Latent TGF-beta binding protein-1 plays an important role in craniofacial development

Abstract Objective: This study aims to replicate the phenotype of Ltbp1 knockout mice in zebrafish, and to address the function of LTBP1 in craniofacial development. Methods: Whole mount in situ hybridization (WISH) of ltbp1 was performed at critical periods of zebrafish craniofacial development to explore the spatial-temporal expression pattern. Furthermore, we generated morpholino based knockdown model of ltbp1 to study the craniofacial phenotype. Results: WISH of ltbp1 was mainly detected in the mandibular jaw region, brain trunk, and internal organs such as pancreas and gallbladder. And ltbp1 colocalized with both sox9a and ckma in mandibular region. Morpholino based knockdown of ltbp1 results in severe jaw malformation. Alcian blue staining revealed severe deformity of Meckel's cartilage along with the absence of ceratobranchial. Three-dimension measurements of ltbp1 morphants jaws showed decrease in both mandible length and width and increase in open mouth distance. Expression of cartilage marker sox9a and muscle marker ckma was decreased in ltbp1 morphants. Conclusions: Our experiments found that ltbp1 was expressed in zebrafish mandibular jaw cartilages and the surrounding muscles. The ltbp1 knockdown zebrafish exhibited phenotypes consistent with Ltbp1 knockout mice. And loss of ltbp1 function lead to significant mandibular jaw defects and affect both jaw cartilages and surrounding muscles.


Introduction
The latent transforming growth factor-β binding protein-1 (LTBP-1) is an extracellular matrix protein that is structurally similar to the fibrillin family. There are several human and mouse Ltbp1 splice variants including a short (Ltbp-1S) and long (Ltbp-1L) form, which may arise from the two separated promoters, alternative RNA splicing, and differential proteolytic processing. 1 LTBP1 is widely expressed among species in variable amounts in different tissues, including heart, lung, liver, placenta, skeletal muscle, and bone. 2,3 Current researches suggest that LTBP1 is a multifunctional protein that bind latent transforming growth factor-β (TGF-β) and regulate its function in bone and other connective tissues. [4][5][6][7][8][9][10] LTBP1 is reported to ease the secretion of latent TGF-β and modulate the activation of latent  It is considered as a stable component of extracellular fibrillar structure that play a critical role in the storage and release of TGF-β as a large latent TGF-β complex. [17][18][19] In addition to regulating the function of TGF-β, LTBP1 function as a structural component of connective tissue microfibrils. 20 Studies focusing on its function as matrix proteins find that LTBP1 colocalize with fibrillin 1 in microfibrillar structures not only in the extracellular matrix of primary osteoblasts but also at the surface of newly forming osteoid and bone, suggesting that LTBP1 play an important role in the formation of bone and connective tissue. 8 The important function of LTBP1 has been investigated by a series of studies. Targeted deletion of exon 1 or 2 led to Ltbp-1L-null mice dying shortly after birth from defects in heart organogenesis. 21 These severe cardiac deformities include persistent truncus arteriosus and interrupted aortic arch, which are associated with imperfect function of cardiac neural crest cells. Similarly, targeted deletion of exon 8 to generate Ltbp1L knockout mice died perinatally, these mice also present heart defects including persistent truncus arteriosus and interrupted aortic arch which are similar to Ltbp1L-null mice. 22 These studies indicate the crucial role of LTBP1 in the development of the cardiovascular system. Another Ltbp1 knockout mice is manipulated by targeted deletion of exon 5, which is the first exon shared by the Ltbp1L and Ltbp1S. 23 These total knockout mice are viable and fertile, they exhibit craniofacial abnormalities consisting of more compact head structure with shorten maxilla and mandible. Furthermore, the total Ltbp1 knockout mice have shortened long bones. Moreover, LTBP1 knockout mice are less prone to hepatic fibrogenesis. 23 These phenomena suggest that loss of LTBP1 function lead to potential changes of the biological activity of TGF-β in fibrogenesis action. Researchers also used the deletion of Ltbp1L exon 5 knockout model to address the function of Ltbp1 in female fertility. 24 Since Ltbp1 knockout female mice showed impaired reproduction with subfertility and ovarian cyst formation, the interruption of TGF-β function which would lead to defective follicular wound healing was suggested as the potential cause.
It is known that LTBP1 is critically required for   Table 2.
Zebrafish embryos at the 1-cell stage were injected with 1 nL of ltbp1-MO at a concentration ranging from 0.1 to 1 mM and a control-MO was used as a negative

Whole mount in situ hybridization
Probes for ltbp1, sox9a, and ckma were cloned by polymerase chain reaction with primers presented in Table 3, whole mount in situ hybridization (WISH) was performed as described. 29 The stained embryos were photographed with a Nikon SMZ1500 stereomicroscope.

Alcian blue staining
Embryos were fixed at 5 DPF, and cartilages were visualized by staining with alcian blue (SigmaAldrich; as described by Kimmel, et al. 30 (1998) without using the 1.67% trypsin to digest the tissue.

Statistical analysis
Statistical analysis was performed using GraphPad     Data are expressed as mean ± standard deviation (SD). Comparison of different groups was performed by unpaired t-test to estimate data measurements.
P-value less than 0.05 (P<0.05) was considered statistically significant.

ltbp1 expresses at mandibular arch skeleton
Since previous studies indicated that LTBP1 is a crucial gene in the early embryo development and it is widely expressed in several systems, we suspected that LTBP1 was expressed in craniofacial tissues. However, the expression pattern of LTBP1 in craniofacial tissues remains unclear. Therefore, we decided to explore the expression pattern of LTBP1 zebrafish ortholog in craniofacial tissues.
According to WISH of ltbp1 on zebrafish embryos, maternal transcripts were detected during the early developmental stage from 1 cell ( Figure 1A) to shield ( Figure 1C). At pharyngula period (1 DPF), ltbp1 expressed at pharyngeal arches, brain, the most part of trunk, and the origin of internal organs ( Figure 1D).
In the developmental stage of protruding mouth (3 DPF), ltbp1 was mainly detected in the mandibular jaw region and brain, but reduced expression also existed in the trunk and internal organs such as pancreas and gallbladder ( Figure 1E, F). We performed 2-color WISH of ltbp1 with cartilage marker sox9a and muscle marker ckma, respectively, to further explore the expression pattern of ltbp1 in the mandibular jaw region. sox9a is a marker of cranial neural crest that differs from cartilage, and sox9a participates in both determination of crest-derived chondrogenic lineages and morphogenesis of cartilage (2-3 DPF) 31 .
ckma is a terminal differentiation marker for skeletal muscle, 32 and ckma was expressed in muscles of the trunk, pectoral fin, head, and heart. 33 According to the observation of the tissue section of zebrafish jaw, ltbp1 colocalized with both sox9a (Figure 1G-N) and ckma ( Figure 1J-L). The results indicated that ltbp1 was expressed in craniofacial tissues, and it was mainly concentrated in the zebrafish jaw cartilages and its surrounding muscles.

Loss of ltbp1 function Results in Severe Jaw Malformation
Based on the fact that ltbp1 is expressed in the zebrafish mandibular jaw region, we speculated

Gene disruption experiments have shown that LTBP1
play roles in several fields, including the development and formation of cardiovascular system, craniofacial system, reproduction system as well as functions in both bone and connective tissue. [21][22][23][24] In this study, we explored the expression pattern of ltbp1 using zebrafish. The results of ltbp1 WISH showed detectable maternal transcripts from 1 cell to shield stage, which indicated that ltbp1 is necessary during the early embryo development. In the pharyngula period (1 DPF), ltbp1 was extensively expressed in the pharyngeal arches, brain, most part of trunk and the origin of some internal organs. In the developmental stage of protruding mouth (3 DPF), ltbp1 expression was mainly in the mandibular jaw region and brain, while reduced expression was also present in the trunk and internal organs such as pancreas and gallbladder.
The ltbp1 WISH results are according with previously reported widespread tissue expression. Since ltbp1 is strongly expressed in the mandibular jaw region at 3 DPF, and the zebrafish jaw region mainly consists of cartilages and muscles. We carried out 2-colour WISH of ltbp1 with cartilage marker sox9a and muscle marker ckma in order to specify the target tissue of ltbp1 expression. The results showed that ltbp1 colocalized with both sox9a and ckma, indicating that ltbp1 concentrates in the zebrafish jaw cartilages and its surrounding muscles.
Considering that the function of a gene is usually associated with its expression pattern, we hypothesize ltbp1 may have its function in the craniofacial development of zebrafish. According to previous studies, different strategies of Ltbp1 knockout mice presented several phenotypes, including heart defects, craniofacial deformities, and impaired reproduction. [21][22][23][24] However, the discrepancy of the variable phenotypes RNA rescue experiments for ltbp1 knockdown in zebrafish using ltbp1 mRNA. (A-C) Embryos were stained with alcian blue at 5 DPF to visualize craniofacial cartilage structures. All views are ventral. Jaw malformation induced by ltbp1 knockdown was significantly rescued by the co-injection of ltbp1 mRNA (D), including reduction in mouth opening distance (F), recover in mandible length (G) and width (H).   shortened maxilla and mandible. We also found that both penetrance and severity of ltbp1-morphants defects of jaw increases as the dosages of ltbp1 MO increases. Meanwhile, most phenotypes were rescued by co-injection of nonhomologous ltbp1 mRNA with ltbp1 MO, therefore the off-targeting effect of ltbp1 MO was ruled out, indicating the specificity of ltbp1 morphant phenotype. Since ltbp1 is expressed in both jaw cartilages and surrounding muscles, we speculated that the jaw deformities could derive from the defects of either or both the two tissues. In order to address possible major aspect, we checked both cartilage and muscle markers in ltbp1-morphants. Both expression of sox9a and ckma was significantly decreased in ltbp1-morphants compared to control-MO and ltbp1 rescued morphants, indicating that knockdown of ltbp1 affected jaw development in both jaw cartilage and muscle aspects.
Considering that some previous studies have found that latent TGF-β is present in the matrix of chondrocytes and that LTBP1 is responsible for storing TGF-β complex in the matrix. 6

Conclusion
Our experiments found that ltbp1 is expressed in zebrafish mandibular jaw cartilages and the surrounding muscles in addition to the previously reported tissues. Also, the ltbp1 knockdown zebrafish presented phenotypes consistent with Ltbp1 knockout mice. Lastly, loss of ltbp1 function lead to significant mandibular jaw defects and affect both jaw cartilages and surrounding muscles.

Conflict of interest
Nothing to declare.

Funding
Funding for this project is provided by the National Natural Science Foundation of China Latent TGF-beta binding protein-1 plays an important role in craniofacial development