A compendium of developmental gene expression in Lake Malawi cichlid fishes
- R. F. Bloomquist†1, 2,
- T. E. Fowler†1,
- J. B. Sylvester1,
- R. J. Miro1 and
- J. T. Streelman1Email author
© The Author(s). 2017
Received: 15 August 2016
Accepted: 26 January 2017
Published: 3 February 2017
Lake Malawi cichlids represent one of a growing number of vertebrate models used to uncover the genetic and developmental basis of trait diversity. Rapid evolutionary radiation has resulted in species that share similar genomes but differ markedly in phenotypes including brains and behavior, nuptial coloration and the craniofacial skeleton. Research has begun to identify the genes, as well as the molecular and developmental pathways that underlie trait divergence.
We assemble a compendium of gene expression for Lake Malawi cichlids, across pharyngula (the phylotypic stage) and larval stages of development, encompassing hundreds of gene transcripts. We chart patterns of expression in Bone morphogenetic protein (BMP), Fibroblast growth factor (FGF), Hedgehog (Hh), Notch and Wingless (Wnt) signaling pathways, as well as genes involved in neurogenesis, calcium and endocrine signaling, stem cell biology, and numerous homeobox (Hox) factors—in three planes using whole-mount in situ hybridization. Because of low sequence divergence across the Malawi cichlid assemblage, the probes we employ are broadly applicable in hundreds of species. We tabulate gene expression across general tissue domains, and highlight examples of unexpected expression patterns.
On the heels of recently published genomes, this compendium of developmental gene expression in Lake Malawi cichlids provides a valuable resource for those interested in the relationship between evolution and development.
KeywordsCichlid fishes Evolution of gene expression Lake Malawi Developmental pathways
Comparative gene expression is a hallmark of the evolution and development research program . This is particularly the case among closely related vertebrate species, like hominids , beach mice , cavefishes , stickleback , and cichlid fishes . In these examples and many others, diversity in key traits evolves via spatial, temporal and/or quantitative variation in gene expression. Despite the importance of changes in gene expression to the evolution of closely related species, comprehensive surveys of spatial expression patterns are typically confined to laboratory models (e.g., zebrafish, ).
Lake Malawi cichlids used for this study included Metriaclima zebra [MZ] and Petrotilapia chitimba “thickbar” [PC]. These species were used owing to their availability and the fact that they belong to the ‘mbuna’ rock dwelling lineage. While Malawi cichlid species share qualitative expression domains across species, those from different ecotypes (mbuna versus ‘non-mbuna’) may exhibit heterochronic and quantitative differences in expression [6, 17]. Adult cichlids were maintained in re-circulating aquarium systems at 28 °C (Georgia Institute of Technology). Fertilized embryos were removed from the mouths of brooding females and staged in days post-fertilization (dpf), according to the Nile tilapia developmental series . Embryos were raised to 4dpf or 6dpf and euthanized with sodium bicarbonate buffered anesthetic MS-222, before fixation in 4% paraformaldehyde. Pre-hatching embryos at 4dpf were dechorionated using fine forceps to achieve proper fixation and reagent penetration.
Primer and probe design
In situ hybridization
Specimens for ISH were fixed a minimum of 48 h in 4% paraformaldehyde at 4 °C and then dehydrated into a graded series of methanol for further fixation and storage at −20 °C O/N. Embryos were rehydrated and permeabilized in 10 μg/mL proteinase K for one hour. They were then refixed in 4% PFA and incubated in prehybridization solution at 70 °C. Embryos were incubated overnight at 70 °C in digoxigenin-labeled antisense riboprobes. The following day, embryos were washed through a graded series of saline-sodium citrate buffer solutions and blocked with blocking solution (5% blocking buffer, 5% goat serum in MABT). Embryos were then hybridized with 1:3000 anti-digoxigenin-AP FAB fragments in blocking buffer overnight at 4 °C. Excess antibody was removed by washing, and color reaction with NBT/BCIP was performed on the AP-conjugated anti-dig antibodies. Gene expression was imaged in whole mount, using a LeicaDFC295 compound light microscope.
Results and discussion
Bone morphogenetic protein and transforming growth factor beta pathway
The transforming growth factor beta (TGF-β) superfamily is a class of cytokines organized into TGF-βs, bone morphogenetic proteins (BMPs), and activin/inhibins that bind to Type I and II serine/threonine kinase receptors . Upon ligand activation, type II receptors phosphorylate type I receptors, leading to SMAD protein activation and ultimately gene regulation. TGF-β/BMPs play a major role in almost every aspect of vertebrate biology, from gastrulation and organization of the body plan, to the genesis of almost every organ, to renewal and adult tissue maintenance [23, 24]. Inasmuch, mutations in the TGF-β superfamily and its regulators have been demonstrated as causative for the evolution of major adaptations.
BMPs are believed to control multiple aspects of cichlid jaw shape and function [25, 26], are in part responsible for evolutionary novelty in beak shape of Darwin’s finches , and have a direct dose-dependent effect on the craniofacial skeleton when transgenically titrated in mice . All of the BMP pathway factors we include are expressed in the jaw once it has formed in the larval stage, and many are also expressed in the pharynx, as indicated in Table 1. bmp2 and bmp4 pattern, generate, shape, and regenerate teeth in mice, squamates, and cichlids [29–35] while a large-effect QTL containing bmp6 has been reported for a doubling of pharyngeal tooth number in stickleback . bmper may regulate tooth number in Malawi cichlids .
Tumor-suppressor smad1 is phosphorylated in response to BMP pathway activation, and regulates transcription. We observe smad1 throughout the brain, fins, eyes, somites/vertebrae, jaw, and pharynx. Transcription factors snai1 and snai2 exhibit distinct expression patterns. snai1 is in all three brain regions, notably along the longitudinal fissure at 6dpf, as well as the vertebrae/somites. snai2 appears around the eyes pretectum, and pectoral fins, along with heavy expression in the pharyngeal arches and somites. We observe sostdc1 in the pharynx and cranial lateral line, and tgfb1 around the eyes, retinae, pharynx, and fins.
Fibroblast growth factor pathway
Much like the TGF-β/BMP pathway, the Fibroblast growth factor (FGF) pathway plays a part in eukaryotic development and homeostasis across ontogeny, and is particularly important for organogenesis and the generation of evolutionary novelty. FGFs and FGF receptors (FGFRs) are part of a larger family of Tyrosine Kinases and high-affinity cell surface receptors known as Receptor-Tyrosine Kinases (RTKs) that function through activation of Ras/MAP kinase and phospholipase-C gamma pathways . Conserved across all metazoans, FGFs have gained redundancy in higher vertebrate genomes, presumably for the formation of complex traits. In Amphioxus, FGF’s coordinate segment reduction, perhaps permissive for the evolution of the vertebrate head . In frog, FGFs work synergistically with BMPs to induce neurulation . By contrast, FGFs have long been recognized as competitors of the BMP pathway in patterning limb outgrowth in mammals , and formation and regeneration of fish fins . Genetic ablation of the FGF antagonists Spry4 and Spry2 produces mice with tusks for incisors , and heterochrony of fgf8 expression in the blind cavefish neural plate contributes to defective retinal morphogenesis . It is apparent that FGFs are one of the key pathways exploited by nature during animal evolution .
Zinc-finger transcription factor sp8 acts under the regulation of fgf10 and Wnt/β-catenin, and has been shown to regulate fgf8 for limb formation during chicken development . We observe sp8 along the spinal region and throughout the brain and olfactory placodes in a pattern similar to that seen in zebrafish . Repressor spry4 and transcription factor twist1 are both expressed along the somites, as well as in the pharynx, fins, eyes, and jaws.
Forkhead box pathway
The Forkhead Box transcription factors share an evolutionary conserved “forkhead” or “winged-helix” 100 amino-acid DNA binding domain. The moniker for the Fox family was coined when the first homolog forkhead (fkh) was identified in Drosophila, with mutant flies exhibiting split heads . Moreover, the helix-turn-helix motif of this domain is comprised of 3 α-helices and two large loops that resemble “wings.” To date, over 100 Fox transcription factors have been identified across eukaryotes and much work has been done to clarify their nomenclature . For example, in humans there are over fifty Fox proteins categorized into 19 subgroups (FOXA to FOXS) .
In vertebrates, FoxP2 has demonstrated roles in vocalization and the ability to learn language. Deletions in FoxP2 result in verbal dyspraxia and a collapse of the communication system at both the neural and muscular levels ; furthermore Foxp2 has evolved episodically in hominids . In cichlids, foxp2 is expressed in distinct foci in the thalamus and telencephalon, as well as in the pharyngeal arches where sound is produced , in the otic placodes and tectum where sound is received and processed, and in the fins.
The Hedgehog pathway executes pervasive roles in embryonic development, stem cell renewal, and cancer biology. Hedgehog proteins are a group of soluble morphogens that include Indian Hedgehog, Desert Hedgehog, and perhaps the most well studied ligand in embryology, Sonic Hedgehog (SHH). These morphogens bind to the transmembrane receptor Patched (Ptch), releasing co-receptor Smoothened (Smo) and permitting activation of Hedgehog signaling. The Hh pathway is involved in the specification and morphogenesis of nearly all animal organs .
dlx1a, dlx2, dlx3b, dlx5, emx3, msx1, nkx2-1 and nkx2-5 belong to the ANTP class and members have been well described as mediators of zebrafish jaw  and lamprey pharynx development , as well as important for fish brain development . In Fig. 7 we note strikingly similar expression of dlx1a and dlx2, and expression of Dlx and Msx genes in the jaws and pharynx. The Dlx and Nkx genes are expressed in the ventral regions of the mid and/or forebrain. emx3 is expressed in the dorsal telencephalon early, and at later stages is seen throughout the brain and trunk.
In the three amino acid loop extension (TALE) superclass we demonstrate expression of irx1b, irx2 and meis2, all three of which are expressed in the eyes and brain. Belonging to the LIM class we present expression of LIM homeobox 2 (lhx2), lhx6 and lhx9. lhx2 and lhx9 exhibit essentially identical expression patterns in the brain, fins, and spinal region, while lhx6 is only expressed in the jaw, pharynx, and preoptic region.
Calcium, endocrine, and insulin signaling
As another example, gad1 and gad2 (also known as gad67 and gad65, respectively) encode enzymes for the production of the neurotransmitter GABA and have known roles in schizophrenia and Parkinson’s disease. As in zebrafish  and other organisms, we note expression of gad1 and gad2 throughout the brain early in pharyngeal and larval stages of development. Plasma membrane transporter glast is expressed in cephalic lateral line placodes, where ion exchange will mediate sensory signaling later in the fully functional organ. In the insulin pathway, we report expression of igfbp5 in lateral line and all brachial arches. isl1, or Insulin gene enhancer protein, binds to insulin enhancer sites to regulate insulin gene expression and has known roles in diabetic disease. It is commonly used as a marker of pancreatic cells early and late in zebrafish ontogeny  and we show is expressed similarly in cichlids, as well as in forebrain neuronal subsets. Factors involved in endocrine signaling, such as bhlhe40, cdkn1a, and th, are all diffusely expressed in the brain. bhlhe40 exhibits notable expression in the eyes at both stages, and additional expression in the pharynx and somites at 6dpf.
Mitogens, stem cell factors and tumor suppressors
Mesenchymal stem cells (mSCs) can be difficult to define due their loose spatial arrangement and degrees of potency. We report expression of celsr1, mcam, pdgf, and vim, which have recently been hypothesized to maintain mSCs [78, 79] in structures including brain, eyes, lateral line, and fins.
Crucial to the dichotomy of stem cell potency is the genetic environment that houses these cells, known as the niche. For instance, a set of key genes known as the Yamanaka factors, cmyc, klf4, oct4, and sox2 are important for maintaining pluripotent stem cells (PSCs), and through retroviral induction can transfate mouse fibroblasts into induced PSCs (iPSCs) . We have cloned oct4, reported as pou5f1 in the Hox panel (Fig. 9). Similar to reports in zebrafish  we see little whole-mount expression of oct4 past neurulation, presumably because of its defined roles in PSC maintenance. However, we report expression of klf4, noted in lateral line, fins, and brain, as well as sox2, noted even at later larval stages in adult organs capable of self-renewal, including teeth, taste buds, and the cephalic lateral line. The ability of sox2 to persist and localize to epithelial stem cell (eSC) niches has been noted before . sox2 has been reported as an eSC marker in a host of adult organs [83, 84], along with bmi1 and lgr5  in the intestine, and tumor suppressor lrig1 as a master regulator of eSCs . We observe lrig1 throughout the brain, spinal region, fins, and eyes. Finally, we report expression of neural crest stem factor sox10 in the pharynx and somites, and mitogenic factor tp63 in the jaw, pharynx, CNS, cephalic lateral line, and fins.
The intercellular Notch signaling cascade is a highly conserved pathway involved in animal cell specification and proliferation . Notch signaling exhibits versatility through a gamut of posttranslational modifications that alter receptor response to ligand. Notch activation occurs primarily by juxtacrine signaling from Delta, Serrate, and Jagged class ligands, which bind the Notch receptor extracellular domain of an adjacent cell. This binding causes proteolytic cleavage of a cytosolic domain to enable it to act as a transcription factor. The Notch pathway is of particular interest in axial patterning during embryogenesis because of this characteristic signal transduction between neighboring cells. In vertebrate models, including chicken , and mouse , temporal regulation of Notch in the pre-somitic mesoderm plays an important role in segmentation. In zebrafish, segmentation can be restored in Notch-deficient embryos via delivery of artificial pulses of Notch . In cichlids, the Notch pathway is involved in patterning and regeneration of teeth , and in the renewing mouse incisor Notch has a role in maintaining the stem niche .
In Drosophila, Notch signaling has been shown to regulate cell fate in the eye by acting at specific ommatidium photoreceptors . We observe expression in the retina (see frontal view) for deltaA, hes1, jag1, notch1 and notch2, as well as expression of deltaB, dlk1, jag2, and notch3 in the eyes. At 4dpf, dlk1 expression is restricted to small areas in all three regions of the brain and dorsal side of the eyes, and by 6dpf this expression has spread to the telencephalon, optic tectum, and hindbrain. hes1 expression at 4dpf is seen in the head and lateral line, and at 6dpf expression is also evident in the vertebral somites and jaw.
Jagged1 is important for endothelial tissue development, and has been correlated with human congenital diseases of the heart . We observe expression of jag1 and jag2 throughout the brain and fins at both developmental stages, and jag2 additionally in the somites and jaw. lnfg is expressed in the brain and eyes, with heavy expression along the central midline. We also observe lnfg in the somites, where it is critical for somite segmentation according to studies performed in mouse . Notch receptors notch1, notch2 and notch3 all exhibit expression along the center midline of the brain and in the jaw, somites, pharynx, and lateral line.
Brain development and neurogenesis
The formation of the brain and nervous system is highly conserved and requires the integration of many, often competing, molecular signals. Cichlid brains evolve diversity via subtle modification of conserved gene regulatory networks [6, 19]. Here we show expression of transcription factors and other components of nervous system development as well as guidance cues involved in neurogenesis and axonal growth.
In Fig. 13, egr4 is expressed throughout the brain at 6dpf while neuronal differentiation factor neurod1 can be seen in the brain and pharyngeal arches. neurod2 expression at 4dpf is localized to the telencephalon and eyes, but at 6dpf this expression appears throughout the brain and nerve cord. neurog1 expression is evident in the brain and the dorsal trunk.
We observe receptors nrp1a and nrpn2a in similar patterns in the eyes and brain, with heavier expression of nrp1a in the fore/midbrain and pharynx. plxna3 is expressed throughout the brain and eyes, while plxna4 is localized to more restricted regions of the eyes and in the dorsal region of the cerebellum. We report expression of semaphorins 3a, 3c, 3e, and 3f in the retinal tissue similar to expression reported in zebrafish , at both the pharyngula and larval stages. All four of these semaphorins are expressed in the early jaw, pharynx, nasal pits, somites, and presumptive optic and otic regions.
The Wingless (Wnt) signaling pathway involves many factors that alter transcription, regulate calcium levels, and affect cell polarity during embryonic development through paracrine and autocrine signal transduction. Wnt ligands initiate the pathway by binding the N-terminal extracellular domain of Frizzled family receptors, which then bind cytoplasmic Dishevelled within the cell to propagate the signal. This pathway is highly conserved across vertebrates and invertebrates, with more than 20 mammalian Wnt ligands identified .
Developmental roles for Wnt signaling have been demonstrated for decades, and knowledge of the effects of this pathway has continued to grow. In 1980, lethal mutations of wingless were shown to affect Drosophila larvae body segments, making boundaries between body axes indistinguishable . This was further demonstrated in Xenopus embryos, which exhibited duplicated axes when injected with mouse Wnt1 RNA , and similar duplication was observed by injection of other Wnt related factors. This pathway is important for regulation of cell fate in self-renewing tissues, including mouse intestinal epithelium , and in zebrafish has been shown to be important in early neural crest development.
Nuclear β-catenin mediates transcriptional activation by transcription factors Lef1 and Tcf. We observe notable expression of lef1 in the midbrain and forebrain in a similar pattern to that of tcf712 in Fig. 15. tcf3 exhibits expression in the brain, eyes, fins, and somites. Additionally, we report R-spondin receptors lgr4 and lgr6 in distinct patterns in the brain, gut, eyes, fins, and somites, and the secreted R-spondin rspo2 in restricted regions of the fore- and hindbrain.
Other developmentally expressed genes
Transmembrane protein ectodysplasin A (eda) acts through receptor edar in ectodermal tissue development. This signaling pair helps pattern early embryonic structures including skin, hair, and teeth, from germ layers, and outlines placode derived structures such as scales and precisely patterned chicken feathers . We observe eda and edar localized to the tooth placodes and fins at these stages, and note expression in and around scales later in development (not shown). Both factors appear to be expressed more heavily in pharyngula stage than at the larval stage. We see krt8 expressed generally across the entire integument at both stages of development.
Vascular endothelial growth factors vegfa and vegfc are involved in angiogenesis, vasculogenesis, and cell migration. Overexpression of this family of genes is seen in the vascularization of cancerous tumors , and is the target of many emerging cancer therapies. We observe expression of vegfa along the midline of the brain, as well as in the hindbrain and somites. We also observe vegfc in the somites, as well as in the olfactory, optic, and otic regions.
Novel expression domains
Here, we provide a set of probes for spatial analysis of gene expression, useful across hundreds of East African cichlid fishes, for studies of evolution and development. Gene expression patterns are captivating, and provide important clues to the evolution of gene regulation. Gene expression is context-dependent, dynamic in space and time. Our compendium of gene expression for early Lake Malawi cichlid development provides examples of (i) expression patterns conserved with many other animals, as well as (ii) expression patterns that can be considered novel, because they haven’t been assayed at these particular spaces and times. We highlight a few of these novel expression domains.
Calcium and endocrine signals (Fig. 10) as well as the stem cell/mitogenic factors (Fig. 17) are rarely studied at these stages, in whole mount. Particularly striking spatially delimited gene expression patterns are observed for many of these genes, including calb2, calb2a, cldn15a, kiss1r, glast, sparc, stra13, bmi1, pdgf, celsr1, klf4, trp63 and vim, suggestive of precise roles in embryonic development. We also observe new expression domains from well-studied genes. Notable from this class are osr2 (Fig. 3; expression in fins) foxp2 (Fig. 5; expression in fins and jaws), hopx (Fig. 7; expression in the pharynx), nrp1a, sema3a, sema3c and sema3e (Fig. 14; expression in fins and jaws). These novel expression domains set the stage for future exploration of function.
Bone morphogenetic pathway
Fibroblast growth factor
- LF :
- MZ :
Quantitative trait loci
Transforming growth factor
We thank Maya Tome and Avery Shook, along with the members of the Streelman lab and anonymous reviewers for their contributions and comments in preparation of this manuscript.
This study was funded in part by grants from the National Institutes of Health (R01GM101095, 2R01DE019637 to J.T.S.; and F30DE023013 to R.F.B.) and the National Science Foundation (IOS1146275 to J.T.S.). The funding bodies had no role in the design of the study, collection of data, analysis of data, interpretation of data or in writing the manuscript.
Availability of data and materials
Sequence data that support the findings of this study have been deposited in GenBank with the primary accession codes KT906433-KT906561, KC633830- KC633846, EU867210-EU867217, KT851376-- KT851399.
All ISH and imaging performed by RFB, TEF, and RJM. Sequences were cloned and probes were generated by RFB, TEF, and JBS. RFB, TEF, and JTS conceptualized and composed the manuscript. All authors read and approved final versions of the article.
The authors declare that they have no competing interests.
Consent for publication
All experiments conducted in relation to this publication were carried out in a humane and ethical manner in accordance with Georgia Institute of Technology policies in strict adherence to IACUC (Institutional Animal Care and Use Committee) protocols A14053 and A14055.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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