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Specification
These sequences allow the genome to direct the specification program during development by determining the specific set of genes expressed in cells of different spatial domains in the developing embryo. Although the instructions that control the developmental specification program are encoded in genomic DNA, how these instructions are executed is poorly understood and remains the most intriguing question in development.
Developmental Gene Regulatory Networks and their Functional Regulatory States
A regulatory state is defined by the total pool of transcription factors (TFs) expressed in a cell's nucleus. Thus, the combinatorial nature of TF genes within regulatory states represents a fundamental feature in their functional control of gene expression and in the common developmental tasks required for regulatory state specification.
Sea Urchin as a Model for Developmental Gene Regulatory Networks
The Unknown Developmental Regulatory States of the Sea Urchin Larva
Studies looking at spatial gene expression are complementary to transcriptomics, which provide constructed maps of embryonic gene expression during development. While each of these studies has its merits in understanding development through gene expression, the specific expression of cell fate genes is a consequence not only of a single regulatory gene or of all expressed genes, but of the combination of transcription factors, regulatory state, expressed in given cell fate.
Summary of Thesis
There, we investigated the regulatory states governed by the underlying GRNs responsible for generating the morphological complexity of the sea urchin larva. We show that the regulatory states established in these areas during development predict the appearance of future morphological structures.
Direct and indirect control of oral ectoderm regulatory gene expression by nodal signaling in the sea urchin embryo. A disruption model of the gene regulatory network for oral and aboral ectoderm specification in the sea urchin embryo.
Abstract
Results
Bilateral clusters of cells expressing opsin3.2 are on the oral side of the apical neurogenic organ. As expected, co-immunostaining of Opsin3.2 and serotonin shows expression in separate cells, indicating that the photoreceptor neurons are not serotonergic (Figure S2.3A).
Figures
Additional regulatory genes expressed in the PRC are shown in Fig. B) WMISH for pax6, six1/2, dach and eya showing expression in the hydropore channel (HC). Summary diagram of a sea urchin larva showing expression of regulatory genes in the PRC and expression of the indicated RDN genes in the hydropore canal.
Discussion
Interestingly, a set of transcription factors involved in eye development in mice and flies are not expressed in larval sea urchin photoreceptors. Although PSED factors are expressed in retinal progenitor cells and are required for vertebrate eye development, there is no evidence that these factors function in the differentiation of ciliary photoreceptor cell types in the vertebrate retina.
The deployment of a retinal determination gene network stimulates directed cell migration in the sea urchin embryo. The ascidian homolog of the vertebrate homeobox gene Rx is essential for ocellus development and function.
Materials and Methods
The cloned genes were amplified by PCR using a primer flanking the inserted region, and the PCR products were used to synthesize RNA probes for WMISH. The whole-mount in situ hybridization (WMISH) protocol for detecting spatial gene expression has been described previously [ 52 ]. Antibody specificity was determined by preabsorbing the immune serum with an approximately equimolar preparation of the protein used to immunize the rats.
Paraffin oil was applied to the open edges of the coverslip to reduce evaporation and the room temperature was maintained at 16°C. Photoreceptor cells were identified based on location and the presence of a short non-motile cilium, and recorded stacks (Stackreg) were outlined with an elliptical selection tool and Z-axis profiles were plotted.
Supplementary Information
Supplementary Figures and Tables
Pigment cell-specific co-immunostaining of Opsin3.2 and SP1 indicating that PRCs are associated with shading pigments. In this orientation, Opsin3.2-expressing cells are on the ventral surface and clusters of pigment cells (Sp1) in the adjacent dorsal ectoderm. Although the pigment cells are not in direct contact with the Opsin3.2-expressing cells, they are positioned to cover the Opsin3.2 cells from light coming from the dorsal surface.
Phylogenetic tree describing the expression of the retinal determination genes in ciliary PRCs, rhabdomeric PRCs, and other cell types. Eya WHL22.168736 GTATTGGAAGAGGGCGTCAA TAATACGACTCACTATAGGGAGAATGACTTGTTACCCGCCAG Dac WHL22.169355 GATGCGAACCTGTTCTACG TAATACGACTCACTATAGGGAGACAATTCAAAAGCTTGTGGCA opsin3.2 WHL22.338995 CGGTAACATCACCGTCCTTT TAATACGACTCACTATAGGGA GACG GAATTTGGAGCTTGATGT opsin2 WHL22.272775 CGTTAATGTCCCATGCTGTG TAATACGACTCACTATAGGGAGACTTTGGGCAAGACAGCAGAT.
Abstract
Introduction
The qualitative nature of regulatory states represents an underlying feature of their function as entities in the control of gene expression (Peter, 2017). Cis-regulatory modules (CRMs) that control developmentally regulated genes require the combinatorial function of multiple, distinct transcription factors (Xu et al., 2014; Yuh et al., 1998). Several studies have resolved this dilemma by combining massive single-cell RNA-seq with reference gene expression patterns based on in situ hybridization data to map sequenced cells back to their embryonic origin (Karaiskos et al., 2017; Satija et al., 2015).
Thus, other approaches involving the use of gene expression analysis by in situ hybridization have been used to annotate regulatory gene expression patterns across different points in developmental time to create a comprehensive atlas of developmental genes and/or TF expression patterns ( Bell et al., 2004 ; Visel, 2004 ; Pollet et al., 2005 ; Tomancak et al., 2007 ; Tassy et al., 2010 ; Diez-Roux et al., 2011 ; Spencer et al., 2011 ; Hammonds et al., 2013 ; Hu et al., 2017). We used Whole Mount in situ hybridization (WMISH) to detect the expression of 260 regulatory genes at five developmental time points and used these data to determine the regulatory states expressed.
Results
We applied this approach to all territories in the larva to determine the expressed regulatory states. Comparison of the regulatory states between the domains of a given territory showed that there are territory-specific combinations of expressed regulatory genes. However, the complexity of the regulatory states in terms of the number of transcription factors expressed differs significantly between the different domains at 72h (Figure 3.6C).
For example, the regulatory states of the apical ectoderm range from 11 (APE 9, distal aboral region of the apical organ) to 54 (APE 4, central medial region of the apical plate) expressed regulatory genes. To address the temporal change of regulatory states throughout development, we assessed the total number of regulatory genes expressed in each territory from 24 h to 72 h.
Figures
State regulatory domains were identified molecularly by manual comparisons of the spatial expression of regulatory genes at 72 h. -D) Schematic representations of the larva are organized by regulatory state domains determined by combinatorial analysis of regulatory gene expression patterns. The seven regulatory state domains within the SKM are organized in individual cells that contribute to different rods of the larval skeleton.
The ciliary band that separates the oral from the aboral ectoderm is organized by several state regulatory domains in the apical parts of the band (CBE1-3) that overlap with the apical plate domains. Within the band, many regulatory state domains are associated in and around the mouth (OE5-9), a single one behind the mouth (OE10), and in single cells behind the mouth (OE14).
Discussion
The identification and molecular characterization of 74 regulatory state domains shows that sea urchin larval morphology and function are associated with expression of distinct regulatory states. Our analysis of regulatory states revealed very few genes with ubiquitous expression among developmentally expressed regulatory genes, while most display specific spatial information. Although the size of regulatory states varies in all domains, they average 27 regulatory genes in larval states.
In principle, the differences between regulatory states expressed in different spatial domains of the sea urchin larva could be reflected by differences in the composition of transcription factor families represented in each regulatory state. The identification of different sets of regulatory genes within regulatory states highlights the importance of combinatorial expression of multiple regulatory genes.
A gene regulatory network controls the binary fate decision of rods and bipolar cells in the vertebrate retina. Effects of the ubiquitous Zelda factor on Bicoid-dependent DNA binding and transcription in Drosophila.
Material and Methods
For each regulatory gene in the dataset, we imaged at least 3 embryos per developmental time point (24h, 36h, 48h, 60h, 72h) and captured a standard set of images from a variety of focal depths taken in a lateral view, with additional images obtained from oral, aboral, apical and anal views depending on the complexity of the expression patterns. Sea Urchin Expression Database (http://mandolin.caltech.edu/ExpressionData/index.php) and Raw Image Archive (http://mandolin.caltech.edu/JonathanImages/index.php) were created to be used as an image repository and as an analytical tool to help identify state regulatory domains and annotate regulatory patterns of gene expression. To calculate pairwise comparisons between two variables, we used the Pearson correlation method using the cor() function in the R “stats” package to generate a matrix of correlation coefficients (r).
For all distance comparisons between regulatory states, we performed hierarchical clustering using the base functions of dist() and hclust() in the standard R library ("stats" package). A distance matrix was calculated from expression values of Regulatory genes in regulatory states using the absolute distance (method = "manhattan") between any two regulatory states.
Supplementary Information .1. Supplementary Figures
Supplementary Tables
Ap4, Tcfap4L WHL22.28828.0 ATCAATGCTGGCTTCCAGTC CTACCGGGCTTCACTCTCTCAG Apa, Hypp_2213 WHL CTACGCAGGGGAAACCTCAAG TGACATGGGCTGTCTGAAACC Arnt WHLGAGACCAGTTAGTCTACTGT xL WHL GCTATCTTCATTCATTCGTCGTC GGTCGAGACACAAGTTTTTCGTAG Ash1 WHL GTTAGGGAAGGGAAAGAAAGTGA CTGTATTTGTGCTGGTACATCCA Ash2 WHL22.96450.4 CTGAACCTGCACAATAGGACCAATAACG. Cebpa WHL CACAAACTGCATATTGTCAGTAG CTAAGCCCTCGACACGTTTCTT Cebpg WHL CCTGTTCCTGACTCATGCTAATG GCTCAAGGATGTTCACAAGTCAC Cic WHL ACTGGTCCACCAGAGAGACACC AGGGGGTGGACTAGAGTAGAT TGGCAACACTTGGTGA Creb WHL GTCCATTCCCATCGCTTCTA GCCAGTGATTTTCCTTTCCA Creb3l1 WHL22.19719.0 AAGCTCTTCTCATCCTCCTCATC AGTGGGTCCCAAGTACACACAAGAGAG Creb3l3 WHL GGACCGTTGTAGTAGT Gabp WHL AACACTCGATTCCTGTTTATTTCC TAATACGACTCACTATAGGGAGAAGCTTCTATAGTTCATGGATGG GataC WHL CCAACAAGTTCCTACACGTTACC TAATACGACTCACTATAGGGAGAGAGTGTGATGATGAGGATAGGATGATGATGGAT CGTGAGG TAATACGACTCACTATAGGGAGATGTTGTATCCATTCATCTTGTGTGG Gbx WHL ACAGATAAGAGTCCCAGTGATCG TAATACGACTCACTATAGGGAGAAGGGGAGACTGTAGATTGAAACG Gcm WHL22.543333.0443333.043333. ATGTCCACTATGTCCTG Gcnf1 WHL TGCCACATACACCCCTACAAG TAATACGACTCACTATAGGGAGATAGCCACATCAATAATCTCCAGTC Xham2 WHL ATCATCATCACACCTGGACTCTC CTACAAGACAGTAGCGGACGAAC.
GlisC WHL22.66475.1 TTCTATTGACTCTCCCCTTTTGG TAATACGACTCACTATAGGGAGACTGTATGGCTTTTCCTCTAAGTGC Grf WHL TTGATGACTTCCTCTCTCAGCTC TAATACGACTCACTATAGGGAGACTGCATTTTCTCCATTTTCACTTTC GscTAGTCGTCGTAGGTCATTAGTCGTCGGT GTGTTGAAAGTGC Hairy2/4 WHL CAAAATGCCTGTGGATACTAAACC TAATACGACTCACTATAGGGAGTAGACTGGAATGGAATGACTTGG Hb 9 WHL TCCGGGTATATGTGTGTCTCGAT GAGACAGACAGACAGAAATGGACA. Hnf1aL WHL TGATGAAAACCCACCGAAGAGACAGC TCATCCACAATGCAAGCTCTTCAGC Hnf4 WHL22.35553.1 TAGCAGCATGCATGAGATGACC GGGCTGTCCATTGAGGTCAGGT Hnf6 WHL CGCTAGAGAGAAGGCCATGAG1CACCCTCCATTA GCCCTCCATTA C TGACCAACTGAGGGATGTGA Hox7 WHL TCGGGGCTGTTCAGGAGG TGAAGGAGACCAGCGAATATAGAG. Pitx2 WHL22.11036.0 TTTGTGTAGCTTTCTCCCCTCTA GAACGGAGATCAAGTGAAGAAGA Pknox WHL TTGATGCCTGACTGACACATAGT TTGCATGTCTTTTCATGTCTCTG Pou4f2 WHL GACATGACTGAACGTCATCAAA CATGTAAGGCAAAGTGA CTGTAAGGCAAAGTGGA2200000000000000000. GAGAGGAG CTTAATGAGTGGCGGAGGAC.
Rhox3 WHL AAGGAACAGACGACTTCGGTACT AAGTGCATTTCCATTAGCGTTCT Riz WHL22.63053.0 TGGAGTTCAGACAGTCACAGATG GATTCACTTTGGGCATTTAAC Rora WHL
