Transcriptomics reveals complex kinetics of dorsal-ventral patterning gene expression in the mandibular arch.

The mandibular or first pharyngeal arch forms the upper and lower jaws in all gnathostomes. A gene regulatory network that defines ventral, intermediate, and dorsal domains along the dorsal-ventral (D-V) axis of the arch has emerged from studies in zebrafish and mice, but the temporal dynamics of this process remain unclear. To define cell fate trajectories in the arches we have performed quantitative gene expression analyses of D-V patterning genes in pharyngeal arch primordia in zebrafish and mice. Using NanoString technology to measure transcript numbers per cell directly we show that, in many cases, genes expressed in similar D-V domains and induced by similar signals vary dramatically in their temporal profiles. This suggests that cellular responses to D-V patterning signals are likely shaped by the baseline kinetics of target gene expression. Furthermore, similarities in the temporal dynamics of genes that occupy distinct pathways suggest novel shared modes of regulation. Incorporating these gene expression kinetics into our computational models for the mandibular arch improves the accuracy of patterning, and facilitates temporal comparisons between species. These data suggest that the magnitude and timing of target gene expression help diversify responses to patterning signals during craniofacial development.

Modeling craniofacial development reveals spatiotemporal constraints on robust patterning of the mandibular arch.

How does pattern formation occur accurately when confronted with tissue growth and stochastic fluctuations (noise) in gene expression? Dorso-ventral (D-V) patterning of the mandibular arch specifies upper versus lower jaw skeletal elements through a combination of Bone morphogenetic protein (Bmp), Endothelin-1 (Edn1), and Notch signaling, and this system is highly robust. We combine NanoString experiments of early D-V gene expression with live imaging of arch development in zebrafish to construct a computational model of the D-V mandibular patterning network. The model recapitulates published genetic perturbations in arch development. Patterning is most sensitive to changes in Bmp signaling, and the temporal order of gene expression modulates the response of the patterning network to noise. Thus, our integrated systems biology approach reveals non-intuitive features of the complex signaling system crucial for craniofacial development, including novel insights into roles of gene expression timing and stochasticity in signaling and gene regulation.

Regulation of long-range planar cell polarity by Fat-Dachsous signaling.

Fat (Ft) and Dachsous (Ds) are large cadherins that bind each other and have conserved roles in regulating planar cell polarity (PCP). We quantitatively analyzed Ft-Ds pathway mutant clones for their effects on ommatidial polarity in the Drosophila eye. Our findings suggest that the Ft-Ds pathway regulates PCP propagation independently of asymmetric cellular accumulation of Ft or Ds. We find that the Ft effector Atrophin has a position-specific role in regulating polarity in the eye, and that asymmetric accumulation of the atypical myosin Dachs is not essential for production and propagation of a long-range PCP signal. Our observations suggest that Ft and Ds interact to modulate a secondary signal that regulates long-range polarity, that signaling by the Ds intracellular domain is dependent on Ft, and that ommatidial fate specification is genetically separable from long-range signaling.

Single-cell transcriptomic analysis of zebrafish cranial neural crest reveals spatiotemporal regulation of lineage decisions during development.

Neural crest (NC) cells migrate throughout vertebrate embryos to give rise to a huge variety of cell types, but when and where lineages emerge and their regulation remain unclear. We have performed single-cell RNA sequencing (RNA-seq) of cranial NC cells from the first pharyngeal arch in zebrafish over several stages during migration. Computational analysis combining pseudotime and real-time data reveals that these NC cells first adopt a transitional state, becoming specified mid-migration, with the first lineage decisions being skeletal and pigment, followed by neural and glial progenitors. In addition, by computationally integrating these data with RNA-seq data from a transgenic Wnt reporter line, we identify gene cohorts with similar temporal responses to Wnts during migration and show that one, Atp6ap2, is required for melanocyte differentiation. Together, our results show that cranial NC cell lineages arise progressively and uncover a series of spatially restricted cell interactions likely to regulate such cell-fate decisions.

Drosophila melanogaster Cad99C, the orthologue of human Usher cadherin PCDH15, regulates the length of microvilli.

Actin-based protrusions can form prominent structures on the apical surface of epithelial cells, such as microvilli. Several cytoplasmic factors have been identified that control the dynamics of actin filaments in microvilli. However, it remains unclear whether the plasma membrane participates actively in microvillus formation. In this paper, we analyze the function of Drosophila melanogaster cadherin Cad99C in the microvilli of ovarian follicle cells. Cad99C contributes to eggshell formation and female fertility and is expressed in follicle cells, which produce the eggshells. Cad99C specifically localizes to apical microvilli. Loss of Cad99C function results in shortened and disorganized microvilli, whereas overexpression of Cad99C leads to a dramatic increase of microvillus length. Cad99C that lacks most of the cytoplasmic domain, including potential PDZ domain-binding sites, still promotes excessive microvillus outgrowth, suggesting that the amount of the extracellular domain determines microvillus length. This study reveals Cad99C as a critical regulator of microvillus length, the first example of a transmembrane protein that is involved in this process.

Fat and Dachsous cadherins.

Fat and Dachsous (Ds) are very large cell adhesion molecules. They bind each other and have important, highly conserved roles in planar cell polarity (PCP) and growth control. PCP is defined as the directionally coordinated development of cellular structures or behavior. Cellular and tissue growth needs to be modulated in terms of rate and final size, and the Hippo pathway regulates growth in a variety of developmental contexts. Fat and Ds are important upstream regulators of these pathways. There are two Fat proteins in Drosophila, Fat and Fat2, and four in vertebrates, Fat1-4. There is one Ds protein in Drosophila and two in vertebrates, Dachsous1-2. In this chapter, we discuss the roles of Fat and Ds family members, focusing on Drosophila and mouse development.