(-/-) mutants and that Alcama interacts with Nadl1.1 during chondrogenesis. Collectively our results support a model by which Alcama on the endoderm interacts with Nadl1.1 on NC to mediate Edn1 signaling and NC differentiation during chondrogenesis.

Importance of SoxE in neural crest development and the evolution of the pharynx.

The neural crest, a defining character of vertebrates, is of prime importance to their evolutionary origin. To understand neural crest evolution, we explored molecular mechanisms underlying craniofacial development in the basal jawless vertebrate, sea lamprey (Petromyzon marinus), focusing on the SoxE (Sox8, Sox9 and Sox10) gene family. In jawed vertebrates, these are important transcriptional regulators of the neural crest, and the loss of Sox9 causes abnormal craniofacial development. Here we report that two lamprey SoxE genes are expressed in migrating neural crest and crest-derived prechondrocytes in posterior branchial arches, whereas a third paralogue is expressed later in the perichondrium and mandibular arch. Morpholino knock-down of SoxE1 reveals that it is essential for posterior branchial arch development, although the mandibular arch is unaffected. The results show that chondrogenic function of SoxE regulators can be traced to the lamprey-gnathostome common ancestor and indicate that lamprey SoxE genes might have undergone independent duplication to have distinct functions in mandibular versus caudal branchial arches. This work sheds light on the homology of vertebrate branchial arches and supports their common origin at the base of vertebrates.

Plasticity of neural crest-placode interaction in the developing visceral nervous system.

The reciprocal relationship between rhombomere (r)-derived cranial neural crest (NC) and epibranchial placodal cells derived from the adjacent branchial arch is critical for visceral motor and sensory gangliogenesis, respectively. However, it is unknown whether the positional match between these neurogenic precursors is hard-wired along the anterior-posterior (A/P) axis. Here, we use the interaction between r4-derived NC and epibranchial placode-derived geniculate ganglion as a model to address this issue. In Hoxa1(-/-) b1(-/-) embryos, r2 NC compensates for the loss of r4 NC. Specifically, a population of r2 NC cells is redirected toward the geniculate ganglion, where they differentiate into postganglionic (motor) neurons. Reciprocally, the inward migration of the geniculate ganglion is associated with r2 NC. The ability of NC and placodal cells to, respectively, differentiate and migrate despite a positional mismatch along the A/P axis reflects the plasticity in the relationship between the two neurogenic precursors of the vertebrate head.