The differentiation and movement of presomitic mesoderm progenitor cells are controlled by Mesogenin 1

Abstract

Somites are formed from the presomitic mesoderm (PSM) and give rise to the axial skeleton and skeletal muscles. The PSM is dynamic; somites are generated at the anterior end, while the posterior end is continually renewed with new cells entering from the tailbud progenitor region. Which genes control the conversion of tailbud progenitors into PSM and how is this process coordinated with cell movement? Using loss- and gain-of-function experiments and heat-shock transgenics we show in zebrafish that the transcription factor Mesogenin 1 (Msgn1), acting with Spadetail (Spt), has a central role. Msgn1 allows progression of the PSM differentiation program by switching off the progenitor maintenance genes ntl, wnt3a, wnt8 and fgf8 in the future PSM cells as they exit from the tailbud, and subsequently induces expression of PSM markers such as tbx24. msgn1 is itself positively regulated by Ntl/Wnt/Fgf, creating a negative-feedback loop that might be crucial to regulate homeostasis of the progenitor population until somitogenesis ends. Msgn1 drives not only the changes in gene expression in the nascent PSM cells but also the movements by which they stream out of the tailbud into the PSM. Loss of Msgn1 reduces the flux of cells out of the tailbud, producing smaller somites and an enlarged tailbud, and, by delaying exhaustion of the progenitor population, results in supernumerary tail somites. Through its combined effects on gene expression and cell movement, Msgn1 (with Spt) plays a key role both in genesis of the paraxial mesoderm and in maintenance of the progenitor population from which it derives.

Fig. 1.

Msgn1 and Spt are essential for tail somite formation. (A-D,E-H) Live zebrafish embryos typical of their genotypic classes. (A′-D″′,E′-H″′′) In situ hybridisation for ntl and cb1045 (xirp2a), myoD (myod1), tbx24 and mespaa expression in uninjected wild-type (wt) siblings and in the genotypes indicated. spt^–/– mutants were derived from a heterozygous spt^+/– cross. msgn1^–/– mutants, spt^+/–;msgn1^–/– mutants and spt^–/–;msgn1^–/– double mutants were derived from a double heterozygous msgn1^+/–;spt^+/– cross. Indicated is the number of embryos (n) observed with the phenotype shown in each panel, and when embryos were derived from mutant crosses the obtained n corresponded to the expected frequencies for each genotype. Asterisk indicates that wt and msgn1 mutants have an undistinguishable phenotype at the 14-somite stage.

Fig. 2.

Msgn1 regulates the transition from the maturation zone to the PSM. (A) Diagram of sequential gene expression as a mesodermal cell progresses from a progenitor state in the dorsal superficial tailbud until incorporation into a somite. (B-B″) Double fluorescent in situ hybridisation for msgn1 (red) and ntl (green) in a wt zebrafish embryo. (C-J′) Expression of ntl (C-H′) and wnt8 (E-J′) in msgn1MO-injected embryos (D,D′,F,F′) and their siblings (C,C′,E,E′) and in embryos overexpressing msgn1 (H,H′,J,J′) and their siblings (G,G′,I,I′). At the 8- to 10-somite stage (H), 55% of msgn1-overexpressing embryos showed an absence of notochord ntl staining and 33% presented notochord breaks, but some expression of ntl was still evident in the tailbud. At the 20-somite stage (H′), 88% of these embryos showed an absence of ntl staining in the tailbud; this 88% consisted of 66% that showed no notochordal ntl expression and 22% that showed some notochord staining. In both H and H′, 12% of the injected embryos appeared wt. Seventy per cent of msgn1OE embryos showed continuing but strongly downregulated wnt8 staining at the 8- to 10-somite stage (J); the remaining 30% exhibited a total absence of wnt8 expression. By the 20-somite stage (J′), expression of wnt8 had disappeared completely. (K-L′) Confocal images of double fluorescent in situ hybridisation showing expression of ntl (green) and tbx6l (red) in a msgn1MO-injected embryo (L,L′) and an uninjected sibling (K,K′) at the 8- to 10-somite stage, in the superficial dorsal progenitor region (K,L) and at the level of the notochord, where ntl and tbx6l expression overlap (K′,L′). (M,N) Expression of tbx24 in a msgn1 mRNA-injected embryo (N) and its sibling control (M) at the 8- to 10-somite stage.

Fig. 3.

A heat shock-driven pulse of Msgn1 inhibits expression of progenitor-specific genes and drives progression through the PSM differentiation program. Batches of zebrafish embryos comprising heat shocked hsp70:HA-msgn1 transgenics along with wt sibling controls were analysed; ∼50% of each batch showed a clear phenotype and were presumed to be the transgenics. (A-D′) Expression of ntl, wnt8, wnt3a and fgf8 after 1 hour of recovery post-heat shock (hpHS) in hsp70:HA-msgn1 embryos (A′-D′) and their wt siblings (A-D) heat shocked for 1 hour at the 13-somite stage. In the transgenics (32/59 embryos), tailbud expression of ntl was severely reduced (29/59 embryos) or totally eliminated (3/59 embryos). Tailbud expression of wnt8 in the transgenics (35/65 embryos) was also strongly reduced, but reduction in expression levels of wnt3a and fgf8 at this early time after heat shock was milder, making transgenics often hard to distinguish from sibling controls. (E,E′) tbx24 expression at 2 hpHS. In the hsp70:HA-msgn1 transgenics (29/76 embryos), tbx24 expression is intensified in the PSM and abnormally extended into the region of formed somites, but is still absent from the tailbud. (F,F′) fgf8 expression at 2 hpHS. In the hsp70:HA-msgn1 transgenics (10/23 embryos), fgf8 is downregulated but still detectable. (G-M′) Expression of ntl, wnt8, wnt3a, fgf8, tbx24 and tbx6l at 7 hpHS. In the hsp70:HA-msgn1 transgenics (G′, n=25/53; H′, n=46/92; I′, n=13/29; J′, n=32/66; K′, n=15/34; M′, n=10/22), the tailbud has now lost all expression of ntl, wnt3, wnt8 and fgf8 and instead expresses tbx24 and tbx6I. Asterisks, arrowhead and hash sign indicate ectopic expression of tbx24 in somites, tailbud and midline, respectively.

Fig. 4.

Transient inhibition of Wnt signalling by Dkk1 allows ectopic activation of tbx24 in the most posterior region of the PSM. Batches of zebrafish embryos comprising heat shocked hsp70:dkk1 transgenics along with wt sibling controls were analysed. tbx24 expression is shown at 4 hours of recovery after a 30-minute heat shock at the 13-somite stage, in lateral (left of each pair) and flatmount dorsal (right of each pair) views. Arrowhead indicates a posterior expansion into the tailbud of tbx24 expression; bracket highlights the reduction of the tailbud region.

Fig. 5.

Msgn1 controls cell movements in the neighbourhood of the tailbud. (A) An 8-somite stage Kaede-injected zebrafish embryo with a patch of tailbud cells freshly photoactivated (red), imaged in the plane of the notochord and adaxial cells [i.e. in the superficial layer of the maturation zone (MZ)]. (B-C″) Dispersion of the photoactivated tailbud cells imaged in this plane in controlMO (B-B″, Movie 1) and msgn1MO (C-C″, Movie 3) morphants. (E) An 8-somite stage embryo with photoactivated tailbud cells (red) imaged in a deeper layer, ∼20 μm ventral to the notochord and adaxial cells (i.e. in a deep layer of the MZ). (F-G″) Dispersion of the photoactivated tailbud cells imaged in this plane in controlMO (F-F″, Movie 2) and msgn1MO (G-G″, Movie 4) morphants. (D,H) z-position of individual Kaede photoactivated tailbud cells tracked over time in controlMO (D) and msgn1MO (H) morphants. Each colour represents an individual cell. Loss of msgn1 leads to failure of ventral diving and of the subsequent lateral dispersion. Scale bars: 50 μm.

Fig. 6.

Msgn1 regulates snail1a expression. (A-A″) Double fluorescent in situ hybridisation for msgn1 (red) and snail1a (green) in a 10-somite stage wt zebrafish embryo, as seen in confocal section. (B-E) Expression of snail1a is increased in msgn1MO morphants (D,E) compared with sibling controls (B,C). (F,G) Expression of snail1a is decreased in hsp70:HA-msgn1 embryos (G) compared with sibling controls (F) at 1 hpHS. (H) An 8-somite stage Kaede-injected embryo with freshly photoactivated cells (red) in the superficial layer of the MZ (region 1), in the posterior PSM (region 2), and in the anterior PSM (region 3). (I) z-position of individual cells photoactivated in region 1 in snail1aMO morphants, tracked over time. Each colour represents an individual cell. Compare with Fig. 5D,H. (J) Mean ventral diving velocities of cells marked by photoactivation in region 1, comparing wt control, msgn1MO and snail1aMO morphants. Mean ± s.d., ^***P<0.0005 versus wt controls (t-test). (K-P′) Still images showing the tracks of posterior and anterior PSM photoactivated cells in wt control (K-L′), msgn1MO-injected (M-N′) and snail1aMO-injected (O-P′) embryos. The boxed regions in K-P are magnified in K′-P′. Scale bar: 50 μm.

Fig. 7.

Loss of Msgn1 affects somite number and size. Somite numbers and somite size in wt zebrafish embryos (blue and light blue), msgn1^–/– single mutants, spt^+/–;msgn1^–/– double mutants and msgn1MO-injected embryos (colours as indicated in E-I), fixed at 36 hours postfertilisation and measured after staining by in situ hybridisation with cb1045 to show somite boundaries. (A) Distribution of the total number of somites in the different genotypes. The peak of the distribution is at 32 somites for wt, 33 somites for msgn1MO, 33 somites for msgn1^–/–, and 34/35 somites for spt^+/–;msgn1^–/–. (B) The mean (±s.d.) number of somites for each genotype. ^*P<0.01, ^**P<0.005, ^***P<0.0005, versus wt; t-test. (C) Size of somites as a function of position along the A/P body axis in msgn1MO morphants and wt siblings. (D) The mean (±s.d.) somite size in msgn1MO morphants and wt siblings. (E-I) Representative stained embryos of each genotype. The red dot in each case marks the thirtieth somite; black dots mark somites posterior to this.

Fig. 8.

Model of Msgn1 function. Progenitor cells in the tailbud are maintained by a positive-feedback loop between Wnt, ntl and Fgf genes. Wnt, Ntl and Fgf (or some combination of these factors) also activate the expression of msgn1, with a particular probability per progenitor cell per unit time (those that are fated to become PSM), driving cells out of the tailbud into the PSM. The presence of Msgn1 and Spt (Tbx16) in these emigrant cells stimulates their directed movement and also switches off expression of the Wnt/ntl/Fgf gene loop, allowing them to progress along the PSM differentiation pathway and activate tbx24. Msgn1 also represses snail1a to exert some or all of its effects on cell movement. The msgn1/spt-Wnt/ntl/Fgf negative-feedback loop might furthermore contribute to the homeostasis of the tailbud progenitor population: for example, an enlargement of the population of progenitor cells will tend to raise tailbud levels of Wnt and Fgf, which will increase the proportion of cells that switch on msgn1/spt and emigrate, bringing population size down again. PSM, presomitic mesoderm.