Normal embryonic development and neonatal digit regeneration in mice overexpressing a stem cell factor, Sall4.

Sall4 encodes a transcription factor and is known to participate in the pluripotency network of embryonic stem cells. Sall4 expression is known to be high in early stage post-implantation mouse embryos. During early post-gastrulation stages, Sall4 is highly expressed in the tail bud and distal limb buds, where progenitor cells are maintained in an undifferentiated status. The expression of Sall4 is rapidly downregulated during embryonic development. We previously demonstrated that Sall4 is required for limb and posterior axial skeleton development by conditional deletion of Sall4 in the T (Brachyury) lineage. To gain insight into Sall4 functions in embryonic development and postnatal digit regeneration, we genetically overexpressed Sall4 in the mesodermal lineage by the TCre transgene and a novel knockin allele of Rosa26-loxP-stop-loxP-Sall4. In significant contrast to severe defects by Sall4 loss of function reported in previous studies, overexpression of Sall4 resulted in normal morphology and pattern in embryos and neonates. The length of limb long bones showed subtle reduction in Sall4-overexpression mice. It is known that the digit tip of neonatal mice has level-specific regenerative ability after experimental amputation. We observed Sall4 expression in the digit tip by using a sensitive Sall4-LacZ knock-in reporter expression. Sall4 overexpression did not alter the regenerative ability of the terminal phalange that normally regenerates after amputation. Moreover, Sall4 overexpression did not confer regenerative ability to the second phalange that normally does not regenerate after amputation. These genetic experiments show that overexpression of Sall4 does not alter the development of the appendicular and axial skeleton, or neonatal digit regeneration. The results suggest that Sall4 acts as a permissive factor rather than playing an instructive role.

Introduction

Sall4 encodes a zinc finger transcription factor [1, 2], and mutations in human SALL4 cause Duane-radial ray syndrome, an autosomal dominant disorder [3, 4]. Patients with this syndrome exhibit various defects, including upper limb deformities, aberrant ocular movements, renal agenesis, unilateral deafness, choanal atresia, external ear malformations, and ventricular septal defects of varying degrees [3, 5]. Sall4+/- mice partially recapitulate human patients’ symptoms, such as anorectal and heart anomalies [6-8]. These reports demonstrate that Sall4/SALL4 is necessary for the development and function of a variety of tissues and organs.

Sall4 expression has been investigated in pluripotent stem cells and mouse embryos. Sall4 expression is detected in early cleavage stages [8-10]. In blastocysts, Sall4 is expressed in the inner cell mass and trophectoderm [6, 9], and contributes to the proliferation of cells in the inner cell mass of blastocysts [6]. In pluripotent mouse embryonic stem (ES) cells, SALL4 binds to AT-rich sequences and represses activities of differentiation-promoting enhancers [11, 12]. After implantation, Sall4 is widely expressed until embryonic day E7.5 [13, 14]. After gastrulation, Sall4 is highly expressed in the posterior part of the body, such as the tail bud and in the presomitic mesoderm, where undifferentiated progenitor cells undergo proliferation and differentiation at E9.5 –E12.5 [13-15]. Sall4 is also expressed in the distal mesenchyme of developing limb buds, where cells are maintained undifferentiated by the signals from the apical ectodermal ridge [13, 15, 16]. In the postnatal stages, Sall4 is expressed in undifferentiated spermatogonia in the testis [17-19], and is essential for the maintenance of undifferentiated spermatogonia [20]. These reports collectively show that Sall4 expression is associated with undifferentiated cells and tissues.

The functions of Sall4 in mouse development have also been investigated. Sall4 null mouse embryos arrest at the peri-implantation stage, indicating its presence is required in the pluripotent epiblast [6, 9]. Conditional deletion of Sall4 in the meso-endoderm lineage and neuromesodermal progenitors using TCre (or Brachyury-Cre) caused tail truncation through early depletion of progenitor cells [21]. The TCre; Sall4 mutants also show defects of the proximal-anterior skeleton in the hindlimb and disorganized vertebrate, posterior to the lumber level [15, 21]. These studies suggest that Sall4 is required for maintenance of undifferentiated progenitor cells and their differentiation in mouse embryos.

In postnatal stages, Sall4 is involved in regeneration in non-mammalian animals. In the axolotl, Sall4 plays a critical role in scar-free cutaneous wound healing through the regulation of collagen gene transcription [22]. In the regenerating limbs of Xenopus tadpoles, Sall4 is detected in the blastema [23, 24]. It has been suggested that Sall4 maintains cells in an undifferentiated state in the blastema and regulates patterning of the regenerating limb at later stages [23, 25]. In the case of mice, it has been demonstrated that fetus, neonates, and adults possess the ability to regenerate digit tips [26, 27]. The regenerative ability is level specific and restricted to the terminal phalange [28, 29]. However, it remains unknown whether Sall4 is expressed in the mouse digit tip and if Sall4 plays a role in digit tip regeneration in mice.

In addition to expression and function in mouse development and regeneration, SALL4 expression is reported in many types of human cancers (reviewed in [30, 31]). SALL4 expression is normally absent in most adult tissues, but is activated in various human cancers. These cancers include leukemia, germ cell tumors, hepatocellular carcinoma, gastric cancer, colorectal carcinoma, esophageal squamous cell carcinoma, breast cancer, endometrial cancer, lung cancer, and glioma [30, 31]. Studies suggest that SALL4 functions in cancer survival, drug resistance, and metastasis. Furthermore, detailed literature review suggests that SALL4 expression increases both mortality and recurrence of cancer, and suggests SALL4 as an important prognostic marker [32].

Sall4 expression patterns and functional studies suggest that Sall4 is necessary in various tissues and organs during embryonic development, in adult germ cells, and contributes to pathological conditions such as cancer. However, the functions of Sall4 in tissue/organ development remain largely unknown in post-implantation mouse embryos due to the early lethality of Sall4 null mutants [6-9]. To gain insight into Sall4 functions, we genetically overexpressed Sall4 by recombination using the TCre transgene. In significant contrast to severe defects in reported Sall4 loss of function studies, our results show that Sall4 overexpression in the T-lineage results in the normal development of mouse embryos and no alteration of digit tip regenerative ability.

Materials and methods

Generation of the inducible Sall4 knock-in allele and mice breeding

The inducible Sall4 expression vector was generated as previously described [33] with replacement of Dmrt1 by the Flag-Sall4a-T2A-HA-Sall4b sequence. The expression cassette was knocked into the Rosa26 locus by homologous recombination in R1 mouse ES cells, as diagrammed in Fig 1. The correctly targeted ES cells were injected into C57BL/6 blastocysts after confirming the normal karyotype, and chimeras were generated. Germline transmission was confirmed by breeding chimeras with C57BL/6 mice and genomic PCR of offspring, and the mouse line was termed as R26-Sall4. The R26-Sall4 mice were bred with C57BL/6 mice at least 3 generations before experimentation.

Fig 1

Sall4 conditional expression transgene.

(A) Targeting vector used to insert the conditional expression construct for Sall4 into the Rosa26 locus in ES cells. (B) Schematic of the targeting strategy. The targeting vector also contains a diphtheria toxin negative selection cassette (PGK-DTA-bGH-PA) with diphtheria toxin expressed from the PGK promoter and followed by a bovine growth hormone poly-adenylation site. Homologous recombination into the Rosa26 locus removed PGK-DTA-bGH-PA and produced the single copy transgene CAG-Stop-Sall4-IRES-EGFP. Cre-mediated recombination deletes the “floxed” stop cassette to generate CAG-Sall4-IRES-EGFP. (C) Correct homologous recombination of the 5’ homology arm of the targeting vector into Rosa26 was confirmed by PCR of ES cell genomic DNA using primers F1 and R1 (indicated in panel B), which amplify between exon 1 of Rosa26 and the CAG promoter. No amplification product was detected in wild-type ES cells, but the predicted 1248 bp amplicon was detected in a targeted ES cell clone (#4). Control DNA was prepared from the tail clip of conditional Dmrt1 expression mouse that was generated using the same knockin system (33). (D) Homologous recombination of the 3’ vector arm was confirmed by Southern blotting using the 3’ probe indicated in panel B. Wild-type ES cell DNA generated in detection of the predicted 11 kb fragment, whereas targeted clones (two right lanes) generated both the 11 kb fragment and a 9 kb fragment corresponding to the targeted allele.

C57BL/6 and CD-1 mice were obtained from the Jackson Laboratory and Charles River, respectively. TCre mice were previously described [34] and backcrossed >12 generations with C57BL/6. For embryo experiments, R26-Sall4 mice were timed mated with TCre mice. For postnatal skeletal analysis and digit regeneration experiments, R26-Sall4 mice were bred with TCre mice. Experimental protocols were approved by the University of Minnesota Animal Care and Use Committee (protocol number: 1706-34894A, 1910-37482A).

ES cell screening and mouse genotyping

To screen targeted ES cells, a DNA fragment described in [33] was labelled using DIG DNA labeling mix (Roche, Cat#11277065910) and genomic Southern hybridization was performed following a standard procedure [35]. Genomic PCR of ES cell DNA and PCR genotyping on tail clip DNA for the R26-Sall4 mice were conducted as previously described [33].

Skeletal preparation and whole mount in situ hybridization

For alcian blue/alizarin red staining, neonates or the postnatal day (P) 21 autopod were fixed in 50% ethanol. Skeletal staining and whole mount in situ hybridization of embryos were performed as previously described [15, 21].

Bone length measurement and statistical analysis

The length of the humerus and femur was measured by using FIJI. Lines were drawn parallel to the long axis of each bone, then perpendicular to those lines such that the lines touched the edges of the bone. The length of the bone was measured by using the distance between the edges of the line parallel to the long axis. The measurement was performed by two independent individuals, and the average value was used for statistical examination, which was performed by One-way ANOVA using JMP software version 16.

Neonatal digit amputation

Neonatal (P0) mice were anesthetized on ice and the digits of the hindlimbs were amputated [36]. The regenerative amputations were performed using microdissection scissors (Fine Science Tools, Cat#15000–00) cross the distal third of the terminal phalange [27]. For non-regenerative amputation, the distal third of the second phalange was amputated.

Western blotting

For embryos, E10.5 embryos were dissected, and EGFP-positive (TCre; R26-Sall4) or negative (TCre; R26-Sall4) embryos were grouped. Embryos were cut at the anterior to the forelimb buds, and the body was used for protein extraction using RIPA buffer with a protease inhibitor cocktail (Thermo, Cat# 1862209). The extract was centrifuged at 14,000g for 10 min at 4°C, and the supernatant was used for Western blotting.

For neonatal digits, the entire digits were combined, protein was extracted similar to the E10.5 embryos, and the lysate was subjected to Western blotting.

Primary antibodies against SALL4 (Abcam, ab29122, 1:1000) and GAPDH (Thermo, AM4300, 1:2000) were used. HRP-labelled goat anti-rabbit IgG (BioRad, 170–6515, 1:3000) and anti-mouse IgG (BioRad, 170–6516, 1:3000) were used as secondary antibodies and the signals were detected by a Chemiluminescence system (Thermo, #34080). Signal intensities of bands were calculated by FIJI.

Results

Generation of a recombination-dependent Sall4 overexpression mouse line

In order to test whether continued expression of Sall4 interferes with normal embryonic development, we sought to express Sall4 by Cre-dependent genetic recombination. We knocked in a Sall4 cassette into the Rosa26 locus as previously described [33]. The Sall4 transgene expression is silenced by the stop sequence in front of the Sall4 transgene, which is flanked by the loxP sequences. By Cre-dependent deletion of the stop sequence, Sall4 is expressed under a strong CAG promoter (Fig 1A and 1B). There are two Sall4 isoforms, Sall4a and Sall4b, which are generated through an internal splicing in exon 2 [37]. A previous study in mouse ES cells provided evidence that SALL4A and SALL4B have overlapping but non-identical binding sites [37]. Because functional differences between SALL4A and SALL4B during mouse development are unknown, we expressed both Sall4a and Sall4b. We inserted the Sall4a-T2A peptide sequence-Sall4b in the expression cassette, followed by the internal ribosome entry sequence and the EGFP sequence. Cre-dependent recombination causes expression of both Sall4a and Sall4b, as well as an EGFP reporter. This expression cassette is knocked into the Rosa26 locus in the R1 mouse ES cells [35]. After electroporation, the ES cells were selected by G418 treatment. Among the 64 clones isolated after G418 selection, six properly targeted embryonic stem cell colonies were confirmed by genomic Southern blotting and genomic PCR (Fig 1C and 1D). After confirming normal karyotype, chimeric mice were generated by injecting one ES cell clone into blastocysts, which were bred with C57BL/6 mice. Germline transmission was confirmed by genomic PCR of offspring. This study was performed using this one strain.

Sall4 overexpression in the Brachyury lineage results in development of normal skeletal pattern

Sall4 expression is rapidly downregulated in post-gastrulating stages, and the high level of expression is confined in the distal limb buds and the tail buds [13, 14]. Sall4 deletion in the Brachyury lineage using TCre caused severe defects in the appendicular and posterior axial skeleton [15, 21]. In order to test whether continued expression of Sall4 affects those skeletons, we overexpressed Sall4 in the Brachyury lineage by crossing R26-Sall4 mice with TCre mice.

At E9.5, strong Sall4 expression is detected in the caudal part of the body and the distal part of the forelimb bud in control embryos (Fig 2A–2C, n = 3). In TCre; R26-Sall4 embryos, strong Sall4 expression was broadly detected in the trunk mesoderm, the entire forelimb buds and in the caudal part of the body (Fig 2D–2F, n = 3). The tissue with high levels of Sall4 expression correlates with the tissue with the EGFP signal that reports recombination and overexpression of the transgenes (Fig 2G and 2H, n = 3). Western blotting with an extract from the embryo (posterior to the anterior-edge of the forelimb bud) also showed an increased level of SALL4 in TCre; R26-Sall4, compared with TCre; R26-Sall4 embryos. Specifically, the data show that SALL4A overexpression is greater than SALL4B overexpression, while SALL4B expression is increased from baseline in the control. The ratio of SALL4A/GAPDH signal intensity was increased 1.7-fold in TCre; R26-Sall4 embryos, compared with TCre; R26-Sall4 embryos. Taken together, these results show that Sall4 is overexpressed by TCre-dependent recombination.

Fig 2

TCre-dependent overexpression of Sall4.

(A-C) Endogenous expression pattern of Sall4 at E9.5. A lateral view of a whole embryo (A), a dorsal view at the forelimb level (B) and a dorsal view of the caudal part of the embryo (C) are shown. (D-F) Sall4 expression in the TCre; R26-Sall4 embryo. A lateral view shows strong expression of Sall4 in the trunk (D). A dorsal view at the forelimb level shows strong expression in the somite and forelimb bud (E). A dorsal view of the caudal part of the embryo shows strong expression in this region (F). (G, H) Lateral views of the TCre; R26-Sall4 embryo. A bright field image (G) and an EGFP image (H) are shown. The EGFP signal is detected in the trunk, similar to strong Sall4 expression in D. (I) Western blotting of lysate from TCre; R26-Sall4 embryos (control) and TCre; R26-Sall4 embryos (overexp.). Stronger bands for SALL4A and SALL4B are detected with extracts from the TCre; R26-Sall4 embryo. Abbreviations. f: forelimb bud, h: head, n: neural tube, t: tail bud.

To assess whether continued Sall4 overexpression affects skeletal patterns, we stained the skeleton of neonates with alcian blue (cartilage) and alizarin red (bone). Our previous study showed that, in TCre; Sall4 cKO mutants, the lumber vertebrae were disorganized and the tail was truncated [21]. The hindlimb skeletal elements were severely small or absent, while the forelimb skeleton looked normal [15]. Compared to the defects in TCre; Sall4 cKO, we did not detect differences in skeletal development between the control (TCre; R26-Sall4) and the overexpression (TCre; R26-Sall4) neonates (Fig 3A–3H). In the Sall4-overexpression neonates, the lumber vertebrae exhibited normal morphology (Fig 3B and 3F), and both forelimbs and hindlimbs developed normally, compared to control neonates (Fig 3C, 3D, 3G and 3H). In order to evaluate whether Sall4 overexpression affected skeletal development in more detail, we measured the length of the humerus and femur in the allelic series: TCre; R26-Sall4, TCre; R26-Sall4, TCre; R26-Sall4, and TCre; R26-Sall4. Statistical examination by One-way ANOVA indicated that there was slight reduction of long bone length in Sall4-overexpression neonates (Fig 3I and 3J). The humerus and femur exhibited 8.8% and 4.7% shorter, respectively, of wild-type littermates (TCre; R26-Sall4). These results indicated that overexpression of Sall4 does not affect bone pattern and has a negligible effect on the long bone length. Given that the bone length between TCre; R26-Sall4 and TCre; R26-Sall4 did not show significant differences, the presence of the Sall4 cassette may mildly affect limb development.

Fig 3

Analysis of neonatal skeletal development after overexpression of Sall4 in the T lineage.

Skeleton of wild-type (A-D, TCre; R26-Sall4) and Sall4-overexpression (E-H, TCre; R26-Sall4) neonates. Lateral views of the entire body (A, E), dorsal views of the lumber region (B, F), forelimbs (C, G) and hindlimbs (D, H) are shown. Scale bar, 5 mm in A and E, 1 mm in B-D, F-H. (I, J) Box plot of the length of the humerus (I) and femur (J) of neonates with the indicated genotypes. WT and Tg indicate wild type and the +/Tg genotype, respectively. Sample numbers are indicated in the panel. Abbreviations. fe: femur, fi: fibula, h: humerus, pg: pelvic girdle, r: radius, sg: shoulder girdle, t: tibia, u: ulna.

Sall4 is expressed in the digit tip

Next, we sought to determine whether Sall4 is expressed in the digit tip, which regenerates after experimental amputation [27]. Because endogenous Sall4 expression is rapidly downregulated in developing embryos and becomes difficult to detect, we made use of a sensitive LacZ reporter, knocked into the Sall4 locus [6]. By whole mount LacZ staining, we observed strong Sall4-LacZ reporter expression in the tail tip and genital organs at E14.5 (blue arrowhead and red arrowhead, respectively, Fig 4A, n = 4), as previously reported [14]. We also observed signals at the digit tip (black arrows, Fig 4A). The staining at the digit tip was observed at E16.5 (Fig 4B, n = 3, Fig 4D, n = 3) and in neonates (Fig 4C, n = 3, Fig 4E, n = 3). These data show Sall4 expression in the digit tip during late gestation to neonatal stages.

Fig 4

Sall4 expression at the digit tip.

(A) Whole-mount LacZ stained Sall4 embryo at E14.5. Blue and red arrowheads point to signals at the tail tip and genital organ, respectively. Arrows point to signals at the digit tip. Scale bar, 1 mm. (B, C) Ventral views of the wild-type digit tip of the hindlimbs after lacZ staining at indicated stages. (D, E) Ventral views of the Sall4 digit tip of the hindlimbs after lacZ staining at indicated stages. Arrows point to the LacZ signals. Scale bar, 1 mm in A, 0.2 mm in B-E.

Sall4 overexpression does not alter the regenerative ability of the digit tip

The neonatal mouse digit regenerates after amputation at the terminal phalange level. In contrast, the digit fails to regenerate after amputation at the second phalange level [27]. We sought to investigate whether overexpression of Sall4 can alter regenerative ability of the digit tip. We first confirmed overexpression of SALL4 in the digit of TCre; R26-Sall4 neonates with Western blotting. Tissue lysate prepared from the entire digits of TCre; R26-Sall4 neonates did not show detectable SALL4. In contrast, digit lysate from TCre; R26-Sall4 neonates showed overexpressed SALL4A and SALL4B (Fig 5A). We amputated the digit tip at the terminal phalange of control (TCre; R26-Sall4, n = 7) and Sall4-overexpression (TCre; R26-Sall4, n = 9) neonates, and stained the digits with alcian blue/alizarin red at day 21. The regenerated digit tip skeleton was comparable between the two groups (Fig 5B and 5C). This result indicates that overexpression of Sall4 did not impair regenerative ability of the digit tip. Next, we amputated at the digit at the second phalange level at the neonate stage. In both control (n = 7) and Sall4-overexpression (n = 9) mice, alcian blue/alizarin red staining showed no regeneration of the amputated digits (Fig 5D and 5E). This result indicates that overexpression of Sall4 does not supply regenerative ability to non-regenerative digits. Overall, these results indicate that Sall4 overexpression does not change regenerative and non-regenerative ability of the neonatal mouse digit tip.

Fig 5

Sall4 overexpression does not alter the regenerative ability of the digit tip.

(A) Western blotting of lysate from TCre; R26-Sall4 and TCre; R26-Sall4 neonatal digits. Arrows point to SALL4A and SALL4B bands. (B, C) Alcian blue/Alizarin red-stained digits from 21 day-old control (TCre; R26-Sall4) and Sall4-overexpression (TCre; R26-Sall4) mice, amputated at the tip of the terminal phalange level at the P0 stage. (D, E) Alcian blue/Alizarin red-stained digits from 21 day-old control (TCre; R26-Sall4) and Sall4-overexpression (TCre; R26-Sall4) mice, amputated at the second phalange level at the neonatal stage. Scale bar: 500 μm in B-E. P2 and P3 in panels B-E indicate the second phalange and the terminal phalange, respectively.

Discussion

Several studies, including ours, investigated functions of Sall4 in animal development. Sall4-/- mouse embryos die at the peri-implantation stage, indicating that Sall4 is required for epiblast survival [6, 9]. Due to early lethality of Sall4-/- embryos, studies with mice were performed by conditional gene inactivation, which identified that Sall4 was required in the development of the neuromesodermal progenitors [21], limb progenitors [15, 38], primordial gem cells [39], oocytes [10], and spermatogonia [17]. In addition, the Sall4+/- allele or Sall4 gene-trap alleles were used to investigate genetic interaction between Sall4 and other genes. For example, Sall1+/-; Sall4+/- mouse embryos do not develop the kidney [6]. The Sall4+/gene-trap background enhances neural tube defects in Sall2-/- mice [40]. Furthermore, analysis of Tbx5+/-; Sall4+/gene-trap embryos demonstrated their interaction for forelimb and heart patterning [16]. In zebrafish and Xenopus systems, studies using morpholino-based knockdowns, and more recently, a CRISPR-Cas9-based genetic knockout approach in zebrafish were conducted [41]. These studies identified a sall4 requirement in pectoral fin development [42], hematopoiesis [43], and posterior neuroectoderm development [44, 45]. In chick embryos, a truncated form of Sall4, which might interfere with endogenous Sall4, was expressed by electroporation to the otic placode for loss-of-function study [46].

Sall4 overexpression approach was also conducted to gain insight into Sall4 function in animal development. For example, full length Sall4 overexpression by plasmid electroporation into the head ectoderm in chick embryos showed that Sall4 can induce invagination of the non-placodal head ectoderm [46]. In this system, a region of interest is targeted by plasmid injection and electroporation, and the expression is lost as cells divide and dilute the electroporated plasmid. In another approach, synthetic sall4 mRNA was injected into blastomeres at the early cleavage stages of Xenopus [45]. With this approach, one can overexpress sall4 in the entire embryos or target specific blastomeres to spatially restrict overexpression. Nonetheless, as cells divide and the injected mRNA is degraded, the overexpressed transcripts are lost over the course of development. In our study, we used Cre-loxP-dependent conditional overexpression of Sall4 in mouse embryos. A Sall4 expression cassette is knocked into the Rosa26 locus with the loxP-stop-loxP sequence, which is commonly performed for conditional overexpression in mice [47, 48]. This genetic recombination approach allows cells to inherit the recombined allele and continue to overexpress Sall4.

We chose to use TCre, which recombines broadly in meso-endoderm and neuromesodermal progenitors [21, 34]. Sall4 is broadly expressed in gastrulating embryos, and as discussed above, conditional deletion of Sall4 in various types of progenitor cells demonstrated that Sall4 is required in embryo development. Moreover, Sall4 expression is rapidly downregulated from the limb bud mesenchyme, somites, and other areas of the embryo [14]. However, Sall4 overexpression did not cause significant developmental defects, and the overall morphology of these embryos and neonates is comparable to wild-type embryos or neonates. The lack of defects was surprising to us, given the conditional loss of Sall4 causes various defects during embryonic development. However, a similar observation was also reported in Xenopus, in which Sall4 was overexpressed by injecting mRNA into animal-dorsal cells of 4-cell stage embryos [45]. Compared to experiments by the same group, where Sall4 loss-of-function by morpholino injection resulted in a change in expression patterns of several neural markers, Sall4 overexpression did not affect the expression pattern of marker genes in early stage Xenopus embryos. In our study, while the overall morphology was not changed by overexpression of Sall4 in the T-lineage, we observed a slight reduction of limb long bone length. The reduction was very subtle, although statistically significant. Based on the comparison of bone length, we speculate that harboring the Sall4 expression cassette, although its expression is silenced, might have a subtle effect on skeletal growth.

Studies in non-mammalian animals suggested that Sall4 is involved in limb regeneration. Sall4 is upregulated during limb regeneration in Xenopus [23, 24] and axolotls [25]. It has been hypothesized that Sall4 keeps cells in an undifferentiated state in the blastema during the early stages of blastema formation [23-25]. Our study showed that Sall4 is expressed in the digit tips of mouse fetuses and neonates. Mouse digit tips can regenerate after amputation at the terminal phalange level where the nail bed is associated [27]. The expression pattern is consistent with the postulated role of Sall4 in regeneration. However, our study showed that Sall4 overexpression does not affect digit tip regeneration. Overexpression of Sall4 did not interfere with the normal regenerative ability of the digit tip, when amputated at the terminal phalange. Moreover, Sall4 overexpression did not provide a regenerative ability, when amputated at the distal part of the second phalange level. One possible interpretation of our results is that Sall4 alone cannot provide limb regenerative ability. Sall4 may require highly regenerative cellular or tissue context to contribute to limb regeneration. Loss of function experiments in Xenopus or axolotls would provide insights into Sall4 function in limb regeneration.

A lack of evident phenotype after Sall4 overexpression suggests that SALL4 requires molecular partners in the experimental setting examined in this study. For example, SALL4 interacts with PLZF and DMRT1 in undifferentiated spermatogonial progenitor cells by coimmunoprecipitation experiments [17, 49]. Our recent study also suggests that SALL4 functions, at least in part, depend on molecular partners. More specifically, SALL4 ChIP-seq in mouse ES cells [12] and in mesoderm tissues from the posterior part of E9.5 embryos exhibited significant difference in SALL4-enriched sequences [21]. Moreover, transcription factor motifs in the SALL4-enriched sequences are also distinct between ES cells and the embryonic tissue. These reports suggest that, in addition to directly binding to DNA [11], SALL4 also acts in a protein complex. In the latter case, Sall4 function may require cell or tissue-specific interacting partners.

Overall, our study in developing embryos and the neonatal digit showed that overexpression of Sall4 does not alter the developmental and regenerative processes that we examined. These results suggest that Sall4 acts as a permissive factor rather than an instructive factor in these developmental and regeneration contexts.

Although we did not observe significant effects by overexpressing Sall4 in the Brachyury lineage, our Sall4 overexpression model may be used in different contexts to gain insights into Sall4 functions in the future. It is suggested that Sall4 blocks differentiation of mouse ES cells [11, 12]. It will be interesting to interrogate whether Sall4 overexpression can alter developmental processes that are not examined in this study, such as neural development. It will also be of interest to test whether Sall4 overexpression can alter regenerative processes that are not examined in this study, as well as cancer cell phenotypes.

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: Chen et al. inserted Sall4 into the Rosa26 locus and overexpressed Sall4 in mice via mesodermal Cre-dependent recombination. Although Sall4a and Sall4b were overexpressed in the caudal part of the embryo and the entire forelimb bud, minimal abnormalities were detected in early embryogenesis and digit regeneration. Although this manuscript describes negative results, the included information and the generated mouse strain are useful for the research community.

Major comments

1. The authors should describe the number of homologous recombinant ES clones and the number of independent mouse strains that were generated and analyzed. If this study involved only one ES clone and one mouse strain, those should be mentioned as limitations of the study; such information is necessary for objective interpretation by readers.

2. Figure 2: A description should be provided concerning the number of biologically independent experiments performed with reproducible results. This point applies to all figures in the manuscript.

3. Figure 2I: Because intensities differ among the loading controls (GAPDH bands), it is difficult to judge the quantitative increase in Sall4 protein expression. The ratio of Sall4 band intensity to GAPDH band intensity should be quantified, and the percentage increase should be provided. In addition, the results should be described in greater detail. For example, Sall4a overexpression is greater than Sall4b overexpression, while Sall4b expression is increased from baseline in the control.

4. Figure 3I, J: the definitions of the dots and double dots should be clarified; they also appear to be mislocated in the graphs. Because there are no significant differences between Cre-WT/Rosa-Tg and Cre-Tg/Rosa-Tg, the effect of Sall4 overexpression is negligible. Instead, the presence of Sall4 cassette itself may mildly affect limb development. Such interpretation should be provided in the Results section, not the Discussion section.

5. The Discussion section largely consists of repetitive descriptions of the results; it also contains many descriptions of loss-of-function studies that are already explained in the Introduction section. The authors should discuss the overexpression studies in greater detail, instead of simply mentioning that Sall4 is a “permissive” factor. Their data indicate that Sall4 overexpression alone is insufficient to evoke significant effects in their setting. They should discuss possible reasons based on the known molecular mechanisms of Sall4. They can compare their results with the previous reports with or without phenotypes; the findings may suggest technical differences or dependency on cellular context. The established mouse strain can also be used to address other biological questions. Such discussion will be informative for readers.

Reviewer #2: This manuscript reports the finding that over expression of both Sall4a and Sall4b do not affect the embryonic development and digit regeneration in the mouse model. The finding shows that, contrary to expectation, Sall4 over expression does not impart regenerative capacity to the limb, despite of its requirement for limb regeneration in amphibian animal models.

I reckon the work merits publication.

Your title emphasizes neonatal digit regeneration, but I am still very curious about whether you have performed P2 amputation experiment in adult stage. I understand that one would expect the non-regeneration of adult P2 phalanges when the neonatal P2 fail to regenerate with Sall4 over expression.

Another question is about Sall4a and Sall4b. You over-expressed both in your mouse models. Is there any difference between Sall4a versus Sall4b, in your expectation, to stimulate digit regeneration? The western results seem to suggest that expression of Sall4a is higher than Sall4b in the digit tip (with the same anti-Sall4). It was reported that Sall4b, but not Sall4a, "binds preferentially to highly expressed loci in ES cells" (doi: 10.1128/MCB.00419-10). You may want to discuss this.

Some suggestions:

1. Line 83, "The regenerative ability is level specific and restricted to the terminal

84 phalangeal bone", I suggest delete "bone".

2.Line 310, legend, "neonatal stage", I suggest indicating the time of digit amputation (P0). "neonatal stage" does not necessarily equal to day 0. For example, “The neonatal period is the first 4 weeks of a child's life".

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Reviewer #2: No

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30 Mar 2022

Responses to the reviewers

Thank you for the critical and helpful comments regarding our manuscript (PONE-D-22-00272) entitled “Normal embryonic development and neonatal digit regeneration in mice overexpressing a stem cell factor, Sall4.”

We have examined the comments carefully and revised our manuscript. We believe that the we have adequately addressed the Reviewers’ concerns and improved the revised manuscript. Below, we provide point-by-point responses.

RESPONSE TO REVIEWER 1

Reviewer #1: Chen et al. inserted Sall4 into the Rosa26 locus and overexpressed Sall4 in mice via mesodermal Cre-dependent recombination. Although Sall4a and Sall4b were overexpressed in the caudal part of the embryo and the entire forelimb bud, minimal abnormalities were detected in early embryogenesis and digit regeneration. Although this manuscript describes negative results, the included information and the generated mouse strain are useful for the research community.

We thank the reviewer for considering “the included information and the generated mouse strain are useful for the research community”. Below, we provide response to each comment.

Major comments

Comment 1. The authors should describe the number of homologous recombinant ES clones and the number of independent mouse strains that were generated and analyzed. If this study involved only one ES clone and one mouse strain, those should be mentioned as limitations of the study; such information is necessary for objective interpretation by readers.

Response: Thank you for pointing out important information that was missing in the original manuscript. We picked up 64 ESC clones after G418 selection, and six of them were properly targeted by genomics PCR and Southern blotting. Among them, we used one clone for injection into the blastocyst after confirming normal karyotype. We have included this information as below (modified/new information is underlined)

Line 185-196: Among the 64 clones isolated after G418 selection, six properly targeted embryonic stem cell colonies were confirmed by genomic Southern blotting and genomic PCR (Fig 1C, D). After confirming normal karyotype, chimeric mice were generated by injecting one ES cell clone into blastocysts, which were bred with C57BL/6 mice. Germline transmission was confirmed by genomic PCR of offspring. This study was performed using this one strain.

Comment 2. Figure 2: A description should be provided concerning the number of biologically independent experiments performed with reproducible results. This point applies to all figures in the manuscript.

Response: Thank you for pointing this out. The number of biologically independent experiments/samples, which were not described are as below.

Fig. 2A-C (n=3), 2D-F (n=3), 2G, H (n=3)

Fig. 4A (n=4), 4B (n=3), 4C (n=3), 4D (n=3), 4E (n=3)

We have included this information in the main text.

Comment 3. Figure 2I: Because intensities differ among the loading controls (GAPDH bands), it is difficult to judge the quantitative increase in Sall4 protein expression. The ratio of Sall4 band intensity to GAPDH band intensity should be quantified, and the percentage increase should be provided. In addition, the results should be described in greater detail. For example, Sall4a overexpression is greater than Sall4b overexpression, while Sall4b expression is increased from baseline in the control.

Response: We have measured the Western blot band intensity by FIJI. The result shows that the ratio of SALL4A/GAPDH signal intensity was increased 1.7-fold in TCre+/Tg; R26-Sall4+/tg embryos, compared to TCre+/+; R26-Sall4+/tg embryos. We have included this information in the text as below.

Regarding SALL4B, it does not show detectable signals in the control sample. So, following the reviewer’s comment, we have included the following description.

Line 239 – 243: Specifically, the data show that SALL4A overexpression is greater than SALL4B overexpression, while SALL4B expression is increased from baseline in the control. The ratio of SALL4A/GAPDH signal intensity was increased 1.7-fold in TCre+/Tg; R26-Sall4+/tg embryos, compared with TCre+/+; R26-Sall4+/tg embryos. Taken together, these results show that Sall4 is overexpressed by TCre-dependent recombination.

Comment 4. Figure 3I, J: the definitions of the dots and double dots should be clarified; they also appear to be mislocated in the graphs. Because there are no significant differences between Cre-WT/Rosa-Tg and Cre-Tg/Rosa-Tg, the effect of Sall4 overexpression is negligible. Instead, the presence of Sall4 cassette itself may mildly affect limb development. Such interpretation should be provided in the Results section, not the Discussion section.

Response: Thank you for pointing this out. The dots were introduced to indicate statistical significance. However, we indicated the p values in the panel, so we removed the dots from Fig 3I and J..

Regarding the interpretation of bone lengths, as suggested by the reviewer, we have included the following description.

Line 281 – 283: Given that the bone length between TCre+/+; R26-Sall4+/Tg and TCre+/Tg; R26-Sall4+/Tg did not show significant differences, the effect of Sall4 overexpression is negligible and the presence of the Sall4 cassette may mildly affect limb development.

Comment 5. The Discussion section largely consists of repetitive descriptions of the results; it also contains many descriptions of loss-of-function studies that are already explained in the Introduction section. The authors should discuss the overexpression studies in greater detail, instead of simply mentioning that Sall4 is a “permissive” factor. Their data indicate that Sall4 overexpression alone is insufficient to evoke significant effects in their setting. They should discuss possible reasons based on the known molecular mechanisms of Sall4. They can compare their results with the previous reports with or without phenotypes; the findings may suggest technical differences or dependency on cellular context. The established mouse strain can also be used to address other biological questions. Such discussion will be informative for readers.

Response: Thank you for this suggestion. We have extended our discussion as below.

First, we have discussed that Sall4 may require highly regenerative cellular or tissue context to contribute to limb regeneration (line 418-421).

Second, we have discussed the possibility that SALL4 requires molecular partner to evoke significant effects in the tissue we examined (line 423 -433).

Third, we have discussed other biological questions that the Sall4 overexpression mouse strain may be useful (line 439 - 445).

We hope that the reviewer acknowledges our new discussion.

RESPONSE TO REVIEWER 2

Reviewer #2: This manuscript reports the finding that over expression of both Sall4a and Sall4b do not affect the embryonic development and digit regeneration in the mouse model. The finding shows that, contrary to expectation, Sall4 over expression does not impart regenerative capacity to the limb, despite of its requirement for limb regeneration in amphibian animal models.

I reckon the work merits publication.

We thank the reviewer for his/her words “I reckon the work merits publication”. Below, we provide response to each comment.

Comment 1: Your title emphasizes neonatal digit regeneration, but I am still very curious about whether you have performed P2 amputation experiment in adult stage. I understand that one would expect the non-regeneration of adult P2 phalanges when the neonatal P2 fail to regenerate with Sall4 over expression.

Response: Thank you for this comment. As we showed in the manuscript, we did not observe regeneration after P2 amputation at the P0 stage. We think it is highly unlikely that the digit regenerates after P2 amputation in the adult stage, and therefore, we did not perform P2 amputation at the adult stage.

Comment 2: Another question is about Sall4a and Sall4b. You over-expressed both in your mouse models. Is there any difference between Sall4a versus Sall4b, in your expectation, to stimulate digit regeneration? The western results seem to suggest that expression of Sall4a is higher than Sall4b in the digit tip (with the same anti-Sall4). It was reported that Sall4b, but not Sall4a, "binds preferentially to highly expressed loci in ES cells" (doi: 10.1128/MCB.00419-10). You may want to discuss this.

Response: As the reviewer indicated, a previous study by the Orkin lab provided evidence that SALL4A and SALL4B have overlapping but non-identical binding sites in the mouse ES cell. However, we do not know functional differences between SALL4A and SALL4B in the Brachyury lineage during embryonic development and neonatal digit regeneration. Therefore, we simultaneously overexpressed both Sall4a and Sall4b in our study.

We have clarified this point in the Result as below (line: 177-180).

A previous study in mouse ES cells provided evidence that SALL4A and SALL4B have overlapping but non-identical binding sites (37). Because functional differences between SALL4A and SALL4B during mouse development are unknown, we expressed both Sall4a and Sall4b.

Some suggestions:

Comment 3 (Suggestion 1). Line 83, "The regenerative ability is level specific and restricted to the terminal phalangeal bone", I suggest delete "bone".

Response: Thank you for the suggestion. We have deleted “bone” from the sentence (new line 86).

Comment 4 (Suggestion 2) .Line 310, legend, "neonatal stage", I suggest indicating the time of digit amputation (P0). "neonatal stage" does not necessarily equal to day 0. For example, “The neonatal period is the first 4 weeks of a child's life".

Response: Thank you for the suggestion. We have indicated the amputation was performed at the P0 stage in line 344 in the revised manuscript.

Submitted filename: Responses to Reviewers.docx

Click here for additional data file.

6 Apr 2022

Normal embryonic development and neonatal digit regeneration in mice overexpressing a stem cell factor, Sall4

PONE-D-22-00272R1

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Reviewers' comments:

8 Apr 2022

PONE-D-22-00272R1

Normal embryonic development and neonatal digit regeneration in mice overexpressing a stem cell factor, Sall4

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