The zebrafish homeobox gene irxl1 is required for brain and pharyngeal arch morphogenesis

Iroquois homeobox‐like 1 (irxl1) is a novel member of the TALE superfamily of homeobox genes that is most closely related to the Iroquois class. We have identified the zebrafish irxl1 gene and characterized its structure. The protein contains a homeodomain that shares 100% sequence identity with other vertebrate orthologs. During embryogenesis, irxl1 is expressed from 18 hours postfertilization onward and prominent expression is detected in the pharyngeal arches. Knockdown of irxl1 by morpholinos results in malformed brain and arch structures, which can be partially rescued by cRNA injection. The heads of the morphants become small and flat, and extensions along the anterior–posterior/dorso–ventral axes are reduced without affecting regional specification. Loss of irxl1 function also causes deficit in neural crest cells which consequently results in partial loss of craniofacial muscles and severe deformation of arch cartilages. These observations suggest that irxl1 may regulate factors involved in brain and pharyngeal arch development. Developmental Dynamics 239:639–650, 2010. © 2009 Wiley‐Liss, Inc.     

Hedgehog signaling regulates dental papilla formation and tooth size during zebrafish odontogenesis

Background : Intercellular communication by the hedgehog cell signaling pathway is necessary for tooth development throughout the vertebrates, but it remains unclear which specific developmental signals control cell behavior at different stages of odontogenesis. To address this issue, we have manipulated hedgehog activity during zebrafish tooth development and visualized the results using confocal microscopy. Results : We first established that reporter lines for dlx2b, fli1, NF‐κB, and prdm1a are markers for specific subsets of tooth germ tissues. We then blocked hedgehog signaling with cyclopamine and observed a reduction or elimination of the cranial neural crest derived dental papilla, which normally contains the cells that later give rise to dentin‐producing odontoblasts. Upon further investigation, we observed that the dental papilla begins to form and then regresses in the absence of hedgehog signaling, through a mechanism unrelated to cell proliferation or apoptosis. We also found evidence of an isometric reduction in tooth size that correlates with the time of earliest hedgehog inhibition. Conclusions : We hypothesize that these results reveal a previously uncharacterized function of hedgehog signaling during tooth morphogenesis, regulating the number of cells in the dental papilla and thereby controlling tooth size. Developmental Dynamics 244:577–590, 2015. © 2015 Wiley Periodicals, Inc.     

Sonic hedgehog from pharyngeal arch 1 epithelium is necessary for early mandibular arch cell survival and later cartilage condensation differentiation

Background: Morphogenesis of vertebrate craniofacial skeletal elements is dependent on a key cell population, the cranial neural crest cells (NCC). Cranial NCC are formed dorsally in the cranial neural tube and migrate ventrally to form craniofacial skeletal elements as well as other tissues. Multiple extracellular signaling pathways regulate the migration, survival, proliferation, and differentiation of NCC. Results: In this study, we demonstrate that Shh expression in the oral ectoderm and pharyngeal endoderm is essential for mandibular development. We show that a loss of Shh in these domains results in increased mesenchymal cell death in pharyngeal arch 1 (PA1) after NCC migration. This increased cell death can be rescued in utero by pharmacological inhibition of p53. Furthermore, we show that epithelial SHH is necessary for the early differentiation of mandibular cartilage condensations and, therefore, the subsequent development of Meckel's cartilage, around which the dentary bone forms. Nonetheless, a rescue of the cell death phenotype does not rescue the defect in cartilage condensation formation. Conclusions: Our results show that SHH produced by the PA1 epithelium is necessary for the survival of post‐migratory NCC, and suggests a key role in the subsequent differentiation of chondrocytes to form Meckel's cartilage. Developmental Dynamics 244:564–576, 2015. © 2015 Wiley Periodicals, Inc.     

13‐cis‐retinoic acid alters neural crest cells expressing Krox‐20 and Pax‐2 in macaque embryos

This study investigates hindbrain and associated neural crest (NCC), otocyst, and pharyngeal arch development in monkey embryos following teratogenic exposure to 13‐cis‐retinoic acid (cRA). cRA was orally administered (5 mg/kg) to pregnant long‐tailed macaques (Macaca fascicularis) between gestational days (GD) 12 and 27. Embryos were surgically collected at desired stages during treatment, analyzed for external morphological changes, and processed for immunohistochemistry. Two transiently expressed nuclear proteins, Krox‐20 and Pax‐2, were used as markers for the target cellular and anatomical structures. Rhombomere (r) expression patterns of Pax‐2 (r4/r6) and Krox‐20 (r3/r5) were maintained after cRA treatment, but r4 and r5 were substantially reduced in size. In untreated embryos, Krox‐20 immunoreactive NCC derived from r5 migrated caudally around the developing otocyst to contribute to the third pharyngeal arch mesenchyme. In cRA‐treated embryos, a subpopulation of NCC rostral to the otocyst also showed Krox‐20 immunoreactivity, but there was a substantial reduction in Krox‐20 post‐otic NCC. Pax‐2 immunoreactive NCC migrating from r4 to the second pharyngeal arch were substantially reduced in numbers in treated embryos. Alteration in the otic anlage included delayed invagination, abnormal relationship with the adjacent hindbrain epithelium, and altered expression boundaries for Pax‐2. cRA‐associated changes in the pharyngeal arch region due to cRA included truncation of the distal portion of the first arch and reduction in the size of the second arch. These alterations in hindbrain, neural crest, otic anlage, and pharyngeal arch morphogenesis could contribute to some of the craniofacial malformations in the macaque fetus associated with exposure to cRA. Anat Rec 255:142–154, 1999. © 1999 Wiley‐Liss, Inc.     

Notch pathway regulation of neural crest cell development in vivo

Background : The function of Notch signaling in murine neural crest–derived cell lineages in vivo was examined. Results : Conditional gain (Wnt1Cre;Rosa^Notch) or loss (Wnt1Cre;RBP‐J^f/f) of Notch signaling in neural crest cells (NCCs) in vivo results in craniofacial, cardiac, and trunk abnormalities. Severe craniofacial malformations are apparent in Wnt1Cre;Rosa^Notch embryos, while less severe skull abnormalities are evident in Wnt1Cre;RBP‐J^f/f mice. Deficient cardiac neural crest migration, resulting in cardiac outflow tract malformations, occurs with increased or decreased Notch signaling in NCCs. Smooth muscle cell differentiation also is impaired in pharyngeal NCC derivatives in both Wnt1Cre;Rosa^Notch and Wnt1Cre;RBP‐J^f/f embryos. Neurogenesis is absent and gliogenesis is increased in the dorsal root ganglia of Wnt1Cre;Rosa^Notch embryos, while neurogenesis is increased and gliogenesis is decreased in Wnt1Cre;RBP‐J^f/f embryos. Conclusions : Together, these studies demonstrate essential cell‐autonomous roles for appropriate levels of Notch signaling during NCC migration, proliferation, and differentiation with critical implications in craniofacial, cardiac, and neurogenic development and disease. Developmental Dynamics 241:376–389, 2012. © 2011 Wiley Periodicals, Inc.     

Zebrafish ift57, ift88, and ift172 intraflagellar transport mutants disrupt cilia but do not affect hedgehog signaling

Cilia formation requires intraflagellar transport (IFT) proteins. Recent studies indicate that mammalian Hedgehog (Hh) signaling requires cilia. It is unclear, however, if the requirement for cilia and IFT proteins in Hh signaling represents a general rule for all vertebrates. Here we examine zebrafish ift57, ift88, and ift172 mutants and morphants for defects in Hh signaling. Although ift57 and ift88 mutants and morphants contained residual maternal protein, the cilia were disrupted. In contrast to previous genetic studies in mouse, mutations in zebrafish IFT genes did not affect the expression of Hh target genes in the neural tube and forebrain and had no quantitative effect on Hh target gene expression. Zebrafish IFT mutants also exhibited no dramatic changes in the craniofacial skeleton, somite formation, or motor neuron patterning. Thus, our data indicate the requirement for cilia in the Hh signal transduction pathway may not represent a universal mechanism in vertebrates. Developmental Dynamics 238:1744–1759, 2009. © 2009 Wiley‐Liss, Inc.     

Zebrafish acvr2a and acvr2b exhibit distinct roles in craniofacial development.

To examine the roles of activin type II receptor signaling in craniofacial development, full-length zebrafish acvr2a and acvr2b clones were isolated. Although ubiquitously expressed as maternal mRNAs and in early embryogenesis, by 24 hr postfertilization (hpf), acvr2a and acvr2b exhibit restricted expression in neural, hindbrain, and neural crest cells (NCCs). A morpholino-based targeted protein depletion approach was used to reveal discrete functions for each acvr2 gene product. The acvr2a morphants exhibited defects in the development of most cranial NCC-derived cartilage, bone, and pharyngeal tooth structures, whereas acvr2b morphant defects were largely restricted to posterior arch structures and included the absence and/or aberrant migration of posterior NCC streams, defects in NCC-derived posterior arch cartilages, and dysmorphic pharyngeal tooth development. These studies revealed previously uncharacterized roles for acvr2a and acvr2b in hindbrain and NCC patterning, in NCC derived pharyngeal arch cartilage and joint formation, and in tooth development.     

Embryonic Development and Skeletogenesis of the Pharyngeal Jaw Apparatus in the Cichlid Nile Tilapia (Oreochromis niloticus)

The evolution of a specialized pharyngeal jaw apparatus (PJA) has been argued to be the key evolutionary innovation that allowed the explosive adaptive radiation of cichlid fishes in East African lakes. Subsequent studies together with recent molecular phylogenies have shown that similar innovations evolved independently several times within the teleosts, which poses the questions: (1) how similar are the developmental mechanisms responsible for these changes in divergent taxa and (2) how did such complex features arise independently in evolution? A detailed knowledge of PJA development in cichlids and other teleosts is needed to address these questions. Here, we provide a detailed account of the development of the PJA in one species of cichlid, the Nile tilapia (Oreochromis niloticus), from the early segmentation and patterning of its embryonic precursors – pharyngeal arches 3 to 7 – to its ossification. We find that pharyngeal segmentation occurs sequentially from anterior to posterior during early segmentation stages through the mid‐pharyngula period. We show a clear combinatorial code of Hox gene expression such that each posterior arch is defined by a distinctive Hox signature. Posterior arch chondrogenesis in tilapia is essentially complete by the end of the hatching period, and most elements become ossified over the next two days. Our results reveal that both the fusion of lower jaw bones and articulation between the neurocranium and upper jaws occur during post‐embryonic development. Abstract end data should be: Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

Developmental and evolutionary significance of the mandibular arch and prechordal/premandibular cranium in vertebrates: revising the heterotopy scenario of gnathostome jaw evolution

The cephalic neural crest produces streams of migrating cells that populate pharyngeal arches and a more rostral, premandibular domain, to give rise to an extensive ectomesenchyme in the embryonic vertebrate head. The crest cells forming the trigeminal stream are the major source of the craniofacial skeleton; however, there is no clear distinction between the mandibular arch and the premandibular domain in this ectomesenchyme. The question regarding the evolution of the gnathostome jaw is, in part, a question about the differentiation of the mandibular arch, the rostralmost component of the pharynx, and in part a question about the developmental fate of the premandibular domain. We address the developmental definition of the mandibular arch in connection with the developmental origin of the trabeculae, paired cartilaginous elements generally believed to develop in the premandibular domain, and also of enigmatic cartilaginous elements called polar cartilages. Based on comparative embryology, we propose that the mandibular arch ectomesenchyme in gnathostomes can be defined as a Dlx1-positive domain, and that the polar cartilages, which develop from the Dlx1-negative premandibular ectomesenchyme, would represent merely posterior parts of the trabeculae. We also show, in the lamprey embryo, early migration of mandibular arch mesenchyme into the premandibular domain, and propose an updated version of the heterotopy theory on the origin of the jaw.     

Twist1 contributes to cranial bone initiation and dermal condensation by maintaining wnt signaling responsiveness

Background: Specification of cranial bone and dermal fibroblast progenitors in the supraorbital arch mesenchyme is Wnt/β‐catenin signaling‐dependent. The mechanism underlying how these cells interpret instructive signaling cues and differentiate into these two lineages is unclear. Twist1 is a target of the Wnt/β‐catenin signaling pathway and is expressed in cranial bone and dermal lineages. Results: Here, we show that onset of Twist1 expression in the mouse cranial mesenchyme is dependent on ectodermal Wnts and mesenchymal β‐catenin activity. Conditional deletion of Twist1 in the supraorbital arch mesenchyme leads to cranial bone agenesis and hypoplastic dermis, as well as craniofacial malformation of eyes and palate. Twist1 is preferentially required for cranial bone lineage commitment by maintaining Wnt responsiveness. In the conditional absence of Twist1, the cranial dermis fails to condense and expand apically leading to extensive cranial dermal hypoplasia with few and undifferentiated hair follicles. Conclusions: Thus, Twist1, a target of canonical Wnt/β‐catenin signaling, also functions to maintain Wnt responsiveness and is a key effector for cranial bone fate selection and dermal condensation. Developmental Dynamics 245:144–156, 2016. © 2015 Wiley Periodicals, Inc.     

Effects of activation of hedgehog signaling on patterning,growth, and differentiation in Xenopus froglet limb regeneration

Regenerating limbs of urodele amphibians and Xenopus tadpole are reconstructed along proximal–distal, anterior–posterior (AP), and dorsal–ventral axes. In contrast, a regenerated limb of the Xenopus froglet does not have digits, and only a simple cartilaginous structure referred to as a “spike” is formed. This suggests that repatterning along the AP axis is absent in the froglet blastema. Previous studies have shown that Shh and its target genes are not expressed in the froglet blastema. In this study, we activated Hedgehog signaling in the froglet blastema and found that target genes of Shh were inducible in the mesenchyme of limb blastema. Furthermore, we found that activation of the signaling had effects on blastema cell proliferation and chondrogenesis and resulted in the formation of multiple cartilaginous structures. These findings indicate that activation of signaling that is absent in the froglet blastema is effective for improvement of limb regeneration ability in the Xenopus froglet. Developmental Dynamics, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of a distant cis‐regulatory element controlling pharyngeal arch‐specific expression of zebrafish gdf6a/radar

Skeletal formation is an essential and intricately regulated part of vertebrate development. Humans and mice deficient in growth and differentiation factor 6 (Gdf6) have numerous skeletal abnormalities, including joint fusions and cartilage reductions. The expression of Gdf6 is dynamic and in part regulated by distant evolutionarily conserved cis‐regulatory elements. radar/gdf6a is a zebrafish ortholog of Gdf6 and has an essential role in embryonic patterning. Here, we show that radar is transcribed in the cells surrounding and between the developing cartilages of the ventral pharyngeal arches, similar to mouse Gdf6. A 312 bp evolutionarily conserved region (ECR5), 122 kilobases downstream, drives expression in a pharyngeal arch‐specific manner similar to endogenous radar/gdf6a. Deletion analysis identified a 78 bp region within ECR5 that is essential for transgene activity. This work illustrates that radar is regulated in the pharyngeal arches by a distant conserved element and suggests radar has similar functions in skeletal development in fish and mammals. Developmental Dynamics 239:1047–1060, 2010. © 2010 Wiley‐Liss, Inc.     

Role of primary cilia and Hedgehog signaling in craniofacial features of Ellis–van Creveld syndrome

Ellis–van Creveld syndrome (EvC) is an autosomal recessive genetic disorder involving pathogenic variants of EVC and EVC2 genes and classified as a ciliopathy. The syndrome is caused by mutations in the EVC gene on chromosome 4p16, and EVC2 gene, located close to the EVC gene, in a head-to-head configuration. Regardless of the affliction of EVC or EVC2, the clinical features of Ellis–van Creveld syndrome are similar. Both these genes are expressed in tissues such as, but not limited to, the heart, liver, skeletal muscle, and placenta, while the predominant expression in the craniofacial tissues is that of EVC2. Biallelic mutations of EVC and EVC2 affect Hedgehog signaling and thereby ciliary function, crucial factors in vertebrate development, culminating in the phenotypical features characteristic of EvC. The clinical features of Ellis–van Creveld syndrome are consistent with significant abnormalities in morphogenesis and differentiation of the affected tissues. The robust role of primary cilia in histodifferentiation and morphodifferentiation of oral, perioral, and craniofacial tissues is becoming more evident in the most recent literature. In this review, we give a summary of the mechanistic role of primary cilia in craniofacial development, taking Ellis–van Creveld syndrome as a representative example.