Welcome

We would like to thank Jian Ming Khor and Gary Wessel for their useful tips and helpful correspondence.

Goals

Functional validation of candidate regulatory variation

Sub aim: Developing a reliable and efficient method in the lab

Main idea: GFP reporter construct with modified regulatory region microinjected into fertilized sea urchin eggs -> output intensity of fluorescence, could be:

• Tissue specific
• Developmental time specific
• Genetic background specific
• Environment specific

Primary gene of interest

ECM3 - extracellular matrix protein 3. LOC579397 / SPU_001796.

• Outlier putatively adaptive cis-regulatory variant.
• Down-regulated in populations most frequently exposed to ph <7.8 & up-regulated in populations less frequently exposed to ph <7.8.
• In a conserved promoter region with L. variegatus (50 Mya).
• High expression during early development
• Important biomineralisation gene.

(a) Expression pattern

(b) Location of ecm3 expression

(c) Emc3 knockout shows abnormal larval skeleton development

Figure 1: Ecm3 facts

Available control constructs

From Mamiko Yajima:

• SM50-GFP construct

From Jimmy Khor¹:

• Sp-anpep_1
• Sp-Clect_25

(a) SM50-GFP

(b) epGFPII backbone

Figure 2: Constructs

(a) epGFPII with Sp-anpep_1

Figure 3: GFP expression upon successful Sp-anpep_1 injection

Available testing constructs

epGFPII backbone with:

• ECM3 promoter region, 1,700 bp upstream TSS
• ECM3 promoter region also with UTR

(a) epGFPII-Ecm3

Figure 4: Contruct with ECM3 promoter

Alternative methods

Challanges

Troubleshooting

Reference:

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1. Khor, J. M., Guerrero-Santoro, J., & Ettensohn, C. A. (2019). Genome-wide identification of binding sites and gene targets of Alx1, a pivotal regulator of echinoderm skeletogenesis. Development, 146(16), dev180653.