Schwend Tyler, Ahlgren Sara C
Integrated Graduate Program, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
BMC Dev Biol. 2009 Nov 30;9:59. doi: 10.1186/1471-213X-9-59.
The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC). Genetic studies in zebrafish and mice have established that the Hedgehog (Hh)-signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in CNCC development. Disruption of the Hh-signaling pathway in humans can lead to the spectral disorder of Holoprosencephaly (HPE), which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia 12. Previous work has uncovered a role for Hh-signaling in zebrafish dorsal neurocranium patterning and chondrogenesis, however Hh-signaling mutants have not been described with respect to the ventral pharyngeal arch (PA) skeleton. Lipid-modified Hh-ligands require the transmembrane-spanning receptor Dispatched 1 (Disp1) for proper secretion from Hh-synthesizing cells to the extracellular field where they act on target cells. Here we study chameleon mutants, lacking a functional disp1(con/disp1).
con/disp1 mutants display reduced and dysmorphic mandibular and hyoid arch cartilages and lack all ceratobranchial cartilage elements. CNCC specification and migration into the PA primorida occurs normally in con/disp1 mutants, however disp1 is necessary for post-migratory CNCC patterning and differentiation. We show that disp1 is required for post-migratory CNCC to become properly patterned within the first arch, while the gene is dispensable for CNCC condensation and patterning in more posterior arches. Upon residing in well-formed pharyngeal epithelium, neural crest condensations in the posterior PA fail to maintain expression of two transcription factors essential for chondrogenesis, sox9a and dlx2a, yet continue to robustly express other neural crest markers. Histology reveals that posterior arch residing-CNCC differentiate into fibrous-connective tissue, rather than becoming chondrocytes. Treatments with Cyclopamine, to inhibit Hh-signaling at different developmental stages, show that Hh-signaling is required during gastrulation for normal patterning of CNCC in the first PA, and then during the late pharyngula stage, to promote CNCC chondrogenesis within the posterior arches. Further, loss of disp1 disrupted normal expression of bapx1 and gdf5, markers of jaw joint patterning, thus resulting in jaw joint defects in con/disp1 mutant animals.
This study reveals novel requirements for Hh-signaling in the zebrafish PA skeleton and highlights the functional diversity and differential sensitivity of craniofacial tissues to Hh-signaling throughout the face, a finding that may help to explain the spectrum of human facial phenotypes characteristic of HPE.
脊椎动物的头部骨骼主要源自颅神经嵴细胞（CNCC）。斑马鱼和小鼠的遗传学研究已证实，刺猬信号通路（Hh信号通路）在颅面发育中起关键作用，部分原因在于该通路在CNCC发育中的作用。人类Hh信号通路的破坏可导致全前脑畸形（HPE）这一谱系障碍，其通常特征为包括中线面部裂和独眼畸形等多种颅面缺陷。先前的研究发现Hh信号在斑马鱼背侧神经颅骨模式形成和软骨形成中发挥作用，然而关于腹侧咽弓（PA）骨骼，尚未有Hh信号突变体的相关描述。脂质修饰的Hh配体需要跨膜受体Dispatched 1（Disp1）才能从Hh合成细胞正确分泌到细胞外区域，在那里它们作用于靶细胞。在此，我们研究缺乏功能性disp1（con/disp1）的变色龙突变体。
con/disp1突变体的下颌和舌弓软骨减少且形态异常，并且所有鳃弓软骨元件均缺失。在con/disp1突变体中，CNCC的特化和迁移至PA原基的过程正常发生，然而disp1对于迁移后CNCC的模式形成和分化是必需的。我们发现disp1是迁移后CNCC在第一弓内正确形成模式所必需的，而该基因对于更靠后的弓中CNCC的凝聚和模式形成是可有可无的。在驻留在形态良好的咽上皮后，PA后部的神经嵴凝聚物无法维持软骨形成所必需的两个转录因子sox9a和dlx2a的表达，但仍继续强烈表达其他神经嵴标记物。组织学显示，驻留在后部弓的CNCC分化为纤维结缔组织，而非成为软骨细胞。用环杷明在不同发育阶段抑制Hh信号的处理表明，在原肠胚形成期间，Hh信号对于第一PA中CNCC的正常模式形成是必需的，然后在咽胚后期，Hh信号用于促进后部弓内CNCC的软骨形成。此外，disp1的缺失破坏了颌关节模式形成标记物bapx1和gdf5的正常表达，从而导致con/disp1突变动物出现颌关节缺陷。
本研究揭示了斑马鱼PA骨骼中Hh信号的新需求，并突出了颅面组织在整个面部对Hh信号的功能多样性和不同敏感性，这一发现可能有助于解释HPE特征性的人类面部表型谱系。
