Deveau VA et al. (2025), Differential requirement of Hand1 during differ...

XB-ART-61730

Dev Biol 2025 Sep 14;525:294-305. doi: 10.1016/j.ydbio.2025.06.005.

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Differential requirement of Hand1 during differentiation of lateral plate mesoderm lineages in Xenopus laevis.

Deveau VA, Willson JA, Carleton JL, Drysdale TA.

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The transcription factor hand1 is expressed in the heart, lateral plate mesoderm (LPM) and neural crest cells during development. Hand1-null mice die early in embryogenesis due to defects in the heart and placenta. This lethality has made our understanding of the range of functions for Hand1 in early development incomplete. In Xenopus, we observed that the hand1 expression pattern initially marked essentially all LPM derivatives and then correlated closely with a subset of specific LPM derivatives later in development. This led us to hypothesize that tight regulation of hand1 is required for normal development of LPM lineages. Using hand1 knockdown and overexpression models in Xenopus, development of LPM derivatives was compared. As in the mouse, we found that hand1 is required for proper heart morphogenesis. Hand1 is also required for the formation of a complex vascular plexus within embryos by maintaining early endothelial cell populations, however, hand1 over expression is not sufficient to induce endothelial cell differentiation. Furthermore, down regulation of hand1 is critical for the development of the hematopoietic lineages. These findings confirm a conserved role of hand1 in heart morphogenesis and suggest regulation of hand1 levels is critical for normal development of specific LPM lineages.

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Species referenced: Xenopus laevis
Genes referenced: aplnr cfd etv2 hand1 hbg1 isl1 nkx2-5 spib tal1 tnni3
GO keywords: blood vessel development [+]
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gRNAs referenced: hand1 gRNA1 hand1 gRNA2 hand1 gRNA3 hand1 gRNA4

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	Graphical Abstract
	Fig. 1. hand1 expression becomes rapidly downregulated in the ventral side of the embryo, similar to that of etv2 expression. Control embryos were fixed at stage 20, 23 and 25 and probed with dig-labelled antisense mRNA probes against hand1, etv2, spib, globin or ami. Both a side view and ventral view of each embryo is shown above. At stage 25, expression of hand1 is lost along the ventral side of the embryo. When the cleared region begins to form, it corresponds to expression of etv2 in that clear zone. The same phenomenon occurs with Spib expressing cells which are initially in a restricted domain on the ventral midline that tightly corresponds to an area on the ventral midline devoid of hand1 staining starting at stage 23. The Spib expressing cells then migrate throughout the embryo. Both expression of globin and ami are not yet present at these early stages.
	Fig. 2. hand1 expression is located in the same region as the developing vasculature, and not expressed upon differentiation of the ventral blood islands. Control embryos were fixed at stage 29, 32, 35 and 37 and probed with dig-labelled antisense mRNA probes against hand1, etv2, spib, globin or ami. Both a side view and ventral view of each embryo are shown above. Expression of hand1 is maintained throughout development and begins to appear again in the ventral region of the embryo around stage 35. etv2 expression begins to disappear as the vascular plexus (network of blue forming linear structures along the flank of the embryo) becomes more defined, and expression of ami first appears, marking the forming vascular plexus. Spib expression is maintained, with cells expressing spib now located throughout the embryo after they migrate away from their origin on the ventral midline seen in Fig. 1). Globin expression begins to appear at stage 29, and cells remain in the ventral blood islands on the ventral side of the embryo. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 3. Loss of hand1 disrupts heart morphogenesis. Schematic showcasing heart looping of a stage 35 embryo, where the linear heart tube has looped to the right. Embryos were fixed at stage 35 probed with dig-labelled antisense mRNA probes against tnni3 transcripts. Shown is a ventral view of the embryos. Control embryos display a similar rightwards heart looping (A–D) while hand1 mutants display disturbed heart looping, with an increase in cardiac edema (E–H). Phenotypes of the heart were classified into 3 groups; normal looping, when the heart was observed to loop to the right, moderate, which was characterized based on the extent of reduced looping, G being an example of moderate looping, where the heart has incomplete heart looping, and lastly, linear, where the heart had no indication of looping morphogenesis occurring (I).
	Fig. 4. Loss of hand1 disrupts vasculature formation. Embryos in both control and hand1 mutant groups were classified into one of four groups based on the complexity of the vascular plexus: normal, mild, moderate and severe, at stage 35 and 37 with either the probe aplnr or ami, with a representative image of each classification shown (A). Percent of embryos in each classification are shown for stage 35 (B) and stage 37 (C). At both stages, there was an increased frequency of mild, moderate and severe phenotypes in hand1 mutants as compared to control embryos.
	Fig. 5. Loss of hand1 decreases vasculature complexity and increases the size of the non-vascularized region. Embryos were fixed at stage 37 and probed with dig-labelled antisense mRNA probes against ami. Complexity was measured based on number of times the vasculature intersected along a line across the middle of the embryo (A). Hand1 mutants showed a significant decrease in number of line intersections as compared to the non-injected and water injected controls (B; one-way ANOVA, p > 0.001). Size of the non-vascularized region was measured as a ratio of x/y for all embryos (C). Embryos are oriented with anterior to the left, posterior to the right. In each case, in the hand1 mutants, the area of the non-vascularized region was increased (D; one-way ANOVA, p > 0.01) with different lowercase letters representing statistically significant differences.
	Fig. 6. Embryos overexpressing hand1 display an increase in hand1 mRNA throughout the embryo, and the mRNA is being translated into protein. Control embryos and hand1 over expressing embryos were fixed at stage 20 and probed with dig-labelled antisense mRNA probes against hand1 transcripts. A side view (A, D, G, J) ventral view (B, E, H, K) and dorsal view (C, F, I, L) are shown of each embryo. Control embryos show normal hand1 expression that appears as a saddle shape along the middle of the LPM (A–C). Embryos over expressing hand1 lose this expression pattern, and instead have hand1 expressed throughout the embryo (broad purple staining, D-F, J-L). Some embryos were also shown to have only increased expression in one half of the embryo, with one side of the embryo displaying the normal hand1 expression pattern (G), while the opposing side had a ubiquitous expression pattern, which is most evident in the ventral (H) and dorsal (I) photos. The one-sidedness suggests that hand1 mRNA was introduced later during the 1-cell stage, and only was present one of the two cells after the first division in those embryos, leading to only half of the embryo showing increased hand1 transcripts later on in development. Myc-tagged-Hand1 protein is correctly translated to yield the Hand1 protein as seen by the 40 kB band in OE embryos at stage 20 and stage 30, which is not detected in the control embryos (M). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 7. Overexpression of hand1 causes defects in heart morphogenesis. Control embryos and embryos injecting with varying amounts of hand1 mRNA (100 pg, 500 pg, 1 ng) were fixed at stage 35 and probed with dig-labelled antisense mRNA probes against tnni3 to visualize heart shape. Phenotypes of the heart were classified into 3 groups; normal looping, moderate, or linear. Percentage of each phenotype is displayed for each experimental group (A). Compared to the control embryos which showcase looping of the heart towards the right (B,C), embryos over expressing hand1 show a range of defects, from loss of looping to leftward looping, along with an increase in cardiac edema (D,E).
	Fig. 8. Overexpression of hand1 decreases the size of globin expression domain. Control embryos and embryo over expressing hand1 were fixed at stage 28 and probed with dig-labelled antisense mRNA probes against globin. Both a side view (A, B, E, F, I, J) and ventral view (C, D, G, H, K, L) of each embryo are shown above. There appeared to be a decrease in globin expression in hand1 over expressing embryos (I–L) as compared to control embryos (A–H). Area of globin expression (a’) was measured as a ratio compared to the area of the whole embryo (a), as illustrated in C. There was a significant decrease in the ratio between a’/a for embryos injected with 500 pg hand1 mRNA as compared to controls (M; one-way ANOVA, p > 0.001).
	Fig. 9. Hand1 overexpression decreases the size of the spib expression domain. Control embryos and embryo over expressing hand1 were fixed at stage 20 and probed with dig-labelled antisense mRNA probes against spib transcripts. Spib expressing cells appear to be more compact when hand1 is over expressed (E–F) as compared to control embryos (A–D), however over expression of hand1 does not appear to ablate spib expression. Area of spib expression (a’) was measured as a ratio compared to the area of the whole embryo (a), as illustrated in A. There was a significant decrease in the ratio between a’/a for both embryos injected with 500 pg hand1 mRNA as compared to controls (G; one-way ANOVA, p > 0.001).
	Figure S1. sgRNA targeting hand1 are effective at mutating Xenopus DNA. DNA was extracted from hand1 knockout embryos and a T7 assay was performed on either pools of five or single embryos. Compared to controls, embryos injected with sg1 resolve 3 bands on the gel indicating the DNA has been mutated, compared to the one band in WT controls (A,B). Chromatogram from Sanger sequencing results of the hand1.S sequence surrounding the target site from control (C) and a sg1 injected embryo (D) are shown. Associated PAM sites for each are lined in black. It is evident that the DNA has undergone mutations near the PAM site in the sg1 injected embryo (D). The program TIDE was used to get an estimation of the percent of in-frame mutations, out-of-frame mutations and DNA with no-editing for each sgRNA target from single embryos. Average mutation types from 3 embryos are reported for each hand1 sgRNA target (E-I).
	Figure S2. Loss of hand1 does not impact differentiation or size of the heart fields. Embryos were fixed at stage 18 and probed with dig-labelled antisense mRNA probes against nkx2-5 and isl1 transcripts. No differences in the primary heart field marker nkx2-5 was found between control (A,B) and hand1 mutant embryos (C). No differences in the secondary heart field marker, isl1, were found between control (D,E) and hand1 mutant embryos (F).
	Figure S3. Loss of hand1 decreases the number of endothelial cells in the LPM. Control embryos and hand1 mutants were fixed at stage 23, 25 and 28 and probed with dig-labelled antisense mRNA probes against etv2. Both side view (A,C,E,G,I,K), and ventral view (B,D,F,H,J,L) enlarged side view (M,N) of embryos are shown above. Control embryos show a large number of endothelial cells within the LPM at all stages (A, E, I, M), forming a fine boarder along the ventral side of the embryo (B,F,J). Hand1 mutants show both a decrease in number of endothelial cells (C,G,K,N), and loss of populated boarder surrounding the ventral blood islands (D,H,L).
	Figure S4. Loss of hand1 does not increase apoptosis within the LPM. Control embryos and hand1 mutants were fixed at stage 28/30 of development, and TUNEL assay was performed to determine the number of apoptotic cells. Embryos are oriented with anterior to the left, posterior to the right. Both control (A-D) and hand1 mutant (E-H) embryos show a normal pattern of apoptosis (small punctate blue spots) for their developmental stages. Although CRISPR injected embryos appear to have an increase number of positive nuclei as compared to controls at stage 28, there is no positive nuclei within the hand1 positive region of the LPM.
	Figure S5. Loss of hand1 does not affect hematopoietic differentiation. Control embryos and hand1 mutants were fixed at stage 20 and stage 34 and probed with dig-labelled antisense mRNA probes against spib and globin transcripts to assess hematopoietic differentiation. No differences in differentiation of lineages expressing spib or globin were observed between the control embryos (A,B,D,E,G,H,J,K) and hand1 mutants (C,F,I,L).
	Figure S6. Overexpression of hand1 does not affect differentiation of the heart field Control embryos and embryos over expressing hand1 were fixed at stage 20 and probed with dig-labelled antisense mRNA probes against nkx2-5. There appears to be no difference in the expression of nkx2-5 between control embryos (A-C) and hand1 over expressing embryos (D-F).
	Figure S7. Overexpression of hand1 has no apparent effect on vascular plexus formation. Embryos were fixed at stage 28 and stage 37 and probed with dig-labelled antisense mRNA probes against etv2 and ami transcripts. Over expression of hand1 (C,F,I,L) does not appear to affect vasculature formation as compared to control embryos (A,B,D,E,G,H,J,K
	aplnr ([gene_name]) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35 & 36, lateral view, anterior left, dorsal up.
	ami ([gene_name]) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35 & 36, lateral view, anterior left, dorsal up.