Zebrafish aplnra functions in epiboly
© Nornes et al; licensee BioMed Central Ltd. 2009
Received: 20 August 2009
Accepted: 19 November 2009
Published: 19 November 2009
The zebrafish, Danio rerio, possesses the paralogous genes aplnra and aplnrb that are duplicates of an ancestral orthologue of the human APLNR gene encoding a G-protein coupled receptor that binds the peptide ligand APELIN and is required for normal cardiovascular function. aplnrb is required for migration of cells contributing to heart development in zebrafish embryos. aplnra is transcribed in a complex pattern during early development but its function in embryogenesis is largely unknown.
Blockage of translation of aplnra mRNA in zebrafish embryos results in retarded or failed epiboly with the blastoderm apparently disconnected from the nuclei of the yolk syncytial layer. Gastrulation is also defective. Failure of correct tail extension is observed with ectopic structures resembling somites positioned dorsal to the spinal cord.
aplnra, unlike its duplicate aplnrb, is essential for normal epiboly, although this function appears to be independent of signalling activated by zebrafish Apelin. The defects in epiboly caused by loss of aplnra activity appear, at least partially, to be due to a requirement for aplnra activity in the yolk syncytial layer.
The human gene APLNR (formerly named AGTRL1) encodes a G protein-coupled receptor related to the angiotensin receptors. Investigation in a variety of model systems has shown that APLNR has roles in both development of the blood vasculature and the heart [1–5] and in regulation of cardiovascular function, e.g. as a potent stimulator of cardiac contractility . It also functions as an arterial and venous dilator . APLNR has also been of interest as a receptor for the Human Immunodeficiency Virus, .
The zebrafish genes aplnra (formerly agtrl1a) and aplnrb (formerly agtrl1b) are paralogous genes derived by duplication of an ancestral teleost orthologue of human APLNR. aplnrb has been shown to be essential for the earliest moments of heart morphogensis (the formation of myocardial progenitors in the heart field and their subsequent migration) and for cell movements during gastrulation [4, 5]. These two genes appear to have somewhat redundant functions in formation of cardiomyocytes . aplnra and aplnrb are transcribed in similar patterns during epiboly and gastrulation suggesting that they may also both play roles in controlling these processes [4, 5, 9]. While loss of Aplnrb protein expression causes defects in gastrulation, no role in epiboly has been observed.
The ligand for human APLNR protein is a short peptide, APELIN (reviewed by ), for which a zebrafish orthologue exists encoded by the gene apln. Zeng et al.  detected apln transcripts (by RT-PCR) first during development after commencement of gastrulation. By mid-gastrulation, apln transcripts can be detected in the notochord by whole mount in situ transcript hybridisation. This argues against a role for zebrafish Apelin-Aplnra/b signalling in epiboly. However, ectopic and early expression of apln mRNA by mRNA injection does produce defects in epiboly  suggesting that inappropriate activation of Aplnra/b receptors might interfere with this morphological movement.
Materials and methods
The work was carried out under the auspices of the Animal Ethics and Institutional Biosafety Committees of the University of Adelaide.
The standard negative control MO sequence (Cont MO), also from Gene Tools, had the sequence: 5'-CCTCTTACCTCAGTTACAATTTATA-3'. Rescue of the phenotype generated by MO3 injection was demonstrated by injection of this MO at the 1-cell stage followed by injection of aplnra mRNA (see later) that had been engineered to lack the MO3-binding site and to contain an optimal Kozak's sequence (5'-GCCAGGATG-3', bold indicates start codon). This mRNA was transcribed from the pCS2+ expression vector containing the aplnra open reading frame using the mMessage mMachine kit (Ambion Inc., Austin, TX, USA). 2-5 nL of morpholino or mRNA were injected per embryo. MO and mRNA injections,  and whole mount in situ transcript hybridisation, , were performed essentially as previously described.
To visualise migration of YSL nuclei relative to the blastoderm margin during epiboly, embryos injected with Cont MO or MO3 were subsequently injected with 1 nL of 0.5 mM Sytox Green fluorescent nucleic acid dye (Molecular Probes®, Invitrogen, Life Technologies Corp. Carlsbad, Ca, USA) into the yolk cell at the sphere stage (4 hours post fertilisation @ 28.5°C, hpf) and then visualised at 6-8 hpf under both epifluorescence and bright field optics using a Zeiss AxioImager Z.1 with ApoTome attachment. Images were captured and processed using a Zeiss AxioCam MR and AxioVision 4.5 software (Carl Zeiss MicroImaging GmbH, Jena, Germany).
Results and Discussion
Aplnra is required for normal epiboly
aplnra function is required for normal epiboly and elongation/extension movements
Age (normal developmental stage)
Apparent Developmental Stagea
8 hpf (75% epiboly)
14 hpf (10 somite)
10 somite stage+
6-10 somite stage
bud-6 somite stage
50% - 75% epiboly
24 hpf (prim-5)
50% - 75% epiboly
Ectopic Cell Massb
In contrast to the striking defective epiboly phenotypes seen with the aplnra-targeted MOs, injection of Cont MO at the highest of these concentrations (1.0 mM) produced a very low level of defects (Figure 1J-L, Table 1). Furthermore, injection of MO3 at the 1-cell stage followed by injection into the yolk cell of an aplnra mRNA engineered to lack MO3's binding site led to effective rescue of the epiboly phenotype (described later). We also examined whether the failure to undergo posterior extension might be caused by cell death in the tailbud. Acridine orange staining of aplnra-injected embryos revealed a slight increase in the number of dying cells with elevated levels found primarily in the head (asterisk, Figure 1I) relative to Cont MO-injected siblings (Figure 1H). Combined, these data support that the defective epiboly phenotype is a specific effect of reducing the translation of endogenous aplnra mRNA, indicating a role for this gene in the regulation of epiboly.
aplnra is expressed in the yolk syncytial layer and the enveloping layer (EVL) . Microtubules within the YSL are required to drive normal epibolic movement and this is achieved through correct connections with the EVL. The published data on aplnrb expression are insufficient to determine whether this gene is also expressed in the YSL and EVL. During early epiboly, aplnra is also expressed in deep cells . At the 32-cell stage, central blastomeres no longer have cytoplasmic connections to the yolk cell . If a MO was injected into the yolk at the 32-cell stage, its distribution would be restricted mainly to the YSL and marginal blastomeres and, at later stages, many cells of the embryo proper would possess no or a lower concentration of the MO. When MO2 was injected into the yolk cell at the 32-cell stage, the embryos still showed defects in epiboly but, in those embryos that managed to complete epiboly, there was a decrease in tail extension defects. This supports that the tail extension defects are primarily due to lack of aplnra function in the embryo proper (Table 1).
Rescue of the aplnra MO epiboly phenotype by co-injection of aplnra mRNA
Extent of epiboly at 8 hpf
MO3 + mRNA
75% epiboly - bud
Total number injected (n)
Loss of aplnra function by injection of MO3 results in decoupling of YSL nuclei from the blastoderm margin as demonstrated by labelling of YSL nuclei with Sytox Green dye (Figure 1F, G). Note that we are unable to state whether this uncoupling is due to loss of aplnra function in the YSL, the EVL or both since we have not examined the behaviour of the EVL when aplnra activity is lost.
Aplnra is required for normal cell movement during gastrulation
Close examination of the gastrula margin and presumptive notochord in aplnra MO-injected embryos using the transcription of tbx16 to highlight these structures revealed several types of disruption (Figure 1A-C). Firstly, the margin was often uneven near the shield, with groups of cells apparently displaced animally from their neighbours (asterices, Figure 1B, C). These "notches" in the margin have previously been seen in cdh1 (hab) mutants , suggesting a problem with cell adhesion. Secondly, isolated, scattered tbx16 positive cells were seen in the hypoblast animal to the marginal expression domain (arrowheads, Figure 1B). Thirdly, although the shield and notochord/spinal cord primordia are normally devoid of tbx16 expressing cells, in aplnra-deficient embryos multiple tbx16 positive cells were found in this domain (arrows, Figure 1B, C). Zeng et al.  showed that apelin is expressed in the notochord during gastrulation and both aplnra  and aplnrb  are expressed in the epithelial adaxial cells that flank this structure. Thus, the loss of aplnra expression appears to allow either mixing of axial and adaxial cells or inappropriate differentiation of cells within axial structures. Furthermore, in many of the embryos surviving to tail extension stages, structures resembling somites were observed lying dorsal to the CNS (Figure 1D, E). These observations of aberrant positioning of cells of possible paraxial origin strongly suggest that multiple cell movements are affected by reduction of aplnra function.
List of Abbreviations Used
zebrafish Apelin receptor a or b gene
zebrafish Apelin receptor a or b protein
hours post fertilisation @ 28.5°C
yolk syncytial layer.
This work was supported by funds from Australian Research Council distributed through The Centre for the Molecular Genetics of Development and by infrastructure funding from The School of Molecular and Biomedical Sciences at The University of Adelaide. The work was carried out under the auspices of the Animal Ethics and Institutional Biosafety Committees of the University of Adelaide.
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