The homozygous variant p.(L180Gfs*74) of LRO-associated MMP21 causes laterality defects.

(A) MMP21 consists of an N-terminal signal peptide (SP), a propeptide with Furin cleavage site (FCS), a catalytic domain (CD) and C-terminal Hemopexin-like repeats. Zn coordination as well as conserved residues of the cysteine-switch (C), catalytic core (HEXXHXXGXXH) and methionine-turn (M) are marked. The position of the frameshift mutation p.(L180Gfs*74) is indicated in the CD. (B) Domain composition of Xenopus Mmp21 is equivalent to that of human MMP21. (C) The pedigree of the female proband (II-6) illustrates that both she and the youngest sibling (II-1) presented with heterotaxy (HTX). Wild-type and mutant MMP21 alleles are indicated by a plus and minus symbol, respectively. (D) During Xenopus development, mmp21 is expressed in the central flow-generating cells of the LRO with noto- and hypochordal fate. The sensory LRO cells with somitic fate, surrounded by endodermal cells, as well as the circumblastoporal collar (CBC), are free of mmp21 transcripts. In later stages, expression of mmp21 is restricted to the tailbud and successively vanishes. Dashed red lines indicate consecutive incision and transversal sectioning. Dashed red line square outlines the magnified region. Dashed black lines display tissue borders.

Loss of Mmp21 disturbs laterality owing to disturbed fluid flow response.

(A) Genome editing of mmp21 through Cas9 ribonucleoprotein (CRNP) injections interferes with situs solitus (SS) and induces high rates of situs inversus (SI) and heterotaxy (HTX). Crispant embryos developed otherwise normally with low mortality and malformation rates. (B) Left-sided pitx2 expression is absent in Mmp21 depleted embryos. Uniquely, sided-injections revealed a bilateral requirement of Mmp21. (C) Speed and directionality of the ciliary driven leftward fluid flow was not affected by a loss of Mmp21. Color-graded gradient time trails depict the displacement of fluorescent microspheres, which were used for analysis, within an 8 s time window in the white-outlined LRO region. (D) The nodal1 domain at the LRO was not reduced in size in mmp21 mutants, but (E) flow-dependent downregulation of dand5 on the left side was compromised.

Mmp21 behaves as a morphogen and acts as fluid flow effector.

(A) Unilateral injections of mmp21 mRNA induce ectopic expression of pitx2 on the right side. This effect was more pronounced on the right side, although higher mmp21 mRNA doses compensate for the difference. (B) The active site mutant (E338A) but not the full HEXXH motif mutant (H337F-E33A-H341F) Mmp21 retained the Nodal-cascade inducing effect. (C) nodal1 expression was not impacted by overexpressed Mmp21, whereas (D) the right dand5 domain was reduced in size to the level of the left side post-flow. (E) C-terminally HA tagged Mmp21 spreads over LRO cells. Co-expressed mGFP marks targeted cells on the right side. F-actin staining indicates cell borders. Dashed white line squares outline the magnified regions, highlighting Mmp21 presence on targeted (expressing) and non-targeted (non-expressing) cells. Orthogonal view shows enrichment of Mmp21 above apical mGFP and F-actin signals. (F) Methylcellulose (MC) increases the viscosity of the extracellular fluid and restricts the field of action of overexpressed and endogenous Mmp21, as judged by the expression pattern of pitx2.

Cirop is a downstream transducer of Mmp21 at LRO cilia.

(A) Cirop is composed of an N-terminal signal peptide (SP), a propeptide, a catalytic domain (CD) and a C-terminal transmembrane domain (TMD). Coordination of the Zn atom as well as the conserved residues of the catalytic site (HEXXH plus C-terminal H) and methionine-turn (M) are depicted. (B) C-terminally HA tagged Cirop localizes to LRO cilia and accumulates at cell borders. Cirop-HA producing cells are marked by co-expressed mGFP. Cell borders are visualized by F-actin staining. Dashed white line squares outline the magnified regions, highlighting the ciliary localization of Cirop-HA. An orthogonal view of the region, indicated by two dashed white lines, shows a single protruding cilium marked by a continuous apical Cirop-HA signal (white arrowheads). (C) Cirop acts downstream of Mmp21, as loss of the Nodal cascade in left-sided cirop f0 mutants can be transformed into right-sided cascade activity by co-injecting the mRNA of diffusible Mmp21. (D) Model showing the key steps of laterality determination in vertebrates with a fluid flow generating LRO: Central LRO cells generate a cilia-driven leftward fluid flow and secrete Mmp21 from their apical side. Flow directs diffusion of Mmp21 to left-sided LRO sensor cells. Sensor cells perceive Mmp21 via Cirop-positive sensory cilia. Cirop-dependent signaling of Mmp21 represses dand5 on mRNA level, attenuating Dand5 protein levels on the left side. Left-sided Nodal1 is released from repression by Dand5 and induces the Nodal signaling cascade in the left lateral plate mesoderm (LPM).

Mature Mmp21 has a predicted tertiary structure with two distinct globular domains.

(A) Removal of the propeptide lowers the molecular weight of Mmp21 to 49.8 kDa. (B) ColabFold was employed to predict the conformation of mature Mmp21, which adopts a bipolar, globular structure formed by the catalytic domain (CD) and the Hemopexin-like beta-propeller. AlphaFill enriched the model with the catalytic zinc atom. The conserved histidine and glutamate residues (H337, E338, H341 and H347) of the catalytic core are displayed with their side chains in red. All other residues N-terminal of the C-terminal CD border were colored in light red, while residues C-terminal of the CD were colored in turquoise. Visualization of the catalytic cleft and core was optimized by concealing the loop between A304 and D311.

Mutagenesis of mmp21 is efficient and creates rescuable laterality phenotype.

(A) Sanger sequencing data of the wild-type and mutagenized mmp21 region is displayed as chromatogram. Signal intensity is given in relative fluorescence units (RFU). Synthego ICE analysis confirms efficient mutagenesis, resulting in various genomic DNA species with small deletions. The sgRNA target site is underscored in black and the protospacer adjacent motif with a dashed red line. Vertical black dashed lines indicate the site of the Cas9-induced DNA double-strand break. (B) Specificity of the mmp21 crispant phenotype was verified by the rescue potential of mmp21 mRNA, which restored left-sided pitx2 expression. (C) Expression of pitx2 was also rescued by a left-sided knockdown of dand5, mediated by a translation blocking morpholino oligomer (TBMO), showing that events downstream of flow-dependent dand5 repression are Mmp21 independent.

Secreted Mmp21 does not affect the midline barrier.

(A) Overexpression of Mmp21 does not reduce the mRNA level of lefty in the midline. (B) Bilateral Nodal cascade activity, caused by elevated Mmp21 levels, is not the result of midline leakage, as nodal1 expression started simultaneously between the left and right side. (C) Inhibition of secretion via Brefeldin A incubation prevents spreading of Mmp21-HA over LRO tissue. Mmp21-HA synthesizing cells are indicated by mGFP, which was used as lineage tracer. Cell borders are marked by F-actin. (D) Diffusion of Mmp21 in LRO-unrelated naïve epithelial cells occurs along cell borders, which are visualized by F-actin. Cell bodies and borders of Mmp21 secreting cells are marked by co-expressed mGFP. Dashed white line rectangles outline the magnified regions, highlighting Mmp21-HA foci between non-secreting cells. Orthogonal view shows apical aggregation of Mmp21-HA.