Antisense MOs targeting FoxH1 translation cause gastrulation defects
To determine the phenotype of zebrafish with disruptions of the entire FoxH1 protein, we used a splice-blocking MO to disrupt splicing from the exon1 donor site of zygotic foxh1
pre-mRNA and translation-blocking MOs to disrupt translation of maternal and zygotic foxh1
transcripts. Injecting 8 ng of the splice-blocking MO did not produce any midline defects, but did cause a predicted loss of zygotic foxH1
mRNA, as judged by RT-PCR (data not shown), and also led to a failure in heart looping (mesocardia) in 93% (N=88) of scored embryos, compared to a background mesocardia rate of 7.1% (N=70) in control MO-injected embryos (data not shown). Mesocardia has previously been reported for surty68b
mutants (Bisgrove et al., 2000
). We have observed a similar low penetrance of midline defects accompanied by highly penetrant mesocardia in zygotic surm768
embryos (data not shown), and therefore conclude that the foxh1
splice-disrupted phenotype is equivalent to recessive sur
Injecting 8 ng per embryo of either of two foxh1
translation-blocking morpholinos (FoxH1 MO1 or FoxH1 MO2), by contrast, caused a severe phenotype characterized by (1) a developmental delay evident by 6 hours post-fertilization (hpf) (), when control embryos are at shield stage (), (2) a compromised ability for embryonic cells to epibolize past the embryonic equator (), and (3) embryonic death around 14–17 hpf () when control embryos are at mid somitogenesis stages (). Thus, interference of maternal and zygotic foxh1
MO injection causes earlier and more severe phenotypes in zebrafish than previously reported for sur
alleles (Pogoda et al., 2000
; Sirotkin et al., 2000
We used several approaches to ensure that the translation-blocking MOs are FoxH1-specific. First, a control MO, whose sequence does not target any known zebrafish genes, was injected at the same dose (8 ng) as the FoxH1 MOs, and found to produce no phenotype (). This indicates that general MO toxicity is not a concern at 8 ng doses.
To test whether the FoxH1 MOs actually target FoxH1, we co-injected FoxH1 MO1 or control MO along with (1) a plasmid (ARE-luc) that requires Smad/FoxH1 complex binding for coupled transcription/translation of luciferase, (2) mRNA encoding the Nodal-related protein, Squint (to stimulate phospho-Smad/FoxH1 complex formation) and (3) a consitutively expressed control plasmid encoding a luciferase variant that can be independently assayed (Huang et al., 1995
; Osada et al., 2000
). These embryos were raised for four hours at 28°C, then lysed and assayed for induced and control luciferase activity. This revealed that FoxH1 MO1 specifically decreases Smad/FoxH1 transcription/translation of luciferase (). Thus, FoxH1 MO1 genuinely disrupts FoxH1 function.
Another critical control is to test whether two MOs targeting different sequences on the same gene can induce the same phenotype. This is done to rule out phenotypes that might arise from chance binding of a MO to a second gene. We therefore injected a second non-overlapping FoxH1 MO (FoxH1 MO2) and obtained very similar morphological perturbations and times of death (compare ).
As further control for the specificity of the FoxH1 MOs, we injected them at lower individual doses (4 ng instead of 8 ng). Each of the two MOs causes a milder phenotype at this lower dose, however co-injecting 4 ng each of the two MOs causes the same phenotype as individual 8 ng injections (data not shown). This ability to produce the same phenotype with lower individual FoxH1 MO doses, which are less likely to cause non-specific defects, further argues that the 8 ng MO phenotypes are FoxH1 specific. The remaining data in this study utilizes FoxH1 MO1.
To further explore the morphological defects of gastrulating embryos injected with FoxH1 MO1, we prepared hematoxylin and eosin-stained sections of 8 hpf FoxH1 morphants and controls, when controls were at 70% epiboly. A comparison of lateral sections through the thickest portions of the animal poles of FoxH1 MO1 morphant () and control embryos () reveals three notable differences. (1) Morphant embryos are thicker and their nuclei (arrows) rounder, although there are a similar number of cellular tiers in control and FoxH1 morphant embryos, indicating that morphant embryos are less compacted along the mediolateral axis. (2) The EVL (dotted line), which is the outermost cell layer, is more ruffled in morphants. (3) There are widespread gaps (arrowheads) between the eosin-rich yolk globules and the embryonic cells of morphant embryos. Thus, at mid-gastrulation FoxH1 morphant embryos display several defects in cellular stratification.
Heterochronic expression of mesoderm markers and loss of neural patterning in FoxH1 morphants
We examined how FoxH1 depletion affects gene expression, using whole-mount in situ
hybridization for selected molecular markers. At 10 hpf, when control embryos were at the bud stage of development, FoxH1 morphant expression of the mesodermal markers no tail
; ), protocadherin 8
; ) and snail1
() was restricted to the vegetal margin, resembling the late-blastula stage (5 hpf) expression pattern for those genes in WT embryos (see, e.g.
, for a comparison of ntl
) (Hammerschmidt and Nusslein-Volhard, 1993
; Schulte-Merker et al., 1994
; Yamamoto et al., 1998
). A similar heterochronic expression pattern was seen for the axial mesoderm markers goosecoid
; ) and sonic hedgehog
; ), which in bud stage (10 hpf) FoxH1 morphants are expressed in a small domain near the margin, similar to the expression of these genes in the organizer of late blastula-stage control embryos (see, e.g.
, for a comparison of gsc
)(Stachel et al., 1993
; Strahle et al., 1996
). The restricted expression of the preceding genes to cells near the margin suggests that the presumptive mesoderm and endoderm cells of FoxH1 morphants may have defects in internalization and/or migration towards the embryo’s animal pole. The absence in mid-gastrula stage FoxH1 morphants of bone morphogenetic protein 4
)-positive mesoderm (, arrowhead) or sox17
-positive endoderm () within the dorsal axis also suggests an internalization/migration defect (Chen et al., 1997
; Kunwar et al., 2003
). We noted one exception to this trend: of twenty foxa2
-stained FoxH1 morphants scored, seven displayed deep anterior staining as shown in , suggesting that dorsal internalization can persist in FoxH1 morphants (Strahle et al., 1993
). But this foxa2
exception is variable: of the remaining thirteen scored embryos, seven had staining that remained much closer to the margin and six had nearly undetectable staining, indicating that foxa2
-positive cells are usually compromised as well (data not shown).
Molecular marker expression in FoxH1-depleted embryos
With respect to neurectoderm development, gastrulation-stage FoxH1 morphants exhibit robust expression for markers of broad territories, as seen for the posterior neural marker hoxb1b
(), the anterior neural marker otx2
() and the early ectoderm marker, gata2
() (Imai et al., 2001
; Kramer et al., 2002
; Vlachakis et al., 2000
). By contrast, more regionalized patterning of the CNS is disrupted, seen in bud-stage FoxH1 morphants as a loss of pax6a
transcripts (), which normally mark the presumptive forebrain and hindbrain, in pax2.1
transcripts (), which normally mark the presumptive midbrain-hindbrain boundary and the pronephros, and krox20
transcripts (), which normally mark the presumptive hindbrain rhombomeres 3 and 5 (Reim and Brand, 2006
; Strahle et al., 1993
; Strahle et al., 1996
Defective gastrulation movements in FoxH1 morphants
As noted, FoxH1 perturbations cause gastrulation movement defects in frogs and in a significant fraction of mouse mutants. Although zebrafish sur
mutants show no substantial alterations in gastrulation movements, FoxH1 morphants have dramatic epiboly defects (compare ). We went on to examine other gastrulation movements in FoxH1 morphants. To assess internalization, we co-injected embryos with either FoxH1 MO1 or control MO, together with Kaede protein, a coral-derived green fluorescent protein that fluoresces red after photocleavage by UV and near-UV light (Ando et al., 2002
). These embryos were incubated until the 40% epiboly stage (~5 hpf) at which time we photolabeled two small groups of cells situated on or near the vegetal margin (). The fate of these labeled cells was documented 95 minutes later. In control embryos, some of the labeled cells had clearly internalized and migrated towards the animal pole (, white brackets). Other labeled cells did not internalize and remained at the margin (, white arrowheads), which itself was displaced towards the vegetal pole as a result of continuing epiboly. In FoxH1 morphants, there was no movement of labeled cells, indicating an absence of internalization as well as a lack of epiboly within the experimental time frame (, white arrowheads). We obtained similar results from eleven independent trials, demonstrating a dramatic overall disruption of internalization in FoxH1 morphants. Because the dorsal-ventral position of labeled cells was random in these trials and because the assay time was limited to 95 minutes, it is possible that subtle instances of partial internalization were missed, for instance on the dorsal side where we frequently detect deep foxa2
staining at a substantial distance from the margin of FoxH1 morphants (). These considerations notwithstanding, our internalization assay demonstrates a widespread disruption of internalization in FoxH1 morphant embryos.
FoxH1-depletion disrupts gastrulation movements
To assess convergence in FoxH1 morphants, we used gsc
-GFP transgenic zebrafish, which have green fluorescent protein (GFP) under the control of the goosecoid
) promoter (Doitsidou et al., 2002
). Gsc is first expressed in the dorsal gastrula organizer and GFP recapitulates this pattern in gsc
-GFP embryos, allowing us to visualize the dorsal side of these embryos. We injected gsc
-GFP embryos with either control MO or FoxH1 MO1 together with photoactivatable-GFP, a GFP variant that has weak fluorescence until photoactivated by violet light (Patterson and Lippincott-Schwartz, 2002
). When injected embyos reached the shield stage, we photolabeled two groups of cells close to the margin situated 180 degrees apart and flanking the dorsal gsc
-GFP-positive cells (). After 2-hours of further incubation, the labeled cells in control MO injected embryos had clearly converged ~40 degrees towards the dorsal midline (). By contrast, labeled cells in FoxH1 morphants showed no detectable convergence (). Similar results were obtained in six independent experiments. We did not directly assay extension movements, however this movement relies both on convergence to populate the midline and on epiboly to create space for elongation, so extension is necessarily compromised as well. In summary, our various analyses reveal a critical role for FoxH1 in all four gastrulation movements.
Microarray analysis identifies potential effectors of the FoxH1 morphant phenotype
We wished to identify molecular alterations that might account for the defects in FoxH1 morphants. Our initial analysis of marker gene expression elucidated some of the dynamics of FoxH1 morphant differentiation, but the variety of genes examined was limited and we failed to identify genes with altered expression prior to the onset of morphological defects. To undertake a broader search for genes with altered expression in early FoxH1 morphants, we used microarrays to compare the late blastula (5 hpf) gene expression profile of FoxH1 MO1-injected embryos with that of control MO-injected embryos. This approach yielded 75 RefSeq genes with significantly reduced expression in FoxH1 morphants and 100 RefSeq genes with significantly elevated expression (P<0.01 and >two-fold change). To identify categories of co-regulated genes within these two sets, we used GeneSifter software.
We identified five over-represented categories of up-regulated genes: genes whose products are involved in cell division and/or regulation of translational initiation, genes whose products localize to chromosomes and/or the nuclear envelope and genes whose products have oxidoreductase activity. We also found three over-represented categories of down-regulated genes: genes encoding intermediate filament components, genes whose products are involved in cytoskeleton organization and biogenesis, and genes whose products are involved in mesoderm development. We cloned genes from these categories to confirm their regulation by in situ hybridization. Our selection was biased towards cytoskeletal proteins and proteins involved in the cell cycle, because these seemed the most likely candidates for causing the observed gastrulation movement defects and developmental delays in FoxH1 morphants.
In this way we confirmed the indicated down regulation of five cytoskeletal genes in FoxH1 morphants at 5 hpf: cytokeratin1
), cytokeratin II (cyt2), keratin
) 18, krt8
(previously called zf-k8
), but not capzb, transgelin2
( and data not shown). Strikingly, all of the downregulated cytoskeletal genes turned out to be keratins with expression patterns restricted to the most superficial layer of cells in the embryo: the EVL (Imboden et al., 1997
; Sagerstrom et al., 2005
; Thisse et al., 2001
; Thisse and Thisse, 2004
). EVL expression is seen, for instance, in a lateral view of cyt1
expression in a control embryo (). The expression of four of these five keratin genes recovers by bud stage in FoxH1 morphants (), whereas cyt1
expression remains low (). We also looked at the cyt1
expression levels in surty68b/ty68b
embryos, and saw no difference from sibling controls (data not shown).
Down-regulation of keratins and up-regulation of cell cycle genes in FoxH1 morphants
We further confirmed the indicated up regulation of two cell cycle genes: her5
, but not cdc45-like, cyclinA1
( and data not shown). To further investigate the possibility of cell cycle alterations in FoxH1 morphants, we looked at mitosis and cell death at bud stage, by anti phospho-histone 3 staining and TUNEL assays respectively, but saw no difference from controls (data not shown). In addition, we assessed the relative numbers of G0/G1, S and G2/M phase cells at various stages using propidium iodine staining and FACS. No differences between FoxH1 morphants and controls were seen during early and mid-gastrulation, but a characteristic increase in the G0/G1 cellular fraction at the expense of the G2/M cellular fraction seen in controls at the two-somite stage (11 hpf) was substantially depressed in FoxH1 morphants (Zamir et al., 1997
). Considering that only two of the cell cycle genes we examined had validated changes in expression, and the only cell-cycle related phenotype we identified was well after the onset of the FoxH1 morphant phenotype, we instead focused our attention on the keratins.
Rescue, epistasis and phenocopy studies
To better understand the requirement for maternal FoxH1, we explored various ways of rescuing, perturbing or phencopying the FoxH1 morphant phenotype. We first tested whether the FoxH1 morphant phenotype could be rescued by co-injecting foxh1
mRNA. To remove the risk of the FoxH1 MO1 interfering with translation of exogenous foxh1
mRNA, or exogenous foxh1
mRNA diluting the effect of the MO, we excluded foxh1
’s 5’ UTR and introduced two silent mutations, eliminating 16 of 25 complementary nucleotides. Co-injecting 150 pg of this foxh1
mRNA with 8 ng of FoxH1 MO1 fails to rescue any morphological defects, but it partially rescues the reduced expression of pax2.1
() and cyt1 (), but not that of pax6.1
(data not shown). The pax2.1
partial rescue is seen as an increased fraction of embryos displaying pax2.1
-positive midbrain-hindbrain boundary precursor cells, while the cyt1
partial rescue is seen as a general increase in transcript levels. While complete rescue would have provided convincing proof of the specificity of the FoxH1 morphant phenotype, our inability to completely rescue does not necessarily reflect non-specificity; it may rather reflect an inability for non-regulated exogenous FoxH1 to compensate for the loss of regulated endogenous FoxH1. In support of this, all of our other controls () have indicated specificity, the gastrulation defects we observe do not fall within the spectrum of typical off-target morpholino effects and difficulty rescuing Foxh1 loss of function has been reported in Xenopus
(Ekker and Larson, 2001
; Howell et al., 2002
; Watanabe and Whitman, 1999
). Although morphological defects in FoxH1 morphants injected with 8 ng of the FoxH1 MO1 cannot be rescued, a milder set of morphological defects resulting from injection of 4 ng of FoxH1 MO1can be rescued. At 24 hpf, embryos injected with 4 ng of FoxH1 MO1 display a dramatic shortening of the body axis and microcephaly and darkened head tissue, indicating necrosis (). Co-injection of 150 pg foxh1
mRNA dramatically reduces the number of embryos displaying this phenotype, instead producing embryos with longer body axes, and larger, less opaque heads (). It is of course possible that this lower-dose rescue represents a qualitative rather than a quantitative rescue.
Rescue of FoxH1 morphant phenotypes by co-injection of foxH1 mRNA
FoxH1 is believed to drive transcription of the zebrafish Nodal-related genes and their antagonists, the lefties. We observed a characteristic heterochronic expression of the Nodal genes squint and cyclops in FoxH1 morphants (data not shown). To directly test whether the FoxH1 morphant phenotype reflects an imbalance in Nodal signaling, we co-injected either squint mRNA or lefty1 mRNA along with FoxH1 MO1, and found that neither was able to visibly alter the morphological phenotype, but these potent mRNAs did produce the expected gain of Nodal function (squint) and loss of Nodal function (lefty1) morphological alterations in sibling embryos in which control MO was co-injected (data not shown). Although squint, lefty1 and (previously) foxh1 mRNA each failed to alter the FoxH1 morphant phenotype, this does not reflect a general inability of FoxH1 morphants to efficiently translate ectopic genes; in our luciferase assays, for instance, embryos injected with either the FoxH1 MO or the control MO produced similar levels of ectopic renilla luciferase (data not shown). We also injected FoxH1 MO1 into maternal-zygotic squinthi975/hi975 embryos, and the phenotype was indistinguishable from the FoxH1 morphant phenotype seen in WT embryos (data not shown). Taken together, our data point to an early requirement for zebrafish FoxH1 that is epistatic to Nodal signaling.
To address whether the FoxH1 morphant phenotype is attributable to the loss of keratin expression, we used two approaches. We attempted to rescue the high dose FoxH1 morphant phenotype by co-injecting cyt1
mRNA, looking for changes in morphology as well as changes in the expression of three molecular markers, but saw no evidence of rescue (data not shown). This may reflect a need for the additional down-regulated keratins or a need to deliver Cyt1 specifically to the EVL. Intriguingly, injection of 200 pg cyt1
mRNA often causes WT embryos to develop with one eye smaller than the other (16%, N=31), perhaps due to unequal distribution of mRNA, and a similar phenotype is seen in a similar fraction of WT embryos (23%, N=39) injected with 150 pg foxh1
mRNA (Fig. S1 A’, B’, C’
We also asked whether removal of keratins from WT embryos could phenocopy the FoxH1 morphant phenotype. Injection of MOs targeting cyt1
translation (Fig. S1
) or splicing (data not shown) caused epiboly defects and decreases in neural marker expression that were consistent with the FoxH1 morphant phenotype, but significantly milder (compare Fig. S1 E–E”” and F–F””; H and I; K and L
Considering that multiple keratins are downregulated in FoxH1 morphants, we went on to ask whether simultaneous depletion of several keratins might produce a better phenocopy, and this is indeed the case. We injected a cocktail of MOs targeting cyt1, cyt2, k18, k8 and k4, comprising a total of 16 ng MO. The resulting phenotype was compared with that seen in embryos injected with 8 ng of FoxH1 MO1, 16 ng of control MO or no MO at all. Because 16 ng is a higher dose of MO than typically used, we compared control-injected and uninjected embryos, revealing that this dose had only mild effects on embryonic development (compare ). By contrast, the keratin MO cocktail produces a severe phenotype that is indistinguishable from the 8 ng FoxH1 morphant phenotype, with respect to the delay in epiboly (compare ) and the time of death (compare ). A striking match between the keratin- and the FoxH1-depleted phenotypes is also seen in the reduced expression of the EVL marker cyt1 (compare ), the reduced expression of the regionalized neural marker pax2.1 (compare ), the heterochronic expression of the paraxial mesoderm marker snail1 (compare ) and the reduced expression of the endoderm and forerunner cell marker sox17 (compare ).