Sonic Hedgehog in immune/embryology Development of Mammals

Introduction

Sonic Hedgehog Homologue (Shh) is a transcriptional regulator and protein product of a morphogen known as Hedgehog. Originally Hedgehog (hh) gene was found in Drosophila. Within this family,  are Desert Hedgehog, Indian Hedgehog and Sonic Hedgehog–all involved in developmental body patterning. Hedgehog is known to have evolved before chordates and vertebrates but has orthologues in mammalian development (Kumar, 1996). Shh is known to play a crucial role in patterning and limb development by intercellular signaling. Shh works in coordination with many gene families and therefor, plays a role in development of many systems.

The video clip below shows a preview of research done on Sonic Hedgehog in chicks and mammals.

Sonic Hedgehog (Shh) in chordates

In humans and mice, Shh generally works with the Patched Protein Receptor. Shh inhibits the repressor function of Patched on Smoothened (SMO), a transcriptional regulator Nakano et al 2004).  Shh, through Smoothened, also targets activation of GLI (Glioblastoma) protein effect cell proliferation in embryos (Dahlen et al, 2004). This page discusses the roles of Shh in development: specifically immunological and neural tube development. Two papers will shed light to these development in these systems.

(Credit: ocskincancer.com) Both Patched (PTC) and SMO are involved in thymocyte development. This gives reason as to why Shh plays a great role in immunology.

Shh involvement in development of the nervous system: According to Lingtingtung Y, and Chiang C

Shh signaling is important in development in many systems in mammals. One of the systems involving Shh is the nervous system, which is comprised of the central and peripheral nervous system. Shh has been experimentally shown to play a role in development of the brain and spinal cord. The neural tube is the precursor to the central nervous system; by affecting cellular signaling in the neural tube, Shh plays a role in development of the brain and spinal cord. Deficiencies in cellular signaling in the neural tube result in a variety birth defects.

Lingtingtung and Chiang propose Shh knockout will result in ventralization of mouse embryos of due to differential expression of motor neurons and interneurons. Shh is expressed in the notochord and floorplate, without Shh signaling, there is a lack of motorneurons, interneurons and a floorplate. Data is displayed below.

(Credit: Lingtingtung et al.)Proposed effect of Shh mutation in neural tube signaling

The Evx1, En1, and Sax1 are neuronal proteins involved in development of the nervous system. RNA In Situ analysis displays the absence of Sax1 and differential localization of Evx1 and En1, proving Shh involvement in neural tube development.

(Credit: Lingtintung et al.) Experimental evidence displaying differential expression of motorneurons and interneurons in Shh-/- and Shh+/+. Motor neuron Sax1 are absent in Shh-/- mutants( A and E), but interneurons En1, and Evx 1 are differentially localized. (B and F, C and G)

Shh knockout in a mouse embryo results in ventralization as shown in the image below, which is produced by gene targeting and deletion of Shh.

(Credit: Lingtintung et al) Morphological differences between wildtype (A-C) and Shh-/- mutant (D-F) Shh-/- embryos display ventrilization and lack of ocular and hypoglossal motor nerves as displayed by *(E and F respectively). While non-motor nerves are present in mutant and wildtype.

Shh involvement in development of the immune system: According to DK Shah et. al.

Shh is also involved in developmental cellular signaling in the immune system. DK Shah et al study Shh involvement in thymocyte development and differentiation. The data is displayed below.

Differentiation and proliferation of thymocytes:

(Credit: DK Shah et al.) Shh involvement in thymocyte developmental signaling

The following article, Reduced thymocyte development in Sonic Hedgehog knockout embryos, discusses the effect of the Shh protein on thymocyte development.

Thymocyte development is dependent on Shh protein dosage.

(Credit: Shah et al.) CD45 receptor is expressed by all cells with a leukocyte lineage. Anti-CD25, and Anti-CD44 staining were gated on CD45 to determine relative thymocyte numbers. Shh mutants. Shh-/- Displayed low numbers of thymocytes in general(A) as well as CD45(B) and CD25(C) thymocytes, suggesting that knockout of the gene reduced thymocyte proliferation.

Since the data displays that Shh-/- mutants have low numbers of thymocyte proliferation, it would be assumed that excessive dosage would result in greater number of thymocytes. However, mmunohistochemical analysis actually showed that excessive dosage results in an arrest in thymocyte development.

(Credit: DK Shah et al.) Thymocyte development is dosage dependent on Shh protein. Immunohistochemical analysis of samples after injecting varying concentrations of Shh are displayed. In all experiments optimal dosage of Shh appears to be 0.0005 micrograms per milliliter, thymocyte proliferation (A, C) is optimal at this concentration and decreases in either direction of concentration manipulation. Proliferation of Tcells to the double positive (DP) stage is also displayed in images (B, E, and F). This optimal concentration is maintained in study of CD4 and CD8 proliferation.

The data shown is convincing as to the effect of Shh on thymocytes, but in contrast to previously cited papers,  there is very little mention of Bmp and Wnt signaling. The pathways are intertwined and the discussion failed to address the effect of manipulation of the Shh protein on the other pathways involved in thymocyte development.

Bibliography

Dahlen A, Fletcher CD, Mertens F, Fletcher JA, Perez-Atayde AR, Hicks MJ, Debiec-Rychter M, Sciot R, Wejde J, Wedin R. et al. Activation of the GLI oncogene through fusion with the beta-actin gene (ACTB) in a group of distinctive pericytic neoplasms: pericytoma with t(7;12) American  Journal of Pathology. 2004;164:1645–1653. doi: 10.1016/S0002-9440(10)63723-6.

Litingtung Y, Chiang C. Control of Shh activity and signaling in the neural tube. Developmental Dynamics 2000;219:143–154. doi: 10.1002/1097-0177(2000)9999:9999<::AID-DVDY1050>3.3.CO;2-H.

Nakano Y, Nystedt S, Shivdasani A. A, Strutt H, Thomas C, et al. Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila. Mech Dev.2004;121:507–518

Shah, D. K., A. L. Hager-Theodorides, S.V. Outram, S.E. Ross, A. Varas, T. Crompton. 2004. Reduced thymocyte development in sonic hedgehog knockout embryos. Journal of Immunology 172: 2296–2306.

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