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Expression of NK cluster genes in the onychophoran Euperipatoides rowelli: implications for the evolution of NK family genes in nephrozoans - PubMed

  • ️Mon Jan 01 2018

Expression of NK cluster genes in the onychophoran Euperipatoides rowelli: implications for the evolution of NK family genes in nephrozoans

Sandra Treffkorn et al. Evodevo. 2018.

Abstract

Background: Understanding the evolution and development of morphological traits of the last common bilaterian ancestor is a major goal of the evo-devo discipline. The reconstruction of this "urbilaterian" is mainly based on comparative studies of common molecular patterning mechanisms in recent model organisms. The NK homeobox genes are key players in many of these molecular pathways, including processes regulating mesoderm, heart and neural development. Shared features seen in the expression patterns of NK genes have been used to determine the ancestral bilaterian characters. However, the commonly used model organisms provide only a limited view on the evolution of these molecular pathways. To further investigate the ancestral roles of NK cluster genes, we analyzed their expression patterns in the onychophoran Euperipatoides rowelli.

Results: We identified nine transcripts of NK cluster genes in E. rowelli, including single copies of NK1, NK3, NK4, NK5, Msx, Lbx and Tlx, and two copies of NK6. All of these genes except for NK6.1 and NK6.2 are expressed in different mesodermal organs and tissues in embryos of E. rowelli, including the anlagen of somatic musculature and the heart. Furthermore, we found distinct expression patterns of NK3, NK5, NK6, Lbx and Msx in the developing nervous system. The same holds true for the NKL gene NK2.2, which does not belong to the NK cluster but is a related gene playing a role in neural patterning. Surprisingly, NK1, Msx and Lbx are additionally expressed in a segment polarity-like pattern early in development-a feature that has been otherwise reported only from annelids.

Conclusion: Our results indicate that the NK cluster genes were involved in mesoderm and neural development in the last common ancestor of bilaterians or at least nephrozoans (i.e., bilaterians to the exclusion of xenacoelomorphs). By comparing our data from an onychophoran to those from other bilaterians, we critically review the hypothesis of a complex "urbilaterian" with a segmented body, a pulsatile organ or heart, and a condensed mediolaterally patterned nerve cord.

Keywords: Gene expression; Homeobox genes; Mesoderm development; NK genes; NK-linked genes; Nephrozoa; Onychophora; Urbilaterian; Velvet worms.

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Figures

Fig. 1
Fig. 1

Maximum likelihood analysis of the NK cluster and NKL genes among metazoans, and summary of the NK gene repertoire in the onychophoran E. rowelli, and the tardigrades H. exemplaris and R. varieornatus. A Maximum likelihood tree of NK cluster and NKL genes to clarify the orthology of E. rowelli, H. exemplaris and R. varieornatus sequences (marked with red, blue and purple arrows, respectively). Bootstrap values > 50 are given at the nodes. Tree scale indicates the number of substitutions per site. B Summary diagram showing the NK gene complements in E. rowelli, H. exemplaris, R. varieornatus, D. melanogaster, S. maritima and P. tepidariorum. Numbers indicate the number of identified transcripts in each species; dashes indicate the absence thereof. 1From Ref. [131]; 2From Ref. [58]; 3From Ref. [132]. See also Additional file 1 for further information on NK gene complements in different bilaterian taxa

Fig. 2
Fig. 2

Genomic scaffolds of the tardigrades H. exemplaris and R. varieornatus showing the location and orientation of NK cluster genes. The NK genes of H. exemplaris are distributed on six, those of R. varieornatus on three different scaffolds. Numbers below the scaffold names indicate the total length of each scaffold. Numbers below or above the brackets show the distances between individual NK genes

Fig. 3
Fig. 3

Comparative transcriptomic analysis of expression of NK cluster genes and the NKL gene NK2.2 in the onychophoran E. rowelli. The diagram illustrates the relative expression levels of all identified NK cluster genes from different transcriptome libraries of partially pooled embryonic stages. The number of transcripts of RPL31 (gene encoding a ribosomal protein) was used as a reference

Fig. 4
Fig. 4

Expression of NK1 at consecutive developmental stages in embryos of the onychophoran E. rowelli. Anterior is up in A, A′, C, D, and left in B, E–J; developing limbs are numbered. Insets in A′ and B show the respective embryos stained with DAPI. A Stage I embryo in lateral view. Note the weak signal in the anterior segments (arrowheads). Inset shows the slime papilla segment and first leg-bearing segment in ventrolateral view. Filled arrowhead points to the transverse segmental furrow. A′ Same embryo as in A, superimposed light micrograph and DAPI image. Dashed lines demarcate transverse furrows. B Stage II embryo in lateral view. C Detail of the same embryo as in B in lateral view. Superimposed light micrograph and DAPI image. Arrowheads indicate the position of transverse furrows. D Detail of stage II embryo in dorsal view. Arrowheads indicate the position of transverse furrows. E Late stage III embryo in lateral view. F Third to fifth developing legs of the same embryo as in E in lateral view. Note the U-shaped domain (asterisks) in all and an additional dorsal domain in the fourth and fifth legs (arrowheads). G Same embryo as in F in ventral view. H Stage IV embryo in lateral view. I Fifth to seventh developing legs of the same embryo as in H in lateral view. Note the U-shaped (asterisks) and an additional dorsal domain (arrowheads). J Same embryo as in H in ventral view. Abbreviations: at, developing antenna; cl, cephalic lobe; de, dorsal extra-embryonic tissue; js, jaw segment; jw, developing jaw; po, proctodeum; sp, developing slime papilla; ss, slime papilla segment; ve, ventral extra-embryonic tissue. Scale bars: A–C, J: 200 µm; E, H: 500 µm; D, F, G, I: 100 µm

Fig. 5
Fig. 5

Expression of NK3 and NK4 at late developmental stages in embryos of the onychophoran E. rowelli. Anterior is left in A, D, D′, E, E′ and up in B, C, F, F′, G; developing legs are numbered. A Stage IV embryo in lateral view. B Head of stage VI embryo in dorsal view. C Anterior region of stage VI embryo in ventral view. Arrowheads point to the expression in the anlagen of ventral nerve cords. D Developing slime papilla and first two legs of a stage IV embryo in lateral view. Anterior and posterior NK3 domains are marked with empty and filled arrowheads, respectively. D′ Third to fifth developing legs of a stage IV embryo in lateral view. Anterior and posterior NK3 domains are marked with empty and filled arrowheads, respectively. E Stage IV embryo in lateral view. Empty arrowheads indicate the expression along the dorsal rims of the lateral germ bands. E′ Superimposed light micrograph and DAPI image of the same embryo as in E. F Posterior end of a stage IV embryo in dorsal view. Arrowheads point to the expression along the dorsal rims of the lateral germ bands. F′ Superimposed light micrograph and DAPI image of the same embryo as in F. G Detail of the 12th and 13th legs in dorsal view. Asterisks indicate the mesodermal NK4 domains. Abbreviations: at, developing antenna; cl, cephalic lobe; cn, central brain neuropil; de, dorsal extra-embryonic tissue; jw, developing jaw; po, proctodeum; sp, developing slime papilla; ve, ventral extra-embryonic tissue. Scale bars: A, E, E′: 500 µm; D, D′, G: 100 µm; B, C, F, F′: 200 µm

Fig. 6
Fig. 6

Expression of NK5 at consecutive developmental stages in embryos of the onychophoran E. rowelli. Anterior is up in A–E, I–K, and left in F–H. A, B–E Expression in the head of subsequent developmental stages in ventral view. Empty and filled arrowheads in A and B point to two separate domains in each cephalic lobe. A′ Same Embryo as in A stained with DAPI. F–H Second to seventh developing legs of a stage VI embryo in dorsolateral view. Arrowheads point to the mesodermal expression in the fourth to seventh legs. I Developing antenna of a stage VI embryo in ventral view. Note the expression in the distal mesoderm (arrowhead). J, K Anterior regions of stage V and VI embryos in ventral view. Note the expression in the ventral nerve cords (asterisks). Abbreviations: at, developing antenna; cl, cephalic lobe; cx, brain cortex; jw, developing jaw; mo, developing mouth; st, stomodeum; sp, developing slime papilla; ve, ventral extra-embryonic tissue. Scale bars: 200 µm

Fig. 7
Fig. 7

Expression of NK6.1 at consecutive developmental stages in embryos of the onychophoran E. rowelli. Anterior is left in A–D, G, H; developing legs are numbered. Insets in A–D show the corresponding embryos stained with DAPI. A Stage I embryo in ventral view. Note the expression around the proctodeum (empty arrowheads) and the closing blastoporal slit (filled arrowheads). Arrows with dashed lines indicate decreasing signal toward the posterior. B Detail of proctodeum (empty arrowheads) and blastoporal slit (filled arrowheads) in the same embryo as in A. C Stage II embryo in lateral view. D Same embryo as in C in ventral view. Note the expression around the proctodeum (arrowheads). E Cross section of a stage II embryo, dorsal is up. Arrowhead point to the NK6.1 expression in the ventrolateral ectoderm. Dotted line demarcates the developing nerve cord. F Same cross section as in E stained with DAPI. G Stage III embryo in lateral view. Note the decreasing signal from anterior to posterior (arrow with dashed line). H Same embryo as in E in ventral view. Small anterior and large posterior domains in the ventral ectodermal thickenings are indicated by arrowheads and asterisks, respectively. Abbreviations: as, antennal segment; at, developing antenna; cl, cephalic lobe; de, dorsal extra-embryonic tissue; js, jaw segment; jw, developing jaw; po, proctodeum; sp, developing slime papilla; ss, slime papilla segment; ve, ventral extra-embryonic tissue. Scale bars: A–D, G, H: 200 µm; E, F: 50 µm

Fig. 8
Fig. 8

Expression of NK6.1 at late developmental stages in embryos of the onychophoran E. rowelli. Anterior is up in A and B; developing legs are numbered. Developing nerve cord is indicated with dashed lines and the neuropil with asterisks in C–D′. A Head of stage IV embryo in ventral view. Signals in the ventral ectodermal thickenings and ventral nerve cords are indicated with asterisks and arrowheads, respectively. B Head of stage V embryo in ventrolateral view. Note the expression in the developing antennae (empty arrowheads) and the ventral nerve cords (filled arrowheads). C Cross section of a stage IV embryo showing the segmental expression in the ventral ectodermal thickenings. Arrowheads point to the expression in the ventral ectoderm and developing nerve cords. D Cross section of the interpedal region of a stage IV embryo. Arrowhead points to the expression in the developing nerve cords. C′ and D′ show DAPI staining of the same cross section as in C and D, respectively. Abbreviations: at, developing antenna; cl, cephalic lobe; sp, developing slime papilla. Scale bars: A, B: 200 µm; C–D′: 50 µm

Fig. 9
Fig. 9

Expression of Msx at consecutive developmental stages in embryos of the onychophoran E. rowelli. Anterior is left in A–C, E–K; developing legs are numbered. Insets in B and F show the respective embryos stained with DAPI. A Stage I embryo in lateral view. B Same embryo as in A in ventrolateral view, superimposed light micrograph and DAPI image. Arrowheads point to the transverse furrows. C Detail of a stage I embryo in ventral view. Arrowheads point to transverse furrows. D Cross section of the first leg-bearing segment, dorsal is left. Arrowhead points to the expression in the mesoderm. E Detail of a stage I embryo in lateral view. Superimposed light micrograph and DAPI image. Transverse furrows are indicated by dashed lines. F Stage III embryo in lateral view. G Detail of the same embryo as in F in ventrolateral view. Anterior and posterior domains in the ventral ectodermal thickenings are marked by empty and filled arrowheads, respectively. H Posterior end of stage III embryo in lateral view. I Stage IV embryo in lateral view. J Detail of the same embryo as in I in ventral view. Arrowheads point to mesodermal domains in the limb buds. K Stage IV embryo in ventrolateral view. Anterior and posterior domains in the ventral ectodermal thickenings are marked with empty and filled arrowheads, respectively. Abbreviations: at, developing antenna; cl, cephalic lobe; de, dorsal extra-embryonic tissue; js, jaw segment; jw, developing jaw; po, proctodeum; sp, developing slime papilla; ss, slime papilla segment; ve, ventral extra-embryonic tissue. Scale bars: A–C, G, H, J, K: 200 µm; F, I: 500 µm; D, E: 100 µm

Fig. 10
Fig. 10

Expression of Msx at consecutive developmental stages in embryos of the onychophoran E. rowelli. Anterior is left in E, F, and up in G, H; developing legs are numbered. A Cross section of a stage III embryo showing the expression in the ventrolateral ectoderm (arrowhead) and developing nerve cord (arrow). B Same cross section as in A stained with DAPI. Developing nerve cord is indicated with dashed lines. C Cross section of a stage IV embryo showing the expression in the ventral ectoderm (arrowhead) and developing nerve cord (arrow). D Same cross section as in C stained with DAPI. Developing nerve cord is indicated with dashed lines, developing neuropil is indicated with asterisks in A and B. E Late stage V embryo in ventrolateral view. Note the strong expression in the distal portion of the developing antennae (inset). F Stage VI embryo in ventrolateral view. Note that the signal is stronger in the slime papilla segment (asterisks) than in the subsequent segments. Note also the decreased expression in the developing antennae (inset) as compared to the embryo in E. G Head of late stage V embryo in dorsal view. H Head of a stage VI embryo in dorsal view. Arrows in G and H point to uncharacterized anterior brain neuropils; arrowheads indicate the putative olfactory lobes. Abbreviations: an, antennal tracts; at, developing antenna; cn, central brain neuropil; cp, connecting piece (cf. Ref. [141]; “medullary connective” sensu Whitington PM and Mayer G [111]); cx, brain cortex; de, dorsal extra-embryonic tissue; jw, developing jaw; mo, mouth; po, proctodeum; sp, developing slime papilla; ve, ventral extra-embryonic tissue. Scale bars: A–D: 50 µm; E, F: 500 µm; G, H: 200 µm

Fig. 11
Fig. 11

Expression of Lbx at consecutive developmental stages in embryos of the onychophoran E. rowelli. Anterior is left in A–B′, and up in C–K; developing legs are numbered. Asterisks point to the proctodeum. Insets in A′ and B′ show the respective embryo stained with DAPI. A Stage 0–I embryo in ventral view. Inset shows the jaw and slime papilla segments in ventrolateral view. A′ Same embryo as in A, superimposed light micrograph and DAPI image. Dashed lines indicate transverse furrows. B Stage I embryo in lateral view. B′ Same embryo as in B, superimposed light micrograph and DAPI image. Arrowheads point to transverse furrows. C Stage II embryo in lateral view. Note the weak signal in the developing antenna (arrowhead). D Head of stage II embryo in ventral view. Note the weak signal in the developing antenna (arrowhead). E Detail of stage II embryo in ventrolateral view. Arrows indicate transverse furrows. F Stage II embryo in lateral view, superimposed light micrograph and DAPI image. Dashed lines demarcate transverse furrows in the posterior segments. G Stage III embryo in ventral view. H Anterior segments of the same embryo as in G in ventral view. I Stage IV embryo in ventral view. J Head of a stage IV embryo in ventral view. Arrowheads point to the expression in the cephalic lobes. K Fifth and sixth developing legs of stage IV embryo in ventral view. Abbreviations: as, antennal segment; at, developing antenna; cl, cephalic lobe; de, dorsal extra-embryonic tissue; js, jaw segment; jw, developing jaw; sp, developing slime papilla; ss, slime papilla segment; st, stomodeum; ve, ventral extra-embryonic tissue. Scale bars: A–G, J: 200 µm; H, K: 100 µm; I: 500 µm

Fig. 12
Fig. 12

Expression of Tlx at consecutive developmental stages in embryos of the onychophoran E. rowelli. Anterior is left in A, C, and up in B, D, E; developing legs are numbered. A Stage III embryo in ventrolateral view. Note the two individual domains in the anterior limb buds (empty and filled arrowheads). Inset shows the same embryo stained with DAPI. B Detail of anterior limb buds of stage III embryo in ventral view. Note the ectodermal (arrows) and mesodermal domains (filled arrowheads) in the tips, and a separate proximal domain (empty arrowheads) in each limb bud. C Anterior end of a stage IV embryo in ventrolateral view. D Detail of the anterior limb buds of a stage IV embryo. Ventrolateral view. Note the ectodermal (arrows) and elongated mesodermal domains (arrowheads). E Developing fourth and fifth legs of a stage IV embryo in ventrolateral view. Note the ectodermal (arrows) and mesodermal domains (filled arrowheads) in the tips, and a separate proximal domain (empty arrowheads) in each limb bud. F Cross section of the developing fifth leg, dorsal is up. Note the ectodermal (arrow) and mesodermal domains (filled arrowhead) in the tips, and a separate proximal domain (empty arrowhead). F′ Same cross section as in F stained with DAPI. Developing nerve cord is indicated with dashed lines in F and F′. Abbreviations: at, developing antenna; cl, cephalic lobe; de, dorsal extra-embryonic tissue; jw, developing jaw; po, proctodeum; sp, developing slime papilla; st, stomodeum; ve, ventral extra-embryonic tissue. Scale bars: A: 500 µm; B, D, E, F, F′: 100 µm; C: 200 µm

Fig. 13
Fig. 13

Expression of NK2.2 at consecutive developmental stages in embryos of the onychophoran E. rowelli. Anterior is left in A, B, D and up in C, E, F, K, L; developing legs are numbered. Empty and filled arrowheads point to the medial and lateral domain in the ventral nerve cords, respectively. A Stage III embryo in lateral view. B Stage IV embryo in ventrolateral view. C Stage III embryo in ventral view. D Stage VI embryo in ventral view. E Stage IV embryo in ventral view. F Stage VI embryo in ventral view. G Cross section of a stage III embryo, dorsal is up. H Same cross section as in G stained with DAPI. I Cross section of a stage IV embryo, dorsal is up. J Same cross section as in I stained with DAPI. Dashed lines indicate developing nerve cords in G–J. K Stage IV embryo in ventral view, detail of the stomodeum. Arrows point to the domains on either side of the stomodeum. L Sagittal section of the head of a stage VI embryo, dorsal is right. Note the expression in the mouth cavity (arrows). Abbreviations: at, developing antenna; cl, cephalic lobe; de, dorsal extra-embryonic tissue; jw, developing jaw; lp, developing lip; mc, mouth cavity; ph, developing pharynx; po, proctodeum; sp, developing slime papilla; st, stomodeum; ve, ventral extra-embryonic tissue; vo, developing ventral organ. Scale bars: A: 200 µm; B, D: 500 µm; C: 50 µm; E–L: 100 µm

Fig. 14
Fig. 14

Comparison of the segment polarity patterns of expression of Msx, Lbx, NK1, NK4 and Tlx in the onychophoran E. rowelli and the annelid Platynereis dumerilii. Diagram of P. dumerilii modified from Ref. [19]. The cavities of somites (= coelomic cavities) are labeled with asterisks. Note that the somites are shifted anteriorly in P. dumerilii, while they align with transverse furrows (arrowheads) in E. rowelli. A, B Note that the expression of Msx, Lbx and NK1 is restricted to the mesoderm in E. rowelli, whereas Msx, Lbx and NK4 show additional ectodermal segment polarity domains in P. dumerilii

Fig. 15
Fig. 15

Alternative scenarios on the evolution of pulsatile organs in nephrozoans. Phylogeny modified from Ref. [16]. A Single origin of the heart and expression of NK4 in cardiac tissue in the last common ancestor of Nephrozoa. According to this scenario, multiple independent losses of hearts occurred in numerous spiralian and ecdysozoan taxa. Additionally, expression of NK4 in cardiac tissue was lost in mollusks. B Convergent evolution of hearts in deuterostomes, panarthropods, annelids and mollusks with either a gain of heart in the onychophoran/arthropod lineage or a secondary loss of the heart in tardigrades. According to this scenario, NK4 might have been recruited independently to fulfill regulatory functions during heart development in vertebrates, panarthropods and annelids, but not in mollusks. Abbreviations: B, Bilateria; D, Deuterostomia; N, Nephrozoa; P, Protostomia

Fig. 16
Fig. 16

Comparison of the expression of mediolateral patterning genes in onychophorans, arthropods, annelids and vertebrates. A Schematic drawing of the ventral ectoderm and nerve cords of a late stage embryo of E. rowelli showing expression of Msx, pax3/7, pax6, NK6 and NK2.2. Bars below show the mediolateral extent of each domain in the ventral ectoderm and nerve cords in early and late developmental stages. Note that the ectodermal expression is interrupted by the ventral extra-embryonic tissue. Empty bars with red crosses indicate the presumed absence of expression in the nerve cord and ventral ectoderm. Asterisks indicate expression data described in Refs [11, 68, 107]. B Expression of Msx, pax3/7, pax6, NK6 and NK2.2 in the neuroectoderm of the fruit fly Drosophila melanogaster, the annelid Platynereis dumerilii and the vertebrate Xenopus laevis. Abbreviations: ec, ventral ectoderm; nc, nerve cord; ve, ventral extra-embryonic tissue. Diagrams in B modified from Ref. [106]. See also Additional file 4 for a comparison of Msx, pax6, NK6 and NK2.2 domains in E. rowelli

Fig. 17
Fig. 17

Alternative scenarios on the evolution of mediolateral patterning of nerve cords among bilaterians. Scenarios A and B are modified from Ref. [17]. A Convergent evolution of similarities of the mediolateral patterning in vertebrates, arthropods and some annelids. The reconstruction of the ancestral morphology of the nervous system in the “urnephrozoan” is still unresolved due to the diversity of nerve cord arrangements in the nephrozoan lineages (white question mark). B The “urnephrozoan” already possessed a medially condensed nerve cord and mediolateral patterning. Thus, the medially condensed nerve cords of vertebrates, arthropods and annelids are homologous, implying multiple losses of mediolateral patterning. C The “urnephrozoan” already possessed a medially condensed nerve cord and mediolateral patterning. The medially condensed nerve cords of vertebrates and annelids are homologous. As a result, multiple independent losses of mediolateral patterning would have occurred, including the ecdysozoan ancestor, which would imply an independent evolution of medially condensed nerve cords in arthropods

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