Expression of Id2 in the developing limb is associated with zones of active BMP signaling and marks the regions of growth and differentiation of the developing digits.
"Inhibitor of DNA binding/differentiation factor 2 (Id2) [is expressed] in the developing chicken limb. Prior to stage 25, Id2 is expressed in the anterior and posterior mesoderm, the AER, and in the early skeletal chondrogenic aggregates. At more advanced stages of limb development Id2 is expressed in the undifferentiated subectodermal and interdigital mesenchyme and exhibits specific domains of expression in the growing digits. These expression domains were closely coincident with zones of activation of BMP-signaling as deduced from the distribution of phosphorylated SMADs 1/5/8. In micromass cultures transcripts of Id2 are associated with the nodules of chondrogenic differentiation. Expression of Id2 both in vivo and in vitro was up-regulated in experiments of BMP-gain-of-function and down-regulated after treatments with BMP-antagonists. Interdigital application of TGFbeta2 transiently upregulates Id2 in coincidence with the inhibition of interdigital cell death and the commitment of the interdigital mesenchyme to form an ectopic digit. Id2 is a molecular mediator of BMP signaling acting in concert with the TGFbeta pathway during the formation of the digits."
"During development Id1, Id2 and Id3 possess overlapping patterns of expression showing only slight differences while Id4 differs from the rest of the family members"<-so if one of the first 3 Id's are pro- or anti-chondrogenic then it's likely that the others are.
This study states that Id is anti-chondrogenic.
"Inhibitors of differentiation (Id) proteins are helix-loop-helix (HLH) transcription factors lacking a DNA-binding domain. Id proteins modulate cell proliferation, apoptosis and differentiation in embryonic/fetal tissue. Perturbation of any of these processes in cells of the developing orofacial region results in orofacial anomalies. Chondrogenesis, a process integral to normal orofacial ontogenesis, is known to be modulated, in part, by Id proteins. In the present study, the mRNA and protein expression patterns of Id1, Id2, Id3 and Id4 were examined in developing murine orofacial tissue in vivo, as well as in murine embryonic maxillary mesenchymal cells in vitro. The functional role of Ids during chondrogenesis was also explored in vitro. Results reveal that cells derived from developing murine orofacial tissue (1) express Id1, Id2, Id3 and Id4 mRNAs and proteins on each of gestational days 12-14, (2) express all four Id proteins in a developmentally regulated manner, (3) undergo chondrogenesis and express genes encoding various chondrogenic marker proteins (e.g. Runx2, Type X collagen, Sox9) when cultured under micromass conditions and (4) can have their chondrogenic potential regulated via alteration of Id protein function through overexpression of a basic HLH factor."
High density decreases Id expression.
"formation of chondrogenic nodules [occurs] in HDM cultures of MEMM cells overexpressing the Id-binding protein E2a/E47"<-meaning there's less free Id's floating around.
"The TWIST family of basic helix-loop-helix transcription factors, Twist-1 and Dermo-1 are known mediators of mesodermal tissue development and contribute to correct patterning of the skeleton. freshly purified human bone marrow-derived mesenchymal stromal/stem cells (MSC) express high levels of Twist-1 and Dermo-1 which are downregulated following ex vivo expansion[we need to mimic this expansion in our epiphyseal stem cells]. Enforced expression of Twist-1 or Dermo-1 in human MSC cultures increased expression of the MSC marker, STRO-1, and the early osteogenic transcription factors, Runx2 and Msx2. overexpression of Twist-1 and Dermo-1 [decreases] the gene expression of bone morphogenic protein-2, bone sialoprotein, osteopontin, alkaline phosphatase and osteocalcin. High expressing Twist-1 or Dermo-1 MSC lines exhibited an enhanced proliferative potential of approximately 2.5-fold compared with control MSC populations that were associated with elevated levels of Id-1 and Id-2 gene expression{so perhaps Twist-1 expressing cells will proliferate until they become denser thus fixing the Id problem}. High expressing Twist-1{however Twist1 overexpression may increase height by increasing cellular proliferation} and Dermo-1 MSC displayed a decreased capacity for osteo/chondrogenic differentiation and an enhanced capacity to undergo adipogenesis."
In the LSJL gene expression study the loads were only applied for 3 days. This was not enough time for Twist1 expressing cells to reach critical mass with cellular expansion before Id could be inhibited and chondrogenesis induced.
This explains why it takes a while to see LSJL results, as initially LSJL proceeds on the BMP-Smad5B-Id pathway until cell density is high enough then after LSJL should induce chondrogenesis. LSJL should have no problems in open plates as there is already high density.
Proper expression of helix-loop-helix protein Id2 is important to chondrogenic differentiation of ATDC5 cells.
"The process of chondrogenesis can be mimicked in vitro by insulin treatment of mouse ATDC5 chondroprogenitor cells. We carried out a large-scale screening through retroviral insertion mutagenesis and isolated a fast-growing ATDC5 clone incapable of chondrogenic differentiation. Inverse-PCR analysis of this clone revealed that the retroviral DNA was inserted into the promoter region of mouse Id2 (inhibitor of DNA-binding protein 2) gene. This retroviral insertion increased Id2 protein levels to twice those found in normal ATDC5 cells. An elevated level of Id2 protein [is] responsible for inhibition of chondrogenic differentiation, ATDC5 cells were infected with a retrovirus to stably express Id2. ATDC5 cells expressing ectopic Id2 exhibited signs of de-differentiation, such as rapid growth, and insulin failed to induce expression of Sox9 (Sry-type high-mobility-group box 9) or matrix genes such as type II collagen (COL2) in these cells. When endogenous Id2 was knocked down by siRNA (small interfering RNA) in ATDC5 cells, expression of Sox9 and COL2 was increased and chondrogenic differentiation was accelerated{and expression of Id2 was reduced in LSJL}. To examine how Id2 is expressed in chondrocytes in vivo, we carried out immunostaining of E16.5 mouse embryos and found that Id2 is expressed in articular chondrocytes and proliferating chondrocytes, but barely detectable in hypertrophic chondrocytes."
Proper expression of helix-loop-helix protein Id2 is important to chondrogenic differentiation of ATDC5 cells.
"The process of chondrogenesis can be mimicked in vitro by insulin treatment of mouse ATDC5 chondroprogenitor cells. We carried out a large-scale screening through retroviral insertion mutagenesis and isolated a fast-growing ATDC5 clone incapable of chondrogenic differentiation. Inverse-PCR analysis of this clone revealed that the retroviral DNA was inserted into the promoter region of mouse Id2 (inhibitor of DNA-binding protein 2) gene. This retroviral insertion increased Id2 protein levels to twice those found in normal ATDC5 cells. An elevated level of Id2 protein [is] responsible for inhibition of chondrogenic differentiation, ATDC5 cells were infected with a retrovirus to stably express Id2. ATDC5 cells expressing ectopic Id2 exhibited signs of de-differentiation, such as rapid growth, and insulin failed to induce expression of Sox9 (Sry-type high-mobility-group box 9) or matrix genes such as type II collagen (COL2) in these cells. When endogenous Id2 was knocked down by siRNA (small interfering RNA) in ATDC5 cells, expression of Sox9 and COL2 was increased and chondrogenic differentiation was accelerated{and expression of Id2 was reduced in LSJL}. To examine how Id2 is expressed in chondrocytes in vivo, we carried out immunostaining of E16.5 mouse embryos and found that Id2 is expressed in articular chondrocytes and proliferating chondrocytes, but barely detectable in hypertrophic chondrocytes."
