Premature Vertebral Mineralization in hmx1-Mutant Zebrafish

H6 family homeobox 1 (HMX1) regulates multiple aspects of craniofacial development, and mutations in HMX1 are linked to an ocular defect termed oculoauricular syndrome of Schorderet–Munier–Franceschetti (OAS) (MIM #612109). Recently, additional altered orofacial features have been reported, including short mandibular rami, asymmetry of the jaws, and altered premaxilla. We found that in two mutant zebrafish lines termed hmx1mut10 and hmx1mut150, precocious mineralization of the proximal vertebrae occurred. Zebrafish hmx1mut10 and hmx1mut150 report mutations in the SD1 and HD domains, which are essential for dimerization and activity of hmx1. In hmx1mut10, the bone morphogenetic protein (BMP) antagonists chordin and noggin1 were downregulated, while bmp2b and bmp4 were highly expressed and specifically localized to the dorsal region prior to the initiation of the osteogenic process. The osteogenic promoters runx2b and spp1 were also upregulated. Supplementation with DMH1—an inhibitor of the BMP signaling pathway—at the specific stage in which bmp2b and bmp4 are highly expressed resulted in reduced vertebral mineralization, resembling the wildtype mineralization progress of the axial skeleton. These results point to a possible role of hmx1 as part of a complex gene network that inhibits bmp2b and bmp4 in the dorsal region, thus regulating early axial skeleton development.

In zebrafish, hmx1-deficient embryos exhibit increased apoptosis in the eyes and brain, in addition to a delayed withdrawal of retinal progenitors from the cell cycle [4]. The "Dmbo" mouse model showed malformations of the squamous temporal bone and hyperplasia of the gonial bone, and failed to develop somatosensory neurons in the geniculate ganglion [5,6].
The majority of the craniofacial bones are formed via direct ossification, while the cranial base, mammal limbs, and axial skeleton structures are formed via endochondral ossification-a process whereby a cartilage template is replaced by bone [7,8].
The dual process of ossification is conserved in teleost fish, including zebrafish. As early as 3 days post-fertilization (dpf) the cranial cartilage starts forming, followed by perichondral bone elements building up on the existing cartilage structures [9][10][11]. Within the same time window, the first bone elements-including the opercle, cleithrum, mandible, and maxilla-form via direct ossification [12,13]. Teleosts' vertebral column is formed through direct mineralization of the notochord, and occurs progressively in a craniocaudal direction [14]. BMP gradient is essential for dorsoventral patterning. and is highly modulated through different mechanisms, including extramembranous regulation. Chordin, follistatin, and noggin negatively regulate BMP signaling by binding BMP ligands and preventing their interaction with transducer receptors localized on the cell membranes of target cells [15][16][17]. BMP expression, following dorsoventral axis formation and regulation, is required for the osteogenic process to proceed [18].
In zebrafish, BMP signaling is crucial between 48 and 72 h post-fertilization (hpf) for head cartilage development and bone mineralization [19].
Disruption of BMP signaling is associated with several human diseases [27], and deletion of BMP2 and BMP4 or BMP2 alone results in a severe chondrodysplastic phenotype [28], while craniofacial defects were reported in conditional knockout mice lacking the BMP type I receptor Alk2 [29]. In zebrafish, a mutation in bmp1a resulted in mature bone with a higher mineral content [30].
Despite the recent reports on the phenotypic defects associated with HMX1 mutation, not much is known about its role in transcriptional regulation. HMX1 binds preferentially to the consensus sequence 5 -CAAGTG-3 located in the promoter region of target genes, and acts as a transcriptional regulator [31]. We previously designed a predictive promoter model [32] and generated two zebrafish models-hmx1 mut10 and hmx1 mut150 mutant linesto screen and identify potential hmx1 target genes [33]. The hmx1 mut10 and hmx1 mut150 mutant lines carry mutations in the SD1 and homeodomain domains, which are implicated in the dimerization and activity of hmx1 [34]. Embryos collected from both mutant zebrafish lines presented eye-related defects in a similar manner to the phenotype observed in human patients, and at present, while there a growing number of articles focus on HMX1-related ocular defects, not much is known about its role during bone formation. With the present work, we aimed at investigating the contribution of hmx1 during skeletogenesis in zebrafish.

Zebrafish and Mouse Maintenance and Breeding
All animal procedures were carried out in accordance with the policies established by the ARVO (Association for Research in Vision and Ophthalmology) Statement for the Use of Animals, and followed FELASA (Federation of European Laboratory Animal Science Associations) recommendations on the use of zebrafish [35]. Experiments were approved by the Veterinary Service of the State of Valais (Switzerland). Zebrafish (Danio rerio) were maintained in a 14 h/10 h light/dark cycle; embryos were kept at 28.5 • C in E3 medium [35]. All embryos at desired stages were kept in Danieau's solution with 0.003% 1-phenyl-2thiourea (Sigma, Buchs, Switzerland) to suppress pigmentation. The zebrafish mutant lines hmx1 mut10 and hmx1 mut150 were previously generated and described [33]. Briefly, hmx1 mut10 zebrafish have a frameshift mutation that generates a termination codon in the SD1 domain, while mutant hmx1 mut150 has an indel mutation that replaces the HD domain; dimerization of SD1 and HD is necessary for the proper function of hmx1.

RNA Extraction, cDNA Synthesis, and RT-PCR
Wildtype and hmx1-knockout embryos at different stages up to 5 dpf were collected and euthanatized. Thirty embryos were obtained from different breeding groups and pooled together. All experiments were repeated three times.
Total RNA was extracted from samples using the RNeasy Micro Kit (Qiagen; Hombrechtikon, Switzerland). Primers specific to the selected genes were designed (Supplementary Table S1), and first-strand cDNA synthesis using the AffinityScript™ Multiple Temperature Reverse Transcriptase Kit (Agilent; Basel, Switzerland) was performed according to the manufacturer's protocol. cDNA was generated (GoScript Reverse Tran-Cells 2022, 11, 1088 3 of 12 scriptase System; Promega; Dübendorf, Switzerland), and real-time RT-PCR (FastStart SYBR Green Master Roche; Rotkreuz, Switzerland) was performed following standard protocols. Gene expression changes were determined using the 2 -∆∆Ct method; relative values were normalized with β-actin.

Statistical Analysis
Averages of the different experiments were expressed as the mean ± SEM.; Student's t-test was used to express the significance of differences between two groups. Significance was set at 0.05.

Alcian Blue and Alizarin Red Staining
Zebrafish embryos at different embryonic stages were euthanatized, rinsed with PBS, and fixed with 4% paraformaldehyde overnight. Specimens were preserved in 100% methanol.

Whole-Mount in Situ Hybridization
Whole-mount staining was performed as previously described [34]. Sense and antisense probes for bmp2b, bmp4, and runx2b were synthetized by transcription of cDNA clones with T7 and SP6 RNA polymerase, using digoxigenin labeling mix. Zebrafish embryos were fixed at 2 dpf in 4% paraformaldehyde. The hybridization reaction was carried out at 68 • C for 14-18 h. Incubation and washing were performed using the BioLane HTI system (Hölle & Hüttner, Tubingen, Germany).

DMH1 Treatment
For BMP signaling inhibition, 10 mM DMH1 stock solution (Sigma, CAS. No: 1206711-16-1) was diluted in DMSO. Embryos at 2 dpf were placed in a 6-well plate with the inhibitor diluted in E3 medium at 50 µM or 100 µM overnight; 0.1% DMSO in E3 was used as a control. DMH1 solution was replaced with E3 medium, and at the desired stage the embryos were collected and fixed. All experiments were performed on 30 individuals per zebrafish group and repeated at least 3 times.

Hmx1 mut10 Zebrafish Present Premature Vertebrae Mineralization
Human patients carrying HMX1 mutations presented defects related to bone development [3]; therefore, we asked whether the mutations generated in hmx1 would affect cranial cartilage and bone development. We performed alcian blue and alizarin red staining on wildtype and hmx1 mut10 embryos. At 5 dpf in hmx1 mut10 , all cranial cartilaginous elements were present and unaffected in their development. Meckel's cartilage, ceratohyal, and branchial arches were formed and fully developed in both wildtype and hmx1 mut10 embryos (Figure 1a,b). Head skeleton formation was initiated subsequently to cartilage formation starting at 3 dpf, and at 5 dpf cranial bones were generally fully formed. Structures formed via intramembranous ossification-such as the cleithrum, anterior notochord, and operculum-were formed and visible at 5 dpf in wildtype and hmx1 mut10 embryos. A morphological inspection showed that there were no differences in bone size, shape, or localization (Figure 1c,d).
formation starting at 3 dpf, and at 5 dpf cranial bones were generally fully formed. Structures formed via intramembranous ossification-such as the cleithrum, anterior notochord, and operculum-were formed and visible at 5 dpf in wildtype and hmx1 mut10 embryos. A morphological inspection showed that there were no differences in bone size, shape, or localization (Figure 1c,d).
In wildtype zebrafish, following the formation of the anterior notochord, mineralization of the vertebrae progresses towards the caudal region, with 70% of the embryos presenting one mineralized vertebra, while the remaining assessed embryos either presented two alizarin-red-stained vertebrae or none at 7 dpf (Figure 1e,f). At the same embryonic stage, hmx1 mut10 zebrafish presented a precocious mineralization, with several additional vertebrae positive for alizarin red; at 8 dpf, the differential mineralization pattern was still maintained, with approximately 80% of embryos presenting five mineralized vertebrae (Figure 1g,h). with alcian blue in wildtype and hmx1 mut10 embryos (a,b) at 5 dpf; no morphological differences in the developing cranial cartilage structures were detected. Cranial bone structures stained with alizarin red in wildtype and hmx1 mut150 at 5 dpf (c,d); wildtype and hmx1 mut10 present regular development of bone structures. At 7 dpf and 8 dpf, alizarin red staining detected an early and progressive mineralization of the vertebrae in hmx1 mut10 (f,h) in comparison to wildtype zebrafish (e,g). cb, ceratobranchial pairs; ch, ceratohyal; mk, Meckel's cartilage; v, vertebrae; c, cleithrum; n, notochord; cb, ceratobranchial 5; en, entopterygoid; o, opercle; d, dentary. Black arrowhead: early mineralization of the vertebrae in hmx1 mut10 (f,h). Bar, (a-c) 250 µm; (e-g) 500 µm.
In wildtype zebrafish, following the formation of the anterior notochord, mineralization of the vertebrae progresses towards the caudal region, with 70% of the embryos presenting one mineralized vertebra, while the remaining assessed embryos either presented two alizarin-red-stained vertebrae or none at 7 dpf (Figure 1e,f). At the same embryonic stage, hmx1 mut10 zebrafish presented a precocious mineralization, with several additional vertebrae positive for alizarin red; at 8 dpf, the differential mineralization pattern was still maintained, with approximately 80% of embryos presenting five mineralized vertebrae (Figure 1g,h).

Hmx1 mut150 Zebrafish Recapitulate Premature Vertebral Mineralization Similarly to Hmx1 mut10 Mutants
Hmx1 presents three conserved regions: the SD1, SD2, and HD domains. Hmx1 mut150 zebrafish carry an indel mutation that replaces the HD domain. Dimerization of HD and SD1 is necessary for hmx1 activity. Alizarin red staining at 5 dpf of hmx1 mut150 embryos showed that at 5 dpf both cartilage (Figure 2a formed. At 7 dpf, hmx1 mut150 -similarly to hmx1 mut10 -presented an early mineralization pattern of the first cranial vertebrae (Figure 2e,f).

Hmx1 mut150 Zebrafish Recapitulate Premature Vertebral Mineralization Similarly to Hmx1 mut10 Mutants
Hmx1 presents three conserved regions: the SD1, SD2, and HD domains. Hmx1 mut150 zebrafish carry an indel mutation that replaces the HD domain. Dimerization of HD and SD1 is necessary for hmx1 activity. Alizarin red staining at 5 dpf of hmx1 mut150 embryos showed that at 5 dpf both cartilage (Figure 2a,b) and bone (Figure 2c,d) structures were formed. At 7 dpf, hmx1 mut150 -similarly to hmx1 mut10 -presented an early mineralization pattern of the first cranial vertebrae (Figure 2e,f).

Osteogenic Signaling during Early Bone Development in Wildtype and Hmx1 mut10 Zebrafish
We aimed at describing the time course of early osteogenesis by profiling bone-related genes. We focused on genes encoding for BMP antagonists and BMPs themselves, in addition to a master transcription factor and a gene involved in osteoblast function and mineralization.
Total RNA was isolated from wildtype and mutant zebrafish embryos collected from 1 dpf to 5 dpf, and RT-PCR analysis was performed. During early development, the antagonists chordin and noggin are expressed in the dorsal region and, thus, restrict bmp activity in the dorsal region [16,36]. RT-PCR analysis of chordin indicated that the antagonist expression did not alter from 1 dpf to 3 dpf in wildtype embryos, while in hmx1 mut10 , chordin expression was greatly reduced at 2 dpf ( Figure 3A). Noggin1 expression, on the other hand, was significantly reduced at 1 dpf and 3 dpf in hmx1 mut10 in comparison to wildtype embryos ( Figure 3B).
The osteogenic differentiation process is initiated upon expression of BMP factorsnamely, bmp2 and bmp4-followed by the specific early marker of osteoprogenitor cells runx2b. Starting at 1 dpf, and continuously until 3 dpf, bmp2b and bmp4 transcripts were significantly greater in hmx1 mut10 compared to wildtype zebrafish; at 5 dpf, bmp2b and bmp4 transcript quantities were stabilized at the same expression level in both wildtype and hmx1 mut10 zebrafish ( Figure 3C,D). In regard to the expression of runx2b, the transcription factor was differentially expressed, and presented a higher RNA level at 2 dpf; spp1 expression followed the expression of runx2b, and it was significantly higher at 3 dpf in Figure 2. Precocious vertebral bone development in hmx1 mut150 zebrafish embryos: Cartilage structures stained with alcian blue in wildtype and hmx1 mut150 embryos (a,b) at 5 dpf; the developing cranial cartilage structures develop properly in hmx1-mutant embryos. Cranial bone structures stained with alizarin red in wildtype and hmx1 mut150 at 5 and 7 dpf (c,f). Wildtype and hmx1 mut150 cranial bones developed regularly at 5 dpf (c,d), while at 7 dpf early mineralization of the vertebrae was detected in hmx1 mut150 , similarly to hmx1 mut10 embryos (e,f). Bar, (a-c) 250 µm; (e-f) 500 µm.

Osteogenic Signaling during Early Bone Development in Wildtype and Hmx1 mut10 Zebrafish
We aimed at describing the time course of early osteogenesis by profiling bone-related genes. We focused on genes encoding for BMP antagonists and BMPs themselves, in addition to a master transcription factor and a gene involved in osteoblast function and mineralization.
Total RNA was isolated from wildtype and mutant zebrafish embryos collected from 1 dpf to 5 dpf, and RT-PCR analysis was performed. During early development, the antagonists chordin and noggin are expressed in the dorsal region and, thus, restrict bmp activity in the dorsal region [16,36]. RT-PCR analysis of chordin indicated that the antagonist expression did not alter from 1 dpf to 3 dpf in wildtype embryos, while in hmx1 mut10 , chordin expression was greatly reduced at 2 dpf ( Figure 3A). Noggin1 expression, on the other hand, was significantly reduced at 1 dpf and 3 dpf in hmx1 mut10 in comparison to wildtype embryos ( Figure 3B).
The osteogenic differentiation process is initiated upon expression of BMP factorsnamely, bmp2 and bmp4-followed by the specific early marker of osteoprogenitor cells runx2b. Starting at 1 dpf, and continuously until 3 dpf, bmp2b and bmp4 transcripts were significantly greater in hmx1 mut10 compared to wildtype zebrafish; at 5 dpf, bmp2b and bmp4 transcript quantities were stabilized at the same expression level in both wildtype and hmx1 mut10 zebrafish ( Figure 3C,D). In regard to the expression of runx2b, the transcription factor was differentially expressed, and presented a higher RNA level at 2 dpf; spp1 expression followed the expression of runx2b, and it was significantly higher at 3 dpf in hmx1 mut10 (Figure 3E,F). Finally, runx2b and spp1 expression patterns detected in mutant zebrafish resembled the pattern observed in wildtype embryos.  In hmx1 mut10 embryos, chordin expression is reduced at 2 dpf (A), while noggin1 expression is lower at 1 dpf and 3 dpf (B). Bmp2b and bmp4 transcripts are significantly different in hmx1 mut10 embryos when compared to wildtype zebrafish from 1 dpf to 3 dpf (C,D). The osteogenic markers runx2b and spp1 are differentially expressed, and are higher in hmx1 mut10 at 2 dpf and 3 dpf, respectively (E,F). Data are expressed as mean of three or more experiments.

Bmp2b and bmp4 Are Expressed in the Dorsal Region in Hmx1 mut10 Embryos
Given the increased expression of skeletal-associated genes during early development, we performed whole-mount in situ hybridization to track the localization of bmp2b, bmp4, and the osteogenic transcription factor runx2b (Supplementary Figure S1). In wildtype embryos, bmp2b and bmp4 expression is inhibited by the antagonists noggin and chordin [37,38] in the dorsal region during early development. We found that prior to the initiation of the osteogenic process, at 2 dpf, there was no signal detected for bmp2b and Figure 3. Hmx1 mut10 embryos present enhanced expression of osteogenic factors: RT-PCR analysis of the BMP antagonist chordin and noggin1 relative to β-actin, the osteogenic inducers bmp2b and bmp4, and the osteogenic markers runx2b and spp1. In hmx1 mut10 embryos, chordin expression is reduced at 2 dpf (A), while noggin1 expression is lower at 1 dpf and 3 dpf (B). Bmp2b and bmp4 transcripts are significantly different in hmx1 mut10 embryos when compared to wildtype zebrafish from 1 dpf to 3 dpf (C,D). The osteogenic markers runx2b and spp1 are differentially expressed, and are higher in hmx1 mut10 at 2 dpf and 3 dpf, respectively (E,F). Data are expressed as mean of three or more experiments.

Bmp2b and bmp4 Are Expressed in the Dorsal Region in Hmx1 mut10 Embryos
Given the increased expression of skeletal-associated genes during early development, we performed whole-mount in situ hybridization to track the localization of bmp2b, bmp4, and the osteogenic transcription factor runx2b (Supplementary Figure S1). In wildtype embryos, bmp2b and bmp4 expression is inhibited by the antagonists noggin and chordin [37,38] in the dorsal region during early development. We found that prior to the initiation of the osteogenic process, at 2 dpf, there was no signal detected for bmp2b and bmp4 in the wildtype dorsal region (Figure 4a,c). Inspection of hmx1 mut10 evidenced strong and localized signals for bmp2b and bmp4 in the dorsal region in the correspondence of the hind-midbrain, along with an increased signal in specific bone-forming domains of the cranial frontal re-gion (Figure 4b,d). Bmp2 induces runx2b expression; therefore, we analyzed the expression pattern of runx2b in zebrafish embryos at the same stage. In wildtype embryos, as expected, runx2b at 2 dpf was expressed in the opercle, ceratohyal, Meckel's cartilage, ceratobranchial cartilage, and cleithrum ( Figure 4e); hmx1 mut10 presented a spread and stronger signal for runx2b in the dorsal region at the same stage. In situ hybridization for runx2b presented a pattern identical to bmp2b and bmp4 expression (Figure 4f). bmp4 in the wildtype dorsal region (Figure 4a,c). Inspection of hmx1 mut10 evidenced strong and localized signals for bmp2b and bmp4 in the dorsal region in the correspondence of the hind-midbrain, along with an increased signal in specific bone-forming domains of the cranial frontal region (Figure 4b,d). Bmp2 induces runx2b expression; therefore, we analyzed the expression pattern of runx2b in zebrafish embryos at the same stage. In wildtype embryos, as expected, runx2b at 2 dpf was expressed in the opercle, ceratohyal, Meckel's cartilage, ceratobranchial cartilage, and cleithrum ( Figure 4e); hmx1 mut10 presented a spread and stronger signal for runx2b in the dorsal region at the same stage. In situ hybridization for runx2b presented a pattern identical to bmp2b and bmp4 expression ( Figure  4f). In situ hybridization detected bmp2b and bmp4 the dorsal region in Hmx1 mut10 embryos: In situ hybridization in wildtype and hmx1 mut10 embryos at 2 dpf. Staining for bmp2b, bmp4, and runx2b. No signal was detected for bmp2b and bmp4 in the dorsal region of wildtype zebrafish, while bmp4 was detected in the heart region ((A,C) red arrows). In hmx1 mut10 , bmp2b and bmp4 are expressed in the dorsal region (B,D). Runx2b in wildtype embryos at 2 dpf is expressed in the bone-forming regions (E). In hmx1 mut10 embryos, runx2b is expressed in the dorsal region, and presents a similar pattern to bmp2b and bmp4 (F). Cl, cleithrum; ch, ceratohyal; cb, ceratobranchial; h, heart; op, opercle. Bar, A-F 100 μm.

Inhibition of bmp Signaling at 2 dpf Reduced the Progression of Vertebral Mineralization in Hmx1 mut10 Embryos
Mutant embryos presenting precocious mineralization of the vertebrae showed localized BMP signaling in the dorsal region during early embryonic development. To confirm the involvement of bmp2 and bmp4 factors in the developing vertebrae in mutant embryos, we investigated the effects of DMH1-an inhibitor of BMP-specific signaling [39]. Overnight treatment with 50 μM or 100 μM concentrations of DMH1 was performed at 2 dpf. Wildtype and hmx1 mut10 were collected at 7 and 8 dpf; alizarin red staining was performed to detect cranial and vertebral mineralization. Wildtype embryos treated with 100 mM DMH1 were viable, and showed reduced mineralization in the cranial region (Supplementary Figure S2A, a,c; S2B, a-d), while hmx1 mut10 reported aberrant development, including cardiac edema, and resulting in their failure to grow (Supplementary Figure S2A, b-d). Alizarin red staining of wildtype embryos treated with 50 μM DMH1 at 8 dpf In situ hybridization in wildtype and hmx1 mut10 embryos at 2 dpf. Staining for bmp2b, bmp4, and runx2b. No signal was detected for bmp2b and bmp4 in the dorsal region of wildtype zebrafish, while bmp4 was detected in the heart region ((A,C) red arrows). In hmx1 mut10 , bmp2b and bmp4 are expressed in the dorsal region (B,D). Runx2b in wildtype embryos at 2 dpf is expressed in the bone-forming regions (E). In hmx1 mut10 embryos, runx2b is expressed in the dorsal region, and presents a similar pattern to bmp2b and bmp4 (F). Cl, cleithrum; ch, ceratohyal; cb, ceratobranchial; h, heart; op, opercle. Bar, A-F 100 µm.

Inhibition of bmp Signaling at 2 dpf Reduced the Progression of Vertebral Mineralization in Hmx1 mut10 Embryos
Mutant embryos presenting precocious mineralization of the vertebrae showed localized BMP signaling in the dorsal region during early embryonic development. To confirm the involvement of bmp2 and bmp4 factors in the developing vertebrae in mutant embryos, we investigated the effects of DMH1-an inhibitor of BMP-specific signaling [39]. Overnight treatment with 50 µM or 100 µM concentrations of DMH1 was performed at 2 dpf. Wildtype and hmx1 mut10 were collected at 7 and 8 dpf; alizarin red staining was performed to detect cranial and vertebral mineralization. Wildtype embryos treated with 100 mM DMH1 were viable, and showed reduced mineralization in the cranial region (Supplementary Figure S2A(a,c),B(a-d)), while hmx1 mut10 reported aberrant development, including cardiac edema, and resulting in their failure to grow (Supplementary Figure S2A(b-d)). Alizarin red staining of wildtype embryos treated with 50 µM DMH1 at 8 dpf showed that the treatment did not lead to reduced cranial mineralization, and that vertebral bone development continued to progress ( Figure 5B). Cranial bone development in hmx1 mut10 treated with 50 µM DHM1 was unaffected, while the precocious vertebral mineralization was inhibited, resulting in a mineralization pattern similar to that of wildtype embryos ( Figure 5B). Wildtype and hmx1 mut10 embryos treated with DMSO did not show altered development or aberrant bone development.
showed that the treatment did not lead to reduced cranial mineralization, and that vertebral bone development continued to progress ( Figure 5B). Cranial bone development in hmx1 mut10 treated with 50 μM DHM1 was unaffected, while the precocious vertebral mineralization was inhibited, resulting in a mineralization pattern similar to that of wildtype embryos ( Figure 5B). Wildtype and hmx1 mut10 embryos treated with DMSO did not show altered development or aberrant bone development. Figure 5. DMH1-mediated inhibition of BMP signaling reduces the progression of vertebral mineralization: Wildtype and hmx1 mut10 embryos were treated with 50 μM DMH1 at 2 dpf and placed in E3 medium at 3 dpf. Alizarin red staining to detect mineralized structures was performed at 7 and 8 dpf (left, lateral view, right ventral view). Wildtype and hmx1 mut10 control embryos treated with DMSO (A). Precocious vertebral mineralization was inhibited in DMH1-treated hmx1 mut10 embryos at 7 dpf (B). At 8 dpf, treated hmx1 mut10 embryos still presented a vertebral mineralization pattern similar to wildtype embryos (B). Bar, (A,B) 500 μm.

Discussion
The HMX gene family is involved in the development of the sensory organs, and mutations in HMX1 result in a set of defects affecting the development of the retina, along with other congenital eye-related defects [1,40]. Patients carrying mutations of HMX1 presented an additional subset of defective maxillomandibular structures and spina bifida [3]. This indicates a possible additional role for HMX1 in the regulation of several developmental processes specific to craniofacial and axial skeleton formation.
We have previously generated two mutated zebrafish lines for hmx1; hmx1 mut10 and hmx1 mut150 carry mutations affecting the SD1 and homeodomain domains-both essential for an active dimeric hmx1 protein [34]. Inspection of hmx1 mut10 at 5 dpf indicated that both maturation of cartilaginous structures and mineralization of the cranial bones were unaffected (Figure 1a-d). In wildtype embryos, vertebral development initiated in a timely manner, while mineralization of the axial skeleton in hmx1 mut10 at the same stages was accelerated, with several vertebrae presenting early mineralization at 7 and 8 dpf (Figure  1e-h). Hmx1 mut10 and hmx1 mut150 zebrafish lines both showed early mineralization of the

Discussion
The HMX gene family is involved in the development of the sensory organs, and mutations in HMX1 result in a set of defects affecting the development of the retina, along with other congenital eye-related defects [1,40]. Patients carrying mutations of HMX1 presented an additional subset of defective maxillomandibular structures and spina bifida [3]. This indicates a possible additional role for HMX1 in the regulation of several developmental processes specific to craniofacial and axial skeleton formation.
We have previously generated two mutated zebrafish lines for hmx1; hmx1 mut10 and hmx1 mut150 carry mutations affecting the SD1 and homeodomain domains-both essential for an active dimeric hmx1 protein [34]. Inspection of hmx1 mut10 at 5 dpf indicated that both maturation of cartilaginous structures and mineralization of the cranial bones were unaffected (Figure 1a-d). In wildtype embryos, vertebral development initiated in a timely manner, while mineralization of the axial skeleton in hmx1 mut10 at the same stages was accelerated, with several vertebrae presenting early mineralization at 7 and 8 dpf (Figure 1e-h). Hmx1 mut10 and hmx1 mut150 zebrafish lines both showed early mineralization of the vertebrae, indicating that mutations affecting either the SD1 or homeodomain domains result in the same altered osteogenic phenotype.
Noggin [41] and chordin activity is required for proper axial skeleton development [16,42]. Whole-embryo RT-PCR analysis showed reduced expression of chordin at 1 dpf and 3 dpf, and of noggin at 2 dpf, in hmx1 mut10 embryos ( Figure 3A-B). Short-term expression of BMP-2 is necessary to induce bone formation [43] by upregulating the expression of the osteogenic markers runxb2 and spp1 [44], and loss of both BMP-2 and BMP-4 results in severe impairment of osteogenesis [45]. Bmp2b and bmp4 were highly expressed in hmx1 mut10 embryos ( Figure 3C-D), followed by an increased sequential expression of runxb2 and spp1 at 2 dpf and 3 dpf when compared to wildtype embryos ( Figure 3E-F).
In situ hybridization showed a strong and broad signal for bmp2b and bmp4 in the dorsal region in hmx1 mut10 embryos. Runx2b presents a dorsal pattern similar to those of bmp2b and bmp4, whereas in wildtype embryos at 2 dpf it was specifically localized to the ventral osteogenic sites (Figure 3).
Treatment with DMH1-a highly selective BMP signaling inhibitor widely used in zebrafish models-limited vertebral mineralization in hmx1 mut10 zebrafish, with no effect on cranial bone development in either hmx1 mut10 or wildtype zebrafish (Figure 4), confirming that early mineralization of the vertebrae occurred in response to the increased bmp2b and bmp4 transcripts [46][47][48]. Noggin and DMH1 have been shown to have a similar effect in regulating the expression of a set of genes involved in neurogenesis [49].
The question of whether hmx1 directly regulates bmp2b and bmp4 could be assessed by employing the heat-shock zebrafish model that we utilized to validate potential hmx1 target genes in vivo [34]; however, we do not believe that this is the case. By accessing the Eukaryotic Promoter Database (https://epd.epfl.ch; accessed 12 December 2020), and using the predictive promoter model, we screened bmp2b and bmp4, as well the noggin1 and chordin regions upstream of the transcription starting site. We did not identify CAAGTG binding sites in bmp2b and bmp4, but several clusters of CAAG binding sites common to humans, mice, and zebrafish were located on the potential promoter region of noggin1 and chordin, (Supplementary Figure S3). It appears more plausible that hmx1 induces bmp2b and bmp4 expression through an indirect mechanism.
Previously, we identified uhrf1 as an hmx1 target gene, and it has been implicated in the regulation of several developmental and homeostatic processes related to zebrafish development. In situ hybridization in hmx1 transgenic and mutant zebrafish showed that uhrf1 expression is modulated in the hindbrain, eye region, and branchial arches [33]. Potentially, uhrf1 could change the methylation pattern of bmp2b and bmp4 and, therefore, induce gene transcription. Reductions in noggin1 and chordin expression could follow as part of a feedback mechanism. In this view, we could analyze the methylome patterns of bmp2b and bmp4 in the dorsal region at critical stages for axial skeletogenesis.
Another potential indirect mechanism could be linked to the dorsoventral polarization process; it is well known that bmp2b and bmp4 are expressed following a gradient pattern across the dorsoventral axis. Noggin1 and chordin are expressed dorsally and, therefore, inhibit BMP expression. Given that potential noggin1 and chordin promoter regions present several hmx1 binding sites, and that we observed a reduced expression in hmx1 mut10 zebrafish, we could assume that in wildtype zebrafish hmx1 contributes to regulating noggin1 and chordin expression, consequently modulating bmp2b and bmp4 expression in the dorsal region. In the mutated hmx1 background, noggin1 and chordin expression is reduced, thus inducing precocious bmp2b, bmp4, and runx2b expression and initiating axial bone formation.
Our work indicates that hmx1 could be a modulator contributing to the maintenance of the BMP gradient in the dorsal region during axial bone development. In vitro studies of hmx1 activity on the chordin and noggin1 promoter regions, coupled with hmx1 heat-shock experiments, could elucidate the contribution of hmx1 during axial development.