Functional analysis of SMOC2 using developmental models

<!-- /* Font Definitions */@font-face {font-family:"&#65325;&#65331; &#26126;&#26397;"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:1 134676480 16 0 131072 0;}@font-face {font-family:"&#65325;&#65331; &#26126;&#26397;"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:1 134676480 16 0 131072 0;}@font-face {font-family:Cambria; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1073743103 0 0 415 0;} /* Style Definitions */p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-fareast-font-family:"&#65325;&#65331; &#26126;&#26397;"; mso-bidi-font-family:"Times New Roman";}a:link, span.MsoHyperlink {mso-style-priority:99; mso-style-parent:""; color:blue; text-decoration:underline; text-underline:single;}a:visited, span.MsoHyperlinkFollowed {mso-style-noshow:yes; mso-style-priority:99; color:purple; mso-themecolor:followedhyperlink; text-decoration:underline; text-underline:single;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-size:10.0pt; mso-ansi-font-size:10.0pt; mso-bidi-font-size:10.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-fareast-font-family:"&#65325;&#65331; &#26126;&#26397;"; mso-hansi-font-family:Cambria;}@page WordSection1 {size:612.0pt 792.0pt; margin:72.0pt 90.0pt 72.0pt 90.0pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;}div.WordSection1 {page:WordSection1;}--><p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%">Musculoskeletal conditions are the leading cause of morbidityand disability, resulting in enormous healthcare costs and working disability.About 150 diseases, syndromes and disorders are grouped in the musculoskeletalconditions, each being progressive and associated with pain (http://www.who.int). One of them is OA, a chronicand progressive joint disorder that is caused by degradation of the articularcartilage and associated defects of the subchondral bone. Although manyresearch groups have investigated the onset and progression of this disablingcondition, the underlying cause or mechanism is far from completely understood.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%">Therefore, our lab analyzed a protein extract from bovinearticular cartilage with in vivochondrogenic activity and isolated the SMOC2 protein. In addition, Smoc2 was shown to be expressed in thejoint interzone of mouse embryonic digits. Recent evidence indicated thatsignaling pathways, which are known to regulate the embryonic stages ofdevelopment, are reactivated during the onset and progression of multipleconditions, including musculoskeletal diseases. Therefore, we useddevelopmental models to analyze the function of SMOC2.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"> <p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%">To investigate the SMOC2 associated signaling pathway(s), weused the zebrafish model system and performed loss and gain of functionexperiments. <p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%">Our LOF analysis indicated that Smoc2 affected myeloiddevelopment by reducing the number of macrophages and neutrophils during theprimitive wave of zebrafish hematopoiesis. This defect could be traced back tothe reduced expression of the myeloid progenitor specific gene spi1b at earlier stages of development. Inaddition, we detected a reduction in Bmp signaling, which was previously shownto regulate spi1b expression. Thissuggests that Smoc2 affects Bmp signaling in the ALPM, potentially bymanipulation of the Bmp gradient.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%">Our GOF analysis indicated that Smoc2 affects the DV patterningand CE movements during gastrulation and the subsequent segmentation stages ofzebrafish development. In addition, Smoc2 appeared to mediate the Wnt5bmediated non-canonical Wnt signaling pathway.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%">Although Smoc2 could affect both the Bmp and the Wnt pathwaydirectly, it is as likely that Smoc2 affects just one of them, whichsubsequently affects the other. Obviously, the interaction and modulation ofthese signaling pathways all depends on the specific microenvironment and thespatio-temporal context of the smoc2expressing cells.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"> <p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%">In order to relate the function of SMOC2 to the homeostasisof the joint, the zebrafish model is less suited. Therefore, we opted for thechicken limb model, as the joints in these limbs are more equal to humanjoints. To overexpress SMOC2 in thechicken limb bud, we cloned SMOC2into the RCAS viral vector that allows for infection of the chicken cells.Localized injection of this RCASBPA-SMOC2virus resulted in an outgrowth defect along the PD axis. Our preliminaryanalysis of the injected limbs indicated a reduced <i style="mso-bidi-font-style:normal">FGF8 and BMP2 expressionin the AER, which are known to regulate the outgrowth of the limb. However, itremains unclear whether SMOC2 directly affects FGF transcription or affects theexpression through a secondary partner, like BMP.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal"> <p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal">As a third animal model, we have used the mouse model toanalyze the phenotype upon loss of SMOC2. Smoc2-/-mouse embryos presented a lethal phenotype during early stages ofdevelopment. As Smoc2 is onlyreported to be expressed in the Reichert’s membrane at those stages,potentially a defect in the Reichert’s membrane underlies the lethal phenotype.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal"> <p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal">In conclusion, our work revealed new insights in theessential function of SMOC2 during zebrafish, chicken and mouse embryonic development,which allows further research to determine the role of SMOC2 within theintricate network of signaling cascades.<!-- /* Font Definitions */@font-face {font-family:"&#65325;&#65331; &#26126;&#26397;"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:1 134676480 16 0 131072 0;}@font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:3 0 0 0 1 0;}@font-face {font-family:Cambria; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1073743103 0 0 415 0;} /* Style Definitions */p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"&#65325;&#65331; &#26126;&#26397;"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}a:link, span.MsoHyperlink {mso-style-priority:99; color:blue; mso-themecolor:hyperlink; text-decoration:underline; text-underline:single;}a:visited, span.MsoHyperlinkFollowed {mso-style-noshow:yes; mso-style-priority:99; color:purple; mso-themecolor:followedhyperlink; text-decoration:underline; text-underline:single;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"&#65325;&#65331; &#26126;&#26397;"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}@page WordSection1 {size:612.0pt 792.0pt; margin:72.0pt 90.0pt 72.0pt 90.0pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;}div.WordSection1 {page:WordSection1;}--><p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal">Musculoskeletal conditions are the leading cause of morbidityand disability, resulting in enormous healthcare costs and working disability.About 150 diseases, syndromes and disorders are grouped in the musculoskeletalconditions, each being progressive and associated with pain (http://www.who.int). One of them is OA, a chronicand progressive joint disorder that is caused by degradation of the articularcartilage and associated defects of the subchondral bone. Although manyresearch groups have investigated the onset and progression of this disablingcondition, the underlying cause or mechanism is far from completely understood.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal">Therefore our lab analyzed a protein extract from bovinearticular cartilage with in vivo chondrogenic activity and isolated the SMOC2protein. In addition, Smoc2 was shownto be expressed in the joint interzone of mouse embryonic digits. Recentevidence indicated that signaling pathways, which are known to regulate theembryonic stages of development, are reactivated during the onset andprogression of multiple conditions, including musculoskeletal diseases.Therefore, we used developmental models to analyze the function of SMOC2.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal"> <p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal">To investigate the SMOC2 associated signaling pathway(s), weused the zebrafish model system and performed loss and gain of functionexperiments. <p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal">Our loss of function analysis indicated that Smoc2 affectedmyeloid development by reducing the number of macrophages and neutrophilsduring the primitive wave of zebrafish hematopoiesis. This defect could betraced back to the reduced expression of the myeloid progenitor specific gene spi1b at earlier stages of development. Inaddition, we detected a reduction in Bmp signaling, which was previously shownto regulate spi1b expression. Thissuggests that Smoc2 affects Bmp signaling in the ALPM, potentially bymanipulation of the Bmp gradient.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal">Our gain of function analysis indicated that Smoc2 affectsthe DV patterning and CE movements during the gastrulation and subsequent segmentationstages of zebrafish development. In addition, Smoc2 appeared to mediate theWnt5b mediated non-canonical Wnt signaling pathway.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal">Although Smoc2 could affect both the Bmp and the Wnt pathwaydirectly, it is as likely that Smoc2 affects just one of them, whichsubsequently affects the other one. Obviously, the interaction and modulationof these signaling pathways all depend on the specific microenvironment and thespatio-temporal context of the smoc2expressing cells.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal"> <p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal">In order to relate the function of SMOC2 to the homeostasisof the joint, the zebrafish model is less suited. Therefore, we opted for thechicken limb model, as the joints in these limbs are more equal to humanjoints. To overexpress SMOC2 in thechicken limb bud, we cloned SMOC2into the RCAS viral vector that allows for infection of the chicken cells.Localized injection of this RCASBPA-SMOC2virus resulted in an outgrowth defect along the proximo-distal axis. Ourpreliminary analysis of the injected limbs indicated a reduced FGF8 expression in the AER, which isknown to regulate the outgrowth of the limb. However, it remains unclearwhether SMOC2 directly affects FGF transcription or affects the expressionthrough a secondary partner, like BMP.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal"> <p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal">As a third animal model, we have used the mouse model toanalyze the phenotype upon loss of SMOC2. Smoc2-/-mouse embryos presented a lethal phenotype during early stages ofdevelopment. As Smoc2 is onlyreported to be expressed in the Reichert’s membrane at those stages,potentially a defect in the Reichert’s membrane underlies the lethal phenotype.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal"> <p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;line-height:150%"><i style="mso-bidi-font-style:normal">In conclusion, our work revealed new insights in theessential function of SMOC2 during zebrafish, chicken and mouse embryonic development,which allows further research to determine the role of SMOC2 within theintricate network of signaling cascades.status: publishe