Functional Substitution Of Shh And Ihh In Mouse Organogenesis

The Hedgehog family?Hedgehog,Hh?is closely related to cell proliferation,differentiation and cell-to-cell communication,and plays a crucial role in embryonic development,homeostasis of mature tissues and tumor development.Mammalian Hh has three homologous genes,?Shh,Ihh and Dhh?.The proteins they encode have two domains,are Hh-N and Hh-C.Shh gene is an important morphogen,which is closely related to the development of neural tube,somites,digestive tract,craniofacial,skeletal axis,anterior and posterior limb buds,and tumor formation.Shh gene is first expressed on the mesoderm and endoderm axis,and its mutation can lead to abnormalities in craniofacial morphology,including mid-cranial line fusion,one-eyed,cerebellar malformation,etc.indicating that Shh plays an extremely important role in the morphogenesis and development of craniomaxillofacial..Ihh is belong to the Hh family and mainly involved in bone development,especially in the development of cartilage.Shh is also involved in regulating the proliferation and differentiation of chondrocytes,especially required for the formation of osteoblasts during the development of cartilage bone.Both IHH and SHH have HH-C end and the Hh-N end structure,and their protein structure shares near 93%identity.Previours studies have demonstrated that the Hh-N end mainly participates in the activation and regulation of Hh signaling pathway,suggesting that Shh and Ihh may have similar biological functions.To explore the specific functions of Shh and Ihh in mouse neural tube development,here,we first mated Shh-Cre mice with Shh^f/f/f mice to obtain Shh^Cre/-mice to generate systemic knockout of Shh gene mice,and the results showed that the knockout Shh mice could lead to monocular deformity,axial pattern defects,neural tube defects and other developmental abnormalities in mice.Next,we mated Shh-Cre mice,Shh^f/f/f mice and pMes-Ihh mice to obtain conditionally overexpressed Ihh at the position of Shh gene expression and the Shh gene was knocked out,we named the three transgenic mice as Shh^Cre/-;pMes-Ihh.the results showed that the defects of Shh knockout mice were partially rescued,the defects of craniofacial midline structure including mouth,bilateral eyes and nose were rescued,and the defects of limbs were totally be rescued.However,overexpressed Ihh could not rescue the defects of neural tube floor,neural tube,dorsal abdominal axial pattern,and brain deformities.To further explore the mechanism why Ihh could not replace Shh to rescue neural tube defects,we mated Shh-Cre mice with R26R-mTmG mice to trace Shh expression pattern.The results showed that in Shh^Cre/-;pMes-Ihh mice,there was no expression of Shh in the neural floor region,indicating that after the knocked out Shh gene,SHH protein is weakly secretion at the notochord and can't reached the neural floor to induce the expression of Shh in the neural floor.That is,in Shh^Cre/-;pMes-Ihh mice,Cre enzyme and Shh were not expressed in the neural floor,therefore Ihh could not be overexpressed in the neural tube floor,and the deformity of the neural tube could not be rescued.Due to Ihh could not be overexpressed in the neural tube,and the effect of Ihh on neural tube development could not be observed in the Shh^Cre/-;pMes-Ihh mice,we used chicken embryo in vitro transplantation technology to study the effects of SHH and IHH on neural tube development.In the HH13 stage of chicken embryo,we first peeled the notochord when the tail neural tube is not fully closed,and placed SHH and IHH proteins agarose beads at the both sides of the neural tube.The results showed that SHH,not IHH could induce the close of neural tube suggesting that Ihh and Shh could not be functionally substitution in the development of neural tube.This study provide a reference for revealing the functional substitution mechanism of Shh and Ihh in mouse organogenesis and the developmental mechanisms of neural tube defects.The normal closure of neural tube is regulated by multiple gene networks,and its in-depth study may provide useful information on the mechanism of neurodevelopmental abnormalities and provide new targets for clinical treatment.