Development of the larval anterior neurogenic domains of Terebratalia transversa (Brachiopoda) provides insights into the diversification of larval apical organs and the spiralian nervous system

Abstract

Background: Larval features such as the apical organ, apical ciliary tuft, and ciliated bands often complicate the evaluation of hypotheses regarding the origin of the adult bilaterian nervous system. Understanding how neurogenic domains form within the bilaterian head and larval apical organ requires expression data from animals that exhibit aspects of both centralized and diffuse nervous systems at different life history stages. Here, we describe the expression of eight neural-related genes during the larval development of the brachiopod, Terebratalia transversa.

Results: Radially symmetric gastrulae broadly express Tt-Six3/6 and Tt-hbn in the animal cap ectoderm. Tt-NK2.1 and Tt-otp are restricted to a central subset of these cells, and Tt-fez and Tt-FoxQ2 expression domains are already asymmetric at this stage. As gastrulation proceeds, the spatial expression of these genes is split between two anterior ectodermal domains, a more dorsal region comprised of Tt-Six3/6, Tt-fez, Tt-FoxQ2, and Tt-otp expression domains, and an anterior ventral domain demarcated by Tt-hbn and Tt-NK2.1 expression. More posteriorly, the latter domains are bordered by Tt-FoxG expression in the region of the transverse ciliated band. Tt-synaptotagmin 1 is expressed throughout the anterior neural ectoderm. All genes are expressed late into larval development. The basiepithelial larval nervous system includes three neurogenic domains comprised of the more dorsal apical organ and a ventral cell cluster in the apical lobe as well as a mid-ventral band of neurons in the mantle lobe. Tt-otp is the only gene expressed in numerous flask-shaped cells of the apical organ and in a subset of neurons in the mantle lobe.

Conclusions: Our expression data for Tt-Six3/6, Tt-FoxQ2, and Tt-otp confirm some aspects of bilaterian-wide conservation of spatial partitioning within anterior neurogenic domains and also suggest a common origin for central otp-positive cell types within the larval apical organs of spiralians. However, the field of sensory neurons within the larval apical organ of Terebratalia is broader and composed of more cells relative to those of other spiralian larvae. These cellular differences are mirrored in the broader spatial and temporal expression patterns of Tt-FoxQ2 and Tt-otp. Corresponding differences in the expression of Tt-hbn, Tt-NK2.1, and Tt-FoxG are also observed relative to their respective domains within the cerebral ganglia of spiralians. Based on these data we argue that the anterior region of the bilaterian stem species included Six3/6, NK2.1, otp, hbn, fez, and FoxQ2 expression domains that were subsequently modified within larval and adult neural tissues of protostome and deuterostome animals.

Figure 1

General aspects of gastrulation and larval development of Terebratalia transversa. Each panel consists of a blastoporal (left) and a corresponding lateral (right) view of a particular developmental stage. (A) Radial gastrula stage, the animal pole (AP) is at the top. (B) Asymmetric gastrula stage showing the shift of the animal pole toward the presumptive anterior of the embryo (ANT). (C) Bilateral gastrula stage, when the blastopore (BP) is a narrow slit. (D) Early trilobed larval stage that begins to demarcate the apical, mantle, and pedicle lobes (AL, ML, and PD, respectively) of the larva. The gut (G) is a blind-ended sac. (E) The late trilobed larval stage has a larger ventral mantle lobe relative to the dorsal side (D) that also bears chaetae (CH).

Figure 2

Cytological and anatomical aspects of the late gastrula and trilobed larva of Terebratalia transversa. (A) Light micrograph of a late trilobed larva with ocelli (OC) on the dorsal side of the apical lobe (AL). Vesicular bodies (VB) and other epidermal cells line the border between the apical and mantle lobe (ML). The mantle lobe has four chaetal sacs with long chaetae (CH). The posterior pedicle lobe (PD) will attach the larva to the substrate at metamorphosis. (B) Ventral view of a bilateral late gastrula stage labeled for acetylated α-tubulin with a long ciliary tuft (AT) produced by specialized cells with recessed ciliary rootlets (ATC). (C, D) Partial frontal z-projections depicting aspects of late larval anatomy such as the cilia of the anterior transverse ciliated band (CB) and sensory neurons (SN1 and SN2) within the apical organ (AO) that send axonal fibers into the central anterior neuropil (NP). The larva is nonfeeding, but does develop a blind-ended gut (G). (E, F) Complete z-projections of the histaminergic nervous system of the late trilobed larva. Cell borders and some larval muscles are stained with phalloidin. There are at least 70 histaminergic cells in the apical organ (AO), approximately 60 histaminergic cells in the broad ventral region of the apical lobe (AVC), and also approximately 30 histaminergic cells (MVC) in a mid-ventral region (V) in the mantle lobe. All scale bars = 25 μm.

Figure 3

Expression patterns of Tt-hbn, Tt-NK2.1, Tt-Six3/6, and Tt-fez in the embryos and larvae of Terebratalia transversa. Abbreviations: ACE, animal cap ectoderm; AL, apical lobe; AT, apical tuft; BP, blastopore; DE, anterior dorsal ectoderm; DN, anterior dorsal ring of ectoderm; ML, mantle lobe; PD, pedicle lobe; VE, anterior ventral ectoderm; VLE, anterior ventrolateral ectoderm. All scale bars = 25 μm.

Figure 4

Expression patterns of Tt-FoxQ2, Tt-otp, Tt-FoxG, and Tt-synaptotagmin 1 in the embryos and larvae of Terebratalia transversa. Abbreviations: AC, spots of animal cap ectoderm; AE, anterior ectoderm; AL, apical lobe; AT, apical tuft; BP, blastopore; CA, anterior zone within the transverse ciliated band; CB transverse ciliated band region; CD, central anterior ectoderm; CP, posterior zone within the transverse ciliated band; DE, anterior dorsal ectoderm; DS, anterior dorsal spots of ectoderm; EG, ectodermal cells around the anterior tip of the foregut; FC, flask cells; LC, ventrolateral expression spots; MC, expression spots on the mid-ventral region of the mantle lobe; ML, mantle lobe; PD, pedicle lobe; VP, ventral posterior ectoderm in the apical lobe; VU, anterior ventral U-shaped region; VS, anterior ventral expression spots. All scale bars = 25 μm.

Figure 5

Diagrams of ectodermal and endodermal (labeled with an asterisk) gene expression domains for Terebratalia transversa at the early trilobed larval stage. All expression domains are based upon single probe in situ hybridizations with NBT/BCIP staining. The extent of domains and regions of overlap in expression among genes were inferred from the position of staining relative to morphological landmarks. The anterior ventral expression domains of Tt-FoxG are not depicted. Abbreviations: AL, apical lobe; AT, apical tuft; CB, region of the developing ciliated band; M, remaining portion of the blastopore; ML, mantle lobe; and PD, pedicle lobe.

Figure 6

Composite expression domains for orthologous genes involved in the patterning of neural and other ectodermal tissues from developmental stages, larval types, or adult forms of representative invertebrate animals. Figure panels based on [5,14-16,41-45,55,56,60-63,66,68,70,71,85-87], see text for details. Abbreviations: AB, aboral tuft of cilia; AL, apical lobe; AT, apical tuft; BP, blastopore; CB, ciliated band; CG, cerebral ganglia; E, adult eyespot; ES, larval eyespot; M, mouth; MB, mushroom body; ML, mantle lobe; MS, mesosome; MT, metatroch; MTS, metasome; NT, neotroch; P, prototroch; PL, pedicle lobe; S, statocyst; T, telotroch; TB, tailbud; VN, ventral nerve cord.