Unexpected novel relational links uncovered by extensive developmental profiling of nuclear receptor expression

Abstract

Nuclear receptors (NRs) are transcription factors that are implicated in several biological processes such as embryonic development, homeostasis, and metabolic diseases. To study the role of NRs in development, it is critically important to know when and where individual genes are expressed. Although systematic expression studies using reverse transcriptase PCR and/or DNA microarrays have been performed in classical model systems such as Drosophila and mouse, no systematic atlas describing NR involvement during embryonic development on a global scale has been assembled. Adopting a systems biology approach, we conducted a systematic analysis of the dynamic spatiotemporal expression of all NR genes as well as their main transcriptional coregulators during zebrafish development (101 genes) using whole-mount in situ hybridization. This extensive dataset establishes overlapping expression patterns among NRs and coregulators, indicating hierarchical transcriptional networks. This complete developmental profiling provides an unprecedented examination of expression of NRs during embryogenesis, uncovering their potential function during central nervous system and retina formation. Moreover, our study reveals that tissue specificity of hormone action is conferred more by the receptors than by their coregulators. Finally, further evolutionary analyses of this global resource led us to propose that neofunctionalization of duplicated genes occurs at the levels of both protein sequence and RNA expression patterns. Altogether, this expression database of NRs provides novel routes for leading investigation into the biological function of each individual NR as well as for the study of their combinatorial regulatory circuitry within the superfamily.

Figure 1. NR Complement in Human, Mouse, Zebrafish, Pufferfish, and the Inferred Complement in the Common Ancestor of Actinopterygian Fish and Mammals (Indicated by “F/M Ancestor”)

Each color corresponds to a specific NR subfamily: light blue, purple, yellow, orange, dark blue, and white for subfamilies 1, 2, 3, 4, 5, 6, and 0, respectively.

Figure 2. Statistical Analysis of NR Expression Patterns in Zebrafish

(A) Proportion (in percent) of NR genes with ubiquitous, restricted, or not-detected expression for each of the studied stages. The proportion of genes with ubiquitous expression during embryonic development is almost constant (approximately 20%), whereas the proportion of genes with a restricted expression pattern increases (from 10% up to 60%). (B) Proportion (in percent) of NR genes with a restricted expression pattern that are expressed in nervous system (brain, spinal cord, and retina) from mid-late somitogenesis (MS) to 48 hpf. At 36 hpf, almost 80% of NR genes with restricted expression patterns are expressed in brain and more than half of them are expressed in the retina at 48hpf. (C) Comparison of the proportion (in percent) of genes expressed in brain and retina from 24 hpf to 48 hpf between NR genes and 1,900 genes, whose expression is described in the ZFIN database. NR genes with a restricted expression pattern show a higher tendency to be expressed in brain and retina.

Figure 3. Expression of NR genes in the CNS

(A–D) Expression in retina, optic tectum, and hindbrain of RARα-A, Reverbα, Reverbβ, and Reverbγ-B, respectively. RORα-B is expressed in one nucleus in ventral diencephalon and in hindbrain rhombomeres (E), RORα-A in retina, optic tectum, epiphysis, and hypophysis (F), RORβ-A in retina, ventral-posterior part of the optic tectum, and in some neuromasts of the posterior lateral line (G), PXR in small diencephalic and telencephalic nuclei as well as in adenohypophysis (H), PNR in epiphysis, ventral part of retina, and some neurons of posterior diencephalon (I), TLL in diencephalon and mesencephalon with more labeling in ventral diencephalon, anterior tegmentum, and optic tectum (J). COUPTFα-A is expressed in the ventral part of the diencephalon, in forebrain ventricular zone, in tegmentum, and hindbrain (K), COUPTFβ displays a similar expression with additional expression in the dorsal half of the diencephalon (L). EAR2-B expression is restricted to dorsoventral stripes in tegmentum, in hindbrain of rhombencephalon, and in spinal cord (M), COUPTFα-B displays an expression in ventral diencephalon, telencephalon ventricular zone, anterior tegmentum, pretectum, and hindbrain (N). ERβ-A is expressed in a small nucleus in the anterior ventral part of the diencephalon (O), while ERRα is expressed in all brain subdivisions except for the forebrain ventricular zone, tegmentum, and dorsal rhombencephalon (P). ERRβ displays a complex expression with a nucleus in ventral telencephalon, nuclei in diencephalon and tegmentum, and an expression in hindbrain (Q), ERRγ has a very similar expression except for the ventral telencephalic nucleus (R). NURR1 is expressed in part of the telencephalon, in a nucleus in anterior diencephalon, in posterior diencephalon and anterior tegmentum, and in the ventral anterior part of rhombencephalon (S). NOR1 is expressed weakly in ventral telencephalon, tegmentum, and hindbrain and strongly in the habenula (T), SF1-A, LRH1, and SF1-B are expressed strongly in the ventral diencephalon (U–W). RXRα-B is expressed at a low basal level in all brain territories with a much stronger intensity in the ventral part of the optic tectum (X). Embryos are in lateral view, anterior to the left, and are 36 hpf except for (A–D, O, W, and X), where they are at 48 hpf. More extensive anatomical descriptions of these expression patterns are presented in Figure S3 and anatomical details are available at ZFIN (http://zfin.org).

Figure 4. Expression of NR Genes in Retina at 72 hpf

(A) Schematic of a zebrafish eye at 72 hpf showing the characteristic multilayered structure. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; ON, optic nerve; PZ, proliferative zone. (B–F; left panels) Schematic showing in blue the various types of expression patterns found for NR genes in retinas at 72 hpf after whole-mount in situ hybridization and section. (B) Genes expressed in the ONL. (C) Genes expressed in the INL. (D) Genes expressed in the dorsal part of the retina. (E) Genes showing expression in the ventral part of the retina. (F) Genes showing expression in the proliferative zone of the retina.

Figure 5. Overlapping Domains of Expression between PGC1 and ERRs

(A–O) Expression of PGC1 in slow muscle fibers, posterior pronephric ducts, mucous cells, epiphysis, and part of the telencephalon and diencephalon (A–C) overlaps extensively with the expression of ERRs. ERRα is coexpressed with PGC1 in slow muscle fibers, posterior pronephric ducts, telencephalon, and mucous cells (D–F). ERRβ is coexpressed with PGC1 in epiphysis and posterior pronephric ducts (G–I), ERRγ in epiphysis, part of the tegmentum, and posterior pronephric ducts and ERRδ in mucous cells. Embryos are at 24 hpf in lateral view anterior on the left except for (C, F, I, L, and O), which are shown at the 14-somite stage. Posterior part of the embryo is presented in dorsal view, anterior to the left. More extensive anatomical descriptions of these expression patterns are presented in Figure S3 and anatomical details are available at ZFIN (http://zfin.org).

Figure 6. Clustering of NR and Coregulator Expression Patterns during Zebrafish Development

A hierarchical clustering procedure was performed to compare the expression profiles of regionally expressed NR genes (Table S2) and the patterns of anatomical structures. The correspondence between the resulting classifications reveals the existence of clusters of genes with hierarchically discriminated expression in time and space during development: early versus late (II/I-III), nervous versus non-nervous (I/III or IIa/IIb), and optical versus spinal versus brain (Ia/Ib/Ic). Abbreviations used for anatomical structures are defined in Table S5.

Figure 7. Relative Rates of Protein Evolution, Coding Sequence Divergence, and Expression Pattern Divergence of the Fish-Specific NR Duplicates

(A) Phylogenetic view of the relative evolutionary rates of the zebrafish duplicates. On the left side, in blue, NR pairs with similar evolution rates (ratio not significantly different from 1). On the right side, in red, NR pairs where one of the duplicates evolved significantly faster than the other, which suggests neofunctionalization. (B) Relation between expression pattern divergence (calculated as explained in the Materials and Methods section) and coding sequence divergence (Ka/Ks ratio) for the pairs with similar evolution rates (blue circles, ns, the same as in (A) except for RORγ, for which Ks is too saturated to be calculated) and for the pairs with an acceleration of the evolutionary rate of one duplicate (red squares, s, the same as in (A) except for PPARα, a nonexpressed pair, and SHP, for which Ks is too saturated to be calculated). s, significant protein sequence acceleration; ns, nonsignificant difference in protein evolution rates (calculated as explained in Materials and Methods). Regression lines are plotted and Pearson correlation coefficients and p-values are indicated. A positive significant correlation is observed for the pairs with a putative “neofunctionalized” duplicate. No significant correlation is detected for the pairs with similar evolution rates of the duplicates.