Since the early nineteenth century, scientists have observed that many animals, from the very simple to the complex, shared similar embryonic morphology and development. They wondered what dictated the developmental direction that a fly, mouse, frog, or human embryo would take.
Near the end of the twentieth century, a particular class of genes was discovered that had this very job. These genes that determine animal structure are called “homeotic genes,” and they contain DNA sequences called homeoboxes. The animal genes containing homeobox sequences are specifically referred to as Hox genes. This family of genes is responsible for determining the general body plan, such as the number of body segments of an animal, the number and placement of appendages, and animal head-tail directionality. The first Hox genes to be sequenced were those from the fruit fly (Drosophila melanogaster). A single Hox mutation in the fruit fly can result in an extra pair of wings or even appendages growing from the “wrong” body part.
While there are a great many genes that play roles in the morphological development of an animal, what makes Hox genes so powerful is that they serve as master control genes that can turn on or off large numbers of other genes. Hox genes do this by coding transcription factors that control the expression of numerous other genes. Hox genes are homologous in the animal kingdom, that is, the genetic sequences of Hox genes and their positions on chromosomes are remarkably similar across most animals because of their presence in a common ancestor, from worms to flies, mice, and humans (Figure 1).
Hox genes are highly conserved genes encoding transcription factors that determine the course of embryonic development in animals. In vertebrates, the genes have been duplicated into four clusters: Hox-A, Hox-B, Hox-C, and Hox-D. Genes within these clusters are expressed in certain body segments at certain stages of development.
One of the contributions to increased animal body complexity is that Hox genes have undergone at least two duplication events during animal evolution, with the additional genes allowing for more complex body types to evolve.
If a Hox 13 gene in a mouse was replaced with a Hox 1 gene, how might this alter animal development?
[reveal-answer q=”319959″]Show Answer[/reveal-answer]
[hidden-answer a=”319959″]The animal might develop two heads and no tail.[/hidden-answer]
Hox gene expression during development of the phoronid Phoronopsis harmeri
Background: Phoronida is a small group of marine worm-like suspension feeders, which together with brachiopods and bryozoans form the clade Lophophorata. Although their development is well studied on the morphological level, data regarding gene expression during this process are scarce and restricted to the analysis of relatively few transcription factors. Here, we present a description of the expression patterns of Hox genes during the embryonic and larval development of the phoronid Phoronopsis harmeri.
Results: We identified sequences of eight Hox genes in the transcriptome of Ph. harmeri and determined their expression pattern during embryonic and larval development using whole mount in situ hybridization. We found that none of the Hox genes is expressed during embryonic development. Instead their expression is initiated in the later developmental stages, when the larval body is already formed. In the investigated initial larval stages the Hox genes are expressed in the non-collinear manner in the posterior body of the larvae: in the telotroch and the structures that represent rudiments of the adult worm. Additionally, we found that certain head-specific transcription factors are expressed in the oral hood, apical organ, preoral coelom, digestive system and developing larval tentacles, anterior to the Hox-expressing territories.
Conclusions: The lack of Hox gene expression during early development of Ph. harmeri indicates that the larval body develops without positional information from the Hox patterning system. Such phenomenon might be a consequence of the evolutionary intercalation of the larval form into an ancestral life cycle of phoronids. The observed Hox gene expression can also be a consequence of the actinotrocha representing a "head larva", which is composed of the most anterior body region that is devoid of Hox gene expression. Such interpretation is further supported by the expression of head-specific transcription factors. This implies that the Hox patterning system is used for the positional information of the trunk rudiments and is, therefore, delayed to the later larval stages. We propose that a new body form was intercalated to the phoronid life cycle by precocious development of the anterior structures or by delayed development of the trunk rudiment in the ancestral phoronid larva.
Keywords: Biphasic life cycle Body plan Brain Head Indirect development Intercalation Life history evolution Lophophorata Lox2 Spiralia.
Conflict of interest statement
Competing interestsThe authors declare that they have no competing interests.
Get full journal access for 1 year
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Runnegar, B. & Pojeta, J. J. Molluscan phylogeny: the paleontological viewpoint. Science 186, 311–317 (1974)
House, M. R. in Cephalopods—Present and Past (eds Wiedmann, J. & Kullmann, J.) 1–16 (Schweizerbart'sche Verlagsbuchhandlung, Stuttgart, 1988)
Lowe, C. J. & Wray, G. A. Radical alterations in the roles of homeobox genes during echinoderm evolution. Nature 389, 718–722 (1997)
Keys, D. N. et al. Recruitment of a hedgehog regulatory circuit in butterfly eyespot evolution. Science 283, 532–534 (1999)
Carroll, S. B., Grenier, J. K. & Weatherbee, S. D. From DNA to Diversity: Molecular Genetics and the Evolution of Animal Design (Blackwell Science, Malden, 2001)
Telford, M. J. Evidence for the derivation of the Drosophila fushi tarazu gene from a Hox gene orthologous to lophotrochozoan. Lox5. Curr. Biol. 10, 349–352 (2000)
Godwin, A. R. & Capecchi, M. R. Hoxc13 mutant mice lack external hair. Genes Dev. 12, 11–20 (1998)
Callaerts, P. et al. HOX genes in the sepiolid squid Euprymna scolopes: implications for the evolution of complex body plans. Proc. Natl Acad. Sci. USA 99, 2088–2093 (2002)
Brusca, R. C. & Brusca, G. J. Invertebrates (Massachusetts, Sinauer, Sunderland, 2003)
Boletzky, S. v. in Cephalopods—Present and Past (ed. Wiedmann, J. K., J.)) 167–179 (Schweizerbart'sche Verlagsbuchhandlung, Stuttgart, 1988)
Budelmann, B. U. in The Nervous Systems of Invertebrates: An Evolutionary and Comparative Approach (eds Breidbach, O. & Kutsch, W.) 115–138 (Birkhauser, Basel, 1995)
Hinman, V. F., O'Brien, E. K., Richards, G. & Degnan, B. M. Expression of anterior Hox genes during larval development of the gastropod Haliotis asinina. Evol. Dev. (in the press)
Shigeno, S., Kidokoro, H., Tsuchiya, K., Segawa, S. & Yamamoto, M. Development of the brain in the oegopsid squid, Todarodes pacificus: an atlas up to the hatching stage. Zool. Sci. 18, 527–541 (2001)
Shigeno, S., Tsuchiya, K. & Segawa, S. Embryonic and paralarval development of the central nervous system of the loliginid squid Sepioteuthis lessoniana. J. Comp. Neurol. 437, 449–475 (2001)
Hartmann, B. et al. Pax6 in the sepiolid squid, Euprymna scolopes: evidence for a role in eye, sensory organ and brain development. Mech. Dev. 120, 177–183 (2003)
Boletzky, S. v. McGraw-Hill Yearbook of Science & Technology 73–76 (McGraw-Hill, New York, 1994)
Young, J. Z. The organization of a cephalopod ganglion. Phil. Trans. R. Soc. Lond. B 263, 409–429 (1972)
Boletzky, S. v., Frösch, D. & Mangold, K. Développement de vésicules associées au complexe brachial chez les Céphalopodes. C.R. Acad. Sci. (Paris) D 270, 2182–2184 (1970)
Sundermann, G. Development and hatching state of ectodermal vesicle-organs in the head of Sepia officinalis, Loligo vulgaris, and Loligo forbesi (Cephalopoda, Decabrachia). Zoomorphology 109, 343–352 (1990)
McFall-Ngai, M. & Montgomery, M. The anatomy and morphology of the adult bacterial light organ of Euprymna scolopes Berry (Cephalopoda: Sepiolidae). Biol. Bull. 179, 332–339 (1990)
Nogi, T. & Watanabe, K. Position-specific and non-colinear expression of the planarian posterior (Abdominal-B-like) gene. Dev. Growth Diff. 43, 177–184 (2001)
Kulakova, M. A., Kostyuchenko, R. P., Andreeva, T. F. & Dondua, A. K. The Abdominal-B-like gene expression during larval development of Nereis virens (polychaeta). Mech. Dev. 115, 177–179 (2002)
Zákány, J. & Duboule, D. Hox genes in digit development and evolution. Cell Tissue Res. 296, 19–25 (1998)
Averof, M. & Patel, N. H. Crustacean appendage evolution associated with changes in Hox gene expression. Nature 388, 682–686 (1997)
Burke, A. C., Nelson, C. E., Morgan, B. A. & Tabin, C. Hox genes and the evolution of vertebrate axial morphology. Development 121, 333–346 (1995)
Moshel, S. M., Levine, M. & Collier, J. R. Shell differentiation and engrailed expression in the Ilyanassa embryo. Dev. Genes Evol. 208, 135–141 (1998)
Nederbragt, A. J., van Loon, A. E. & Dictus, W. J. A. G. Expression of Patella vulgata orthologs of engrailed and dpp-BMP2/4 in adjacent domains during molluscan shell development suggests a conserved compartment boundary mechanism. Dev. Biol. 246, 341–355 (2002)
Wanninger, A. & Haszprunar, G. The expression of an engrailed protein during embryonic shell formation in the tusk-shell, Antalis entalis (Mollusca, Scaphopoda). Evol. Dev. 3, 312–321 (2001)
Seaver, E. C., Paulson, D., Irvine, S. Q. & Martindale, M. Q. The spatial and temporal expression of Ch-en, the engrailed gene in the polychaete Chaetopterus, does not support a role in body axis segmentation. Dev. Biol. 236, 195–209 (2001)
Holland, C. H. The nautiloid cephalopods: a strange success. J. Geol. Soc. Lond. 144, 1–15 (1987)
Hoxb9 overexpression in adrenal cortical cells
HOX genes have been implicated in the initiation and progression of many cancers. 11 Our and other studies have shown that Hoxb9 is expressed at the early stages of adrenal cortical development. 14,33 To investigate if HOXB9 expression is associated with ACC, we analysed patient gene expression data from the Cochin cohort that contains normal adrenal (NAd), adrenocortical adenoma (ACA) and ACC samples. 3 In this dataset, HOXB9 expression was higher in ACC samples with the difference with ACA being significant, but not with NAd (Fig. 1a). Consensus clustering of mRNA expression has been used to subgroup ACC patients into those that have aggressive disease, C1A, and those with the indolent disease, C1B. In both the Cochin dataset and in ACC samples from The Cancer Gene Atlas (TCGA), HOXB9 expression was significantly higher in C1A compared to C1B (Fig. 1b). Consistent with HOXB9 expression being associated with aggressive disease, analyses of TCGA and Cochin patients into high and low HOXB9 expression showed that ACC patients with high HOXB9 expression had a poorer survival prognosis (Fig. 1c). Together these data suggest that elevated HOXB9 expression in ACC may play a role in tumour progression to aggressive disease.
a HOXB9 gene expression in normal human adrenals (NAd), adrenocortical adenoma (ACA) samples, and adrenocortical carcinoma (ACC) samples from the Cochin cohort. Statistical analysis is one-way ANOVA Turkey’s pairwise test, ***P = 0.0004. b HOXB9 expression in ACC samples from patients with aggressive C1A or indolent C1B-type disease from TCGA and Cochin cohorts. Statistical analysis is a Wilcoxon test, ***P = 0.00053, *P = 0.04. c Kaplan–Meier survival curves for ACC patients from the TCGA and Cochin cohorts that had either high or low HOXB9 expression. d Schematic of the Sf-1:Hoxb9 transgenic construct used to increase Hoxb9 expression in adrenal glands. bGH pA is a bovine growth hormone polyA sequence. e qRT-PCR of Hoxb9 on wild-type and Hoxb9 t/g adrenal glands. The data represent mean ± SD from three biological repeats. f HOXB9 immunohistochemistry on sections of female wild-type and Hoxb9 t/g adrenal glands. ZG is zona glomerulosa. g Wet weights of male and female adrenal glands from wild-type and Hoxb9 t/g animals. The data represent mean ± SD from four samples. h Haematoxylin and eosin (H&E) stain on sections from wild-type and Hoxb9 t/g adrenal glands. c is the cortex, m is the medulla. i Ki67 immunohistochemistry on sections of wild-type and Hoxb9 t/g female adrenal glands. j Bar chart of the percentage of Ki67-positive cells in wild-type and Hoxb9 t/g male and female adrenal glands. The data represent the mean ± SD from three biological repeats. k qRT-PCR of Sf-1 on wild-type and Hoxb9 t/g adrenal glands. The data represent the mean ± SD from three biological repeats. Student’s t test, **P < 0.01, *P < 0.05. Hoxb9 t/g indicates Sf-1:Hoxb9 transgenic.
To investigate the effect of high levels of HOXB9 in adrenal cortical cells, we generated transgenic mice carrying a BAC construct that contained the Hoxb9 cDNA inserted into the Sf-1 locus (Sf-1:Hoxb9 mice, referred to as Hoxb9 t/g, Fig. 1d). Quantitative RT-PCR (qRT-PCR) expression analysis showed an increase of Hoxb9 levels in the adult adrenals of transgenic mice (Fig. 1e). This was confirmed in antibody staining studies, which showed an expanded domain of HOXB9 expression, reflecting the pattern of the Sf-1 promoter sequence driving Hoxb9 (Fig. 1f and Supplementary Fig. S1A). In the normal adrenal HOXB9 was primarily expressed in zona fasciculata (ZF), while in transgenic animals expression was also found in the outer cortical zona glomerulosa (ZG) cells. HOXB9-expressing cells were also found in the medulla of transgenic glands from 3-month-old female mice that were not present in control animals (Supplementary Fig. S1B). Phenotypic analysis of the adrenals of transgenic mice at 3 and 18 months of age showed no obvious changes in size or structure compared to wild-type adrenal glands (Fig. 1g, h, Supplementary Fig. S1C, D). Ki67 staining revealed no difference in the number of proliferating cells between transgenic and wild-type animals (Fig. 1i, j and Supplementary Fig. S1E). We next analysed the expression of a known embryonic HOX target gene, Sf-1, markers of adrenal gland stem/progenitor cell function, and a WNT signalling target. In both female and male adrenal glands from transgenic mice, Sf-1 expression was increased threefold, while there was no change in the expression of Shh, Patched (Ptch) or Axin2 (Fig. 1k and Supplementary Fig. S1F). This suggests that elevated Hoxb9 is not able to induce neoplastic development but can promote Sf-1 expression in the adult gland. To determine if adrenal gland zonation was disrupted by elevated Hoxb9 expression, we performed immunohistochemical (IHC) staining and qRT-PCR analyses with cell-type markers for ZG (Dab2 and Cyp11b2), ZF (Cyp11b1), adrenal medulla (TH) and X zone (20α-HSD, gene name Akr1c18, and Pik3c2g) on male and female adrenals (Fig. 2). These data showed that transgenic animals had no change in adrenal cortical ZG or ZF markers (Fig. 2a, b). Instead, glands from 3-month-old female transgenic mice had a bigger foetal derived X zone with Sf-1-positive cells infiltrating the medulla (Fig. 2c, d and S2A). The larger X zone in transgenic animals behaved as in the wild-type in that it was only found in prepubertal males (Supplementary Fig. S2B) and it regressed in older females (Fig. 2c).
a Dab2, Cyp11b2 and Cyp11b1 immunohistochemistry on sections from wild-type and Hoxb9 t/g male and female adrenal glands. b qRT-PCR of Cyp11b1 and Cyp11b2 on wild-type and Hoxb9 t/g adrenal glands. The data represent the mean ± SD from three biological repeats. c Tyrosine Hydroxylase (TH) and 20α-HSD immunohistochemistry on sections from wild-type and Hoxb9 t/g 3- and 12-month-old female adrenal glands. d qRT-PCR of Akr1c18 and Pik3c2g on female wild-type and Hoxb9 t/g adrenal glands. The data represent the mean ± SD from three biological repeats. m is the medulla, arrows indicate positive cells. Student’s t test, *P < 0.05. Hoxb9 t/g indicates Sf-1:Hoxb9 transgenics.
Elevated Hoxb9 cooperates with mutant Ctnnb1 during tumour formation
To investigate if HOXB9 can promote tumour formation we bred Sf-1:Hoxb9 mice with mice containing the activating conditional Ctnnb1 deletion allele and a construct with Cre recombinase driven by Cyp11a1-regulatory sequences (Ctnnb1 mutant mice, referred to as ABC). As expected, adrenals from Ctnnb1 mutant mice showed tumour formation characterised by increased organ size, which was larger in the female (Figs. 1g and 3b). These tumours had a lack of zonal structure, loss of medullar cells and high expression of the ZG marker Dab2 throughout the tumour (Supplementary Fig. S3A, B). Six-month-old Ctnnb1 mutant mice that also carried the Sf-1:Hoxb9 transgene (double-mutant mice, referred to as ABC Hoxb9 t/g) showed an increase in adrenal size, which was restricted to male mice (Fig. 3a, b). Haematoxylin and eosin staining showed no obvious morphological difference between Ctnnb1 and double mutants (Fig. 3c). As expected, antibody staining for β-catenin and the WNT signalling downstream marker Lef1 in Ctnnb1 mutants showed high expression in the majority of cells (Fig. 3c). This staining pattern was unchanged in double-mutant tumours showing that elevated Hoxb9 had no major effect on this pathway (Fig. 3c). Proliferation, as measured by Ki67 staining, was higher in 6-month-old double-mutant male mice, with no changes in apoptosis, as measured by Caspase 3 staining (Fig. 3d–g). No signs of invasive disease were observed in double-mutant animals (Fig. 3c, arrows). Adrenal tumours from double-mutant mice expressed higher levels of Hoxb9 mRNA and protein than single Ctnnb1 mutants, confirming expression of the transgene (Fig. 3h, i). As Sf-1:Hoxb9 adrenals showed an increase in Sf-1 expression, we investigated the levels of this gene in the tumours. qRT-PCR analysis showed that Sf-1 transcript was higher in both female and male double-mutant adrenal tumours relative to Ctnnb1 mutants, but there was no difference in the levels of SF-1 protein expression between the genotypes (Supplementary Fig. S3C, D).
a Bright-field images of male ABC and ABC Hoxb9 t/g adrenal glands. b Wet weight of 6-month-old female and male ABC and ABC Hoxb9 t/g adrenal glands. The data represent the mean ± SD from four tumours. c H&E, β−catenin and Lef1 immunohistochemistry on sections from ABC and ABC Hoxb9 t/g adrenal tumours. Inset shows high-power magnification. Arrow indicates capsule. d Ki67 immunohistochemistry on sections from 6-month-old ABC and ABC Hoxb9 t/g adrenal tumours. e Bar chart of the percentage of Ki67-positive cells in ABC and ABC Hoxb9 t/g adrenals. The data represent the mean ± SD from three biological repeats. f Active Caspase 3 immunohistochemistry on sections from ABC and ABC Hoxb9 t/g male tumours. Arrows indicate positive cells. g Bar chart of the percentage of Caspase 3-positive cells in ABC and ABC Hoxb9 t/g male adrenals. The data represent the mean ± SD from three biological repeats. h qRT-PCR of Hoxb9 on ABC and ABC Hoxb9 t/g adrenal tumours. The data represent mean ± SD from three biological repeats. i Western blot analysis of Hoxb9 on ABC and ABC Hoxb9 t/g adrenal tumours from female animals. Adrenals from two animals of each genotype are shown. Vinculin is used as a loading control. ABC indicates Ctnnb1 mutant tumours, ABC Hoxb9 t/g indicates double-mutant tumours. Student’s t test, **P < 0.01, *P < 0.05.
To investigate the pathways activated in the double mutants we performed RNA-seq on RNA derived from adrenal tumours from 3-month-old Ctnnb1 and double-mutant female and male mutant mice. Comparative analysis identified differentially expressed genes in the tumours of double-mutant mice compared to Ctnnb1 mutants, with a higher number in males (533 genes altered, Benjamini–Hochberg adjusted P < 0.1) than in the same comparison in females (66 genes altered, Benjamini–Hochberg adjusted P < 0.1) (Fig. 4a and Supplementary Tables S2–S5). For both sexes, genes were differentially up- and downregulated (males 232 genes up, 322 genes down females 47 up, 19 genes down), consistent with evidence suggesting that HOX proteins can act as transcriptional activators and repressors.
a Heatmap of the top 50 differentially expressed genes identified from RNA-seq data of male ABC Hoxb9 t/g adrenal tumours compared to male ABC adrenal tumours. b Comparison of differentially expressed genes in male and female ABC Hoxb9 t/g adrenal tumours compared to ABC adrenal tumours. c Comparison of differentially expressed genes in male ABC Hoxb9 t/g tumours compared in male ABC tumours to the genes differentially expressed genes in female ABC tumours compared to male ABC tumours. d STRING database network of differentially expressed genes in male ABC Hoxb9 t/g tumours compared to male ABC tumours identifies angiotensin signalling. e qRT-PCR of Fos, Fosb and Junb in male ABC and ABC Hoxb9 t/g tumours. The data represent mean ± SD from three biological repeats. f IHC of Fosb in 3-month-old male ABC Hoxb9 t/g and ABC adrenal tumours. g GSEA of RNA-seq data from male ABC Hoxb9 t/g and male ABC adrenal tumours. E2F targets normalised enrichment score (NES) = 1.57, FDR q = 0.069, G2M checkpoint NES = 1.70, FDR q = 0.043. h qRT-PCR of Cdk1, Ccnb1, Ccnb2, Ccne1 and Knstrn in female and male ABC and ABC Hoxb9 t/g adrenal tumours. The data represent mean ± SD from three biological repeats. i Cell cycle genes upregulated in male ABC Hoxb9 t/g tumours and female ABC tumours (compared to ABC males). Student’s t test, **P < 0.01, *P < 0.05. ABC indicates Ctnnb1 mutant tumours, ABC Hoxb9 t/g indicates double-mutant tumours.
Comparative analysis of the differentially expressed genes between double mutants and Ctnnb1 mutant female and male tumours showed very few common genes altered in both sexes (three genes upregulated, and six genes downregulated in both females and males) (Fig. 4b). Validating our qRT-PCR result, Sf-1 was increased in double mutants of both sexes. Interestingly, many genes that were differential between male double mutants and Ctnnb1 mutants were shared with those that were different between male and female Ctnnb1 mutant animals (52 upregulated genes and 30 downregulated genes) (Fig. 4c). These data suggest that Hoxb9 acts to promote tumorigenic pathways that are repressed in the male adrenal.
Pathway analysis of the differentially expressed genes using the STRING protein–protein interaction database identified angiotensin signalling enriched in double-mutant male tumours compared to Ctnnb1 mutant tumours, including Agtr1b, Egr1, Nr4a1 and members of the Fos/Jun family Fos, Fosb and Junb (Fig. 4d and Supplementary Table S4). 34 Interestingly, Cyp11b2 expression, a target of this pathway, was not changed in these tumours. qRT-PCR was used to validate these results for the Fos/Jun family, and antibody staining showed widespread expression of Fosb in double-mutant tumours (Fig. 4e, f). Gene set enrichment analysis of the RNA-seq data from male Ctnnb1 and double-mutant tumours revealed enrichment in cell cycle genes in tumours with elevated Hoxb9, consistent with the increase in proliferation in double mutants (Fig. 4g and Supplementary Table S6). These data were validated using qRT-PCR for Cdk1, Ccnb1, Ccnb2, Ccne1 and Knstrn, which showed an increase in these genes in male double mutants but not in females (Fig. 4h). We next checked if the pathways we identified were also altered in female Ctnnb1 mutant tumours, compared to males of the same genotype, as these tumours shared a large number of altered genes with male double mutants (Fig. 4c). We found 15 cell cycle regulatory genes (E2F targets or G2M checkpoint Hallmark genes) upregulated in female Ctnnb1 tumours compared to Ctnnb1 male tumours, including Ccne1 and Cdk1 (Fig. 4i). These data suggest a core set of cell cycle genes that are elevated in tumours with high Hoxb9 are also expressed at high levels in female Ctnnb1 tumours, compared to males.
HOX factors as potential drug targets in ACC
Our transgenic mouse data indicates that high Hoxb9 expression can promote cell proliferation within a tumour context. We next wanted to determine if HOXB9 can promote proliferation in human ACC cells. Consistent with our mouse studies, overexpression of HOXB9 in the human adrenal cortical tumour cell line H295R led to a small but significant increase in cell number (Supplementary Fig. S4A, B, C). To determine if HOXB9 expression correlates with proliferation in human samples we analysed its expression in two ACC datasets, TCGA and Cochin, with the proliferation markers MKI67, CCNE1 and an established proliferation gene signature (Wassef et al. 32 ) (Fig. 5a). We found a significant correlation of HOXB9 with MKI67 and the proliferation signature in the Cochin ACC dataset but not TCGA. To investigate if other HOX genes are implicated in proliferation in human ACC we performed these correlations with all members of the HOX gene family. Several HOX genes showed a significant correlation with all proliferation markers in both datasets, including HOXC9, HOXC10, HOXC11, HOXC13 and HOXD13 (MKI67 P < 0.002, proliferation gene signature P < 0.001, CCND1 P < 0.05) (Fig. 5b, Supplementary Fig. S5, Supplementary Tables S7 and S8). An analysis of the correlation of HOX gene expression with the expression of all other HOX members in the TCGA ACC dataset showed HOXB9 expression significantly correlated with the expression of these HOX genes (HOXC9 r = 0.377 P = 0.000604, HOXC10 r = 0.474 P = 9.81 −06 , HOXC11 r = 0.427 P = 8.60 −05 , HOXC13 r = 0.455 P = 1.16 −06 and HOXD13 r = 0.453 P = 2.69 −05 ) (Supplementary Tables S7, S8 and Supplementary Fig. S6).
a Correlation of HOXB9 expression with MKI67, a proliferation gene signature, and CCNE1 in ACC patient samples from the TCGA and Cochin cohorts. b Correlation of HOXC10 expression with MKI67, a proliferation gene signature, and CCNE1 in ACC patient samples from the TCGA and Cochin cohorts. c HOX gene expression in ACC samples from patients with aggressive C1A or indolent C1B-type disease from the TCGA cohort. Statistical analysis a Wilcoxon test, ***P < 0.001. d HOX gene expression in normal human adrenals (NAd), adrenocortical adenoma (ACA) samples, and adrenocortical carcinoma (ACC) samples from the Cochin cohort. Statistical analysis is one-way ANOVA Turkey’s pairwise test, ***P < 0.001, **P < 0.01, *P < 0.05. e Kaplan–Meier survival curves for ACC patients from the TCGA and Cochin cohorts that had either high or low HOXC10 or HOXD13 expression.
To establish if these genes were implicated in disease progression, we performed correlations between HOX gene expression and ACC C1A versus C1B status (Fig. 5c and Supplementary Fig. S7), and ACC versus ACA and NAd (Fig. 5d), and found that higher levels of HOX genes correlate with ACC and aggressive disease. Analysis of overall and disease-free survival between ACC patients with high and low HOX gene expression showed a correlation between high HOX levels and poor prognosis (Fig. 5e, Supplementary Figs. S8 and S9). These data argue that HOX genes can be drivers of aggressive ACC disease.
To investigate if adrenal tumour growth is dependent on HOX genes, we performed siRNA knockdown studies of HOX genes expressed in H295R cells. 13 Knockdown of HOXA11, but not HOXA10 or HOXA13, led to reduced growth of H295R cells, supporting a role of HOX genes in promoting adrenal tumour cell proliferation (Fig. 6a, b). Analysis of HOXA11 paralogues after HOXA11 knockdown showed a modest reduction in HOXC11 expression, while HOXD11 expression was not detected in H295R cells in control or siRNA-treated samples (Fig. S4D). Our HOX gene expression correlation analysis showed a strong correlation of HOX genes within clusters, including HOXA10 with HOXA11 and HOXA13, and weaker associations with their paralogues (Supplementary Tables S7, S8 and Supplementary Fig. S6). Analysis of HOXA11 expression in the TCGA ACC dataset showed that it significantly correlated with Ki67 expression (P = 0.0023, r = 0.337) the proliferation gene signature (P = 0.00053, r = 0.381) and CCNE1 (P = 0.0010, r = 0.363) expression in these tumours.
a qRT-PCR of HOXA10, HOXA11 or HOXA13 in H295R cells treated with a non-targeting (NT) siRNA or a siRNA targeting HOXA10, HOXA11 or HOXA13. The data represent the mean ± SD from three biological repeats. b Growth curve of H295R cells treated with a NT siRNA or a siRNA targeting HOXA10, HOXA11 or HOXA13. c qRT-PCR of PBX1 in untreated H295R cells, cells treated with a NT siRNA, or a siRNA targeting PBX1. The data represent the mean ± SD from three biological repeats. d Growth curve of untreated H295R cells, cells treated with a NT siRNA, or a siRNA targeting PBX1. e Survival fraction of PC3, H295R, ABC (Ctnnb1 mutant) mouse adrenal cells, ATC1 and ATC7 cells treated with HTL001 or CXR9. Cell viability was measured using Cell TitreGlo. f Caspase 3/7 activity for H295R cells treated with 5 μM HTL001 (IC50), DMSO or 5 μM CXR9 for 24 h. The data represent the mean ± SD from three biological repeats. One-way ANOVA, **P < 0.01, ***P < 0.001.
As we have shown that several HOX genes correlate with proliferation markers and aggressive disease in ACC we chose to target PBX1, a transcription factor that cooperates with HOX proteins to regulate target gene expression and has been implicated in adrenal development and function. 15 siRNA knockdown of PBX1 in H295R cells led to reduced levels of expression and to a marked reduction in cell proliferation (Fig. 6c, d). H295R cells harbour a CTNNB1-activating mutation, and our qRT-PCR studies showed that PBX1 knockdown had no effect on WNT signalling, as measured by the expression levels of downstream targets AXIN2 and LEF1 (Supplementary Fig. S4E). In order to further investigate if HOX factors can act as drug targets in ACC, we used a developed antagonist peptide, which interferes with the interaction between HOX and PBX proteins (HTL001 30 ). Our drug response studies showed that H295R cell growth was highly inhibited by HTL001 but not by a control peptide (CXR9) (Fig. 6e). The IC50 of HTL001 in H295R cells was lower than that of another responsive cell line, the prostate cancer line PC3 (5.54 μM versus 30.11 μM). To investigate if mouse adrenal tumours were also sensitive, we analysed three models: two cell lines derived from adrenal tumours driven by SV40 T antigen, ATC1 (containing an activating Ctnnb1 mutation 35 ) and ATC7 and primary cells derived from adrenal tissue from the Ctnnb1 mutant mice described above. These cells were treated with the antagonist HOX–PBX peptide and found to be responsive, with ATC7 cells being the most sensitive (IC50 23.11 μM) (Fig. 6e). Cell death assays on H295R cells confirmed an increase in apoptosis in cells treated with the antagonist (Fig. 6f).
We apologize to those whose recent publications we were unable to cite owing to space limitations. N.I. was supported by a long-term European Molecular Biology Organization fellowship and by a fellowship from the Human Frontier Science Program Organization. The research of C.G. was supported by grants from the European Research Council (ERC-2008-AdG no. 232947), the Centre national de la recherche scientifique, the European Network of Excellence EpiGeneSys, the Agence Nationale de la Recherche and the Association pour la Recherche sur le Cancer.
Special offers and product promotions
From the Back Cover
Hox Genes: Methods and Protocols explores techniques and methodologies which arose from or were successfully applied to the study of Hox genes and Hox proteins, at the intersection of experimental embryology, genetics, biochemistry, physiology, evolutionary biology, and other life sciences. This detailed volume begins with a section on discovery and functional analysis of Hox genes, and then it continues onward to discuss mode of action and biomedical applications of Hox proteins. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Expert and practical, Hox Genes: Methods and Protocols serves as an ideal guide to researchers striving to move forward in this dynamic and exciting area of study.
We acknowledge the staff of the Orpheus Island Research Station for their assistance in field work and the contribution of the Great Barrier Reef Project consortium (https://data.bioplatforms.com/organization/about/bpa-great-barrier-reef) in the generation of data used in this publication. We also thank Patrick Schaeffer and Lionel Hebbard for commenting on an early version of the manuscript.
Photo acknowledgements for Fig. 1:
Panels b1–b3 Australian Institute of Marine Science, (2017). AIMS Coral Fact Sheets -Goniastrea aspera. Viewed 23 November, 2017 http://coral.aims.gov.au/factsheet.jsp?speciesCode=0187.
Panels c2–c3 Australian Institute of Marine Science, (2017). AIMS Coral Fact Sheets -Fungia fungites. Viewed 23 November, 2017
Panels d1–d3 Australian Institute of Marine Science, (2017). AIMS Coral Fact Sheets-Galaxea fascicularis. Viewed 23 November, 2017 http://coral.aims.gov.au/factsheet.jsp?speciesCode=0185.
Panel e Courtesy Andrew Baird.
Panel f “Acropora digitifera” Courtesy MDC Seamarc Maldives Licensed under Creative Commons International Attributions 4.0.
Panel g “Porites lutea”, Australian Institute of Marine Science, Photograph by Dr. Paul Muir (2014). Licenced under Creative Commons Attributions 3.0 Australia. Available at http://eatlas.org.au/media/1626.
Panel h “Aiptasia pallida” Courtesy Ricardo González-Muñoz, Nuno Simões, José Luis Tello-Musi, Estefanía Rodríguez under Creative Commons Attributions 3.0.
Panel i Courtesy Chiara Sinigaglia.
The authors gratefully acknowledge the support for the Great Barrier Reef Project enabled by funding from Bioplatforms Australia through the Australian Government National Collaborative Research Infrastructure Strategy (NCRIS), Rio Tinto, a private family Foundation and the Great Barrier Reef Foundation. The work was also supported in part by of the Australian Research Council through Grant CE140100020 to DJM and to SF via the ARC Centre of Excellence for Coral Reef Studies at James Cook University.
Availability of data and materials
The sequencing datasets (genome and transcriptome sequencing data) generated by the present study are publicly available at the European Nucleotide Archive (ENA). The accession numbers are PRJEB23333, PRJEB23312, and PRJEB23371 for Galaxea, Fungia, and Goniastrea respectively [116,117,118]. Genome assembly and annotation are publicly accessible through Reefgenomics data repository [119,120,121]. Functional annotations supporting the conclusions of this article are included within the article and its additional files. Protein sequences used for gene phylogeny construction are included in the additional files.
Additional whole genome data used for comparative analyses are available from the following resources. Acropora digitifera data were obtained from the NCBI ftp site  with the assembly accession GCF_000222465.1 and annotation release ID 100. The Acropora millepora genome was assembled and annotated by author SF. Genome-related data for this species have been deposited to NCBI under the accession number PRJNA473876 . Porites lutea genome data are publicly available via the Reefgenomics data repository . Nematostella vectensis genome data were downloaded from Ensembl genome metazoan release 29 . Aiptasia genome v1.0 data were obtained from the Reefgenomics data repository .
In this study, we describe two novel echinoderm Hox genes with possible implications for Hox cluster evolution and the origin of the unusual body plan of Echinodermata. We named these genes Hox11/13d and e to indicate their relationship to the Hox11/13 genes previously described from both echinoderms and hemichordates. Our results increase the typical complement of echinoderm Posterior Hox genes to six, closer in number to the seven Posterior genes of the “archetypal” chordate amphioxus . Additional, divergent sequences such as AbdB-like in S. kowalevskii and an as yet unnamed, unique Posterior Hox-like sequence we found in O. spiculata (data not shown) raise the possibility of even more unexplored, lineage-specific diversity in this iconic gene family.
The problem of Posterior Hox gene phylogeny in deuterostomes has endured ever since Ferrier et al.  first articulated it. Although that study largely focused on the problem as it pertains to chordates, the relationships of Hox11/13b+ genes in ambulacrarians are equally difficult to resolve. Our work confirms that this problem cannot be easily solved with the addition of more taxa and sequences: despite including complete homeodomains and flanking regions of all types of 11/13 protein from all echinoderm classes and three hemichordates, our phylogenetic analyses still yield poorly resolved trees with conflicting topologies. Rather than illuminating its evolutionary history, the mosaic distribution of conserved sequence motifs outside the homeodomain (Additional files 3and 4 Fig. 6) indicates a high level of evolutionary flexibility in this clade.
Hox genes are best known for their conserved roles in patterning the bilaterian AP axis. In echinoderms, the ancestral AP axis is obscured by the pentameral symmetry of the adult body, but a spatially ordered “Hox vector” can still be discerned in the larval somatocoels of both echinoids [30, 35] and crinoids . This vector incorporates Hox7-Hox11/13b in echinoids and Hox5-Hox9/10 in crinoids, although Hara et al.  were unable to clone Hox11/13b from M. rotundus. Separate from this linear expression pattern, some Hox genes are also expressed in radial patterns in the adult rudiment such radial expression has been reported for Hox3 in S. purpuratus  and Hox3, 5 and 11/13b in P. japonica .
The conspicuous absence of Anterior and some Central Hox genes from the somatocoelar Hox vector, together with the rearrangement of the sea urchin Hox cluster with Hox1–3 at the “posterior” end of the cluster , prompted Mooi and David  to hypothesise a link between cluster rearrangement and what they termed the axial, radially symmetrical region of a developing echinoderm adult. Building on Duboule’s  discussion of Hox cluster organisation and ordering as a possible evolutionary constraint, Mooi and David  and David and Mooi  suggested that the translocation of Anterior Hox genes in echinoderms permitted a delay in their expression and a dissociation from the AP axis, allowing their novel deployment as part of the developmental toolkit for radial adult structures. The above hypothesis predicted that the 5′ translocation of Anterior Hox genes would be ancestral to living echinoderms. However, the recent publication of Hox cluster data from sea stars  and sea cucumbers  suggests that it is, in fact, a peculiarity of echinoids and therefore not associated with the origin of pentameral symmetry .
All of the echinoderm Hox clusters described above fell into the “Disorganized (D)” category of Duboule’s  classification, meaning they were intact but relatively loosely organised, with losses, inversions and/or rearrangements within the cluster. Our findings reveal two novel genes that appear completely detached from the main Hox cluster even in the echinoderm species with the least disorganized cluster described to date . Given that Hox11/13d and e both occur in every extant echinoderm class, the ancestral echinoderm Hox cluster may have been “Split (S)” sensu Duboule  instead of merely disorganized. Linkage data from crinoids, which form the sister group to all other living echinoderms, will be essential for testing this idea. A mostly intact Hox cluster which a subset of Posterior Hox genes have nonetheless escaped from is also seen in the annelid Platynereis dumerilii  how closely this loss of cluster integrity parallels the situation in echinoderms remains to be seen.
Hox11/13d is expressed in embryonic stages of several echinoderms, likely an unusual trait for a Hox gene in this clade [30, 36]. Interestingly, the limited spatial expression data that exist for Hox11/13d  hint that despite its departure from the cluster, this gene may exhibit spatially coordinated expression with Hox11/13b, appearing in a domain more vegetal than the latter. Spatial collinearity of Hox gene expression is known to be at least partially independent of clustering. Residual spatial collinearity may persist even after complete Hox cluster disintegration [41, 42]. Conversely, Hox genes in canonically ordered clusters may evolve expression domains that break collinearity, as seen with Hox6 and Hox14 in the “archetypal” chordate amphioxus . Thus, the regulatory, developmental and evolutionary significance of Hox11/13d and e being outside the Hox cluster is difficult to predict without more information on their expression and function.
Nothing is currently known about the expression and developmental roles (if any) of Hox11/13e. Unlike Hox11/13d, it is not present in any of the developmental transcriptomes we searched, suggesting that any developmental expression would happen at late stages that may be crucial for the development of the pentameral adult, or restricted domains that limit its detectability in transcriptome surveys. While Hox11/13d has a very similar homeodomain to Hox11/13b and c (Fig. 2) and shares several conserved motifs with the hemichordate members of the 11/13b+ group (Additional files 3 and 4), Hox11/13e has a highly distinctive homeodomain, so divergent that its original discoverers were not even certain it was a Hox gene . In light of this, the high level of conservation seen in both its homeodomain and C-peptide across different echinoderm clades (Fig. 2) is intriguing, and so is the similarity of the C-peptide to Hox11/13d. As an echinoderm-specific Hox gene that is both highly unique and very conserved within echinoderms, Hox11/13e may prove especially interesting with regard to the evolution of the unusual body plan of this phylum.
Our discovery of two previously unrecognised (except for a brief mention of one of them in ref. ) Hox genes in the well-studied genome of the “model” echinoderm S. purpuratus highlights the continued need for in-depth studies focused on individual gene families in the age of big data. Such deep surveys may be particularly vital for Posterior Hox genes, whose higher levels of sequence divergence compared to most Anterior and Central Hox genes can make them difficult to catch in general homeodomain searches .
The improved taxon sampling resulting from the proliferation of “non-model” genome sequencing projects creates an unprecedented opportunity to chart the distribution of unusual members of key gene families such as Hox11/13e, an essential first step in understanding their role in body plan evolution. In combination with expression and functional studies, such surveys may shed new light on the origin of lineage-specific innovations.
BT made the majority of the experiments, wrote the manuscript, performed statistical analysis. T. V designed experiments, performed the expression analysis of the Hox genes, wrote the manuscript. Á R performed vulval analysis in ceh-13 and synMuv mutant animals. EA performed the phylogenetic analysis of the nematode Hox genes. FM designed experiments, wrote the manuscript. KTV supervised the work, designed experiments, performed cloning experiments, wrote the manuscript. All authors read and approved the final manuscript.