Clustered brachiopod Hox genes are not expressed collinearly and are associated with lophotrochozoan novelties

Abstract

Temporal collinearity is often considered the main force preserving Hox gene clusters in animal genomes. Studies that combine genomic and gene expression data are scarce, however, particularly in invertebrates like the Lophotrochozoa. As a result, the temporal collinearity hypothesis is currently built on poorly supported foundations. Here we characterize the complement, cluster, and expression of Hox genes in two brachiopod species, Terebratalia transversa and Novocrania anomala. T. transversa has a split cluster with 10 genes (lab, pb, Hox3, Dfd, Scr, Lox5, Antp, Lox4, Post2, and Post1), whereas N. anomala has 9 genes (apparently missing Post1). Our in situ hybridization, real-time quantitative PCR, and stage-specific transcriptomic analyses show that brachiopod Hox genes are neither strictly temporally nor spatially collinear; only pb (in T. transversa), Hox3 (in both brachiopods), and Dfd (in both brachiopods) show staggered mesodermal expression. Thus, our findings support the idea that temporal collinearity might contribute to keeping Hox genes clustered. Remarkably, expression of the Hox genes in both brachiopod species demonstrates cooption of Hox genes in the chaetae and shell fields, two major lophotrochozoan morphological novelties. The shared and specific expression of Hox genes, together with Arx, Zic, and Notch pathway components in chaetae and shell fields in brachiopods, mollusks, and annelids provide molecular evidence supporting the conservation of the molecular basis for these lophotrochozoan hallmarks.chaetae | shell fields | Lophotrochozoa | Wiwaxia | Hox cluster

Possible co-option ofengrailedduring brachiopod and mollusc shell development

“…Recent studies have advanced our understanding of brachiopod embryogenesis to molecular levels [17][18][19][20][21][22] by applying molecular analyses to traditional morphological and experimental embryology [1,[23][24][25]], yet the molecular basis of shell development in brachiopods remains unclear. In the lingulid Lingula anatina and the rhynchonellid Terebratalia transversa, embryonic shells (i.e.…”

A technical review and guide to RNA fluorescence in situ hybridization

“…Zebrafish (Danio rerio) embryos and the annelid Platynereis dumerilii can be suitably fixed in 4% PFA for 2 h at room temperature (Jékely & Arendt, 2007;Steinmetz et al, 2011) but can alternatively be fixed overnight at 4 C (Oxtoby & Jowett, 1993;Lauter, Söll & Hauptmann, 2011a, 2011bMarra et al, 2017). Arms of the brittle star (Amphiura filiformis) are also sufficiently fixed in 4% PFA overnight at 4 C. Embryos of the brachiopods Terebratalia transversa and Novocrania anomala should be fixed in 4% formaldehyde for 4 h (Schiemann et al, 2017;Gąsiorowski & Hejnol, 2019). The starlet sea anemone (Nematostella vectensis) and an acoelomorph worm (Convolutriloba longifissura) have been successfully fixed for ISH with 3.7% formaldehyde supplemented with 0.3% glutaraldehyde which is another strong cross-linking agent (Finnerty et al, 2003;Martindale, Pang & Finnerty, 2004;Hejnol & Martindale, 2008).…”

“…Samples are typically subjected to increasingly stringent washes in SSC buffer containing formamide and a detergent (Table S1; Jackson, Herlitze & Hohagen, 2016;Thiruketheeswaran, Kiehl & D'Haese, 2016). Increased stringency can be achieved through sequential washes with incrementally reduced salt concentrations while the wash temperature is matched to the hybridization temperature (Martindale, Pang & Finnerty, 2004;Hejnol & Martindale, 2008;Jackson, Herlitze & Hohagen, 2016;Schiemann et al, 2017;Gąsiorowski & Hejnol, 2019). At the end of washing, the goal is to allow only the specific and stable hybrids to remain.…”

“…Amplification of FISH signal was first achieved through the use of fluorochrome-labeled tyramides that would accumulate at the site of the in situ hybrid due to the use of hapten-labeled probes and anti-hapten antibodies conjugated to horseradish peroxidase (Raap et al, 1995). This method of tyramide signal amplification for FISH is still frequently used to great effect in many sample types including whole-mount invertebrate embryos (Martín-Durán et al, 2016;Schiemann et al, 2017;Gąsiorowski & Hejnol, 2019) as well as vertebrate embryos and organs (Lauter, Söll & Hauptmann, 2011a, 2011bLegendre et al, 2013;Row & Martin, 2017). A more recent development for FISH signal amplification was introduced by Choi et al (2010), expanded on by Marras, Bushkin & Tyagi (2019) and is based on the hybridization chain reaction (HCR) introduced by Dirks & Pierce (2004).…”

Unravelling spiral cleavage

“…Each defined by a relatively distinct body plan, some of these groups are among the most diversified animal clades, such as Platyhelminthes, Mollusca and Annelida. Not surprisingly, there are countless examples of morphological innovations in Spiralia, some of them among the most iconic in the animal tree of life, such as molluscan shells (Wanninger and Wollesen, 2018), and others less known but equally exciting, such as annelid and brachiopod chaetae (Schiemann et al, 2017), and molluscan and brachiopod cartilage (Tarazona et al, 2016). What distinguishes spiralians from other vastly diverse animal groups, such as arthropods and vertebrates, is that, to a large extent, this morphological diversity emerges through the same early spiral cleavage program.…”